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claran

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modular-s2orc

like 3

2014-10-01T00:00:00.000Z

1974-09-01T00:00:00.000Z

26347958

s2

Carcinogens in rat milk. Transfer of ingested diethylnitrosamine into milk by lactating rats. Mothers of 5-day old rats were given diethylnitrosamine (DEN) (130 mg/kg body weight) by stomach tube. The milk removed from the stomachs of the suckling young contained 5, 16 and 36 parts/106 of DEN at 2, 4 and 6 hours respectively after they started suckling the treated mothers. After 49 hours, DEN was no more detectable in the milk. ALKYLNITROSAMINES are among the most versatile and potent carcinogens, able to induce tumours in various organs of several animal species (Magee and Barnes, 1967;Druckrey et al., 1967). No direct evidence is available to indicate whether nitrosamines could be carcinogenic in man, but in workers exposed industrially to dimethylnitrosamine (DMN), acute liver damage and cirrhosis have been observed (Freund, 1937); there is little doubt that under certain conditions nitroso compounds could represent health hazards (Magee, 1971). Search for nitrosamines in foodstuffs is fraught with difficulties, due to the possibility of false positives, artefacts or losses in the course of the analytical procedures. Volatile nitrosamines have been detected in various foodstuffs (Ender et al., 1964;Hedler and Marquardt, 1968;Fazio et al., 1971;Fong and Walsh, 1971;Crosby et al., 1972 and others). In view of the great susceptibility of the very young to mnany carcinogens, including the alkylnitrosamines (for references see Magee and Barnes, 1967;Druckrey et al., 1967;Schoental, 1974a), particular attention is required to ensure that milk, the main foodstuff of the young, does not contain carcinogenic nitroso compounds. Cow's milk and its products were tested by several groups of workers but gave variable results. Traces of diethylnitrosamine (DEN) have been detected in pasteurized milk and in Tilsit cheese (Hedler and Marquardt, 1968) and of DMN in several varieties of cheese (Crosby et al., 1972). On the other hand, when extracts from fat-free heat dried milk were examined (after oxidation to the respective nitramines) by electron capture gas chromatography on Rheoplex 400 columns, a peak coinciding with the oxidation products of DMN has been observed. However, when re-chromatographed on OV-1 column, the material from milk differed from the reference sample of the oxidation product of DMN (Reineccius and Coulter, 1972). Another approach to the subject is to determine whether and under which experimental conditions volatile alkyliiitrosamines, administered to lactating animals, would appear in the milk, in what concentration and whether this could account for the induction of tumours in the offspring. The possibility that carcinogenic metabolites of the parent compound (Blattmann and Preussman, 1973) may be excreted in the milk has also to be considered. In our exploratory experiments, when nursing rats were given a few doses of DEN (130 mg/kg bodv weight) various tumours, including aesthesioneuroepitheliomata developed in the suckling young (Schoental and Appleby, 1973;Schoental, 1974b). In the present communication we report the finding of free DEN in the milk removed from the stomach of the suckling young at various times within a few hours after administration of DEN to their mothers. EXPERIMENTS AND RESULTS Three white, mother rats with their litters (8-11 each) random bred from the Wistar-Porton strain were received from the M.R.C. Laboratory Animals Centre, Carshalton 5 days after parturition. Two of the mothers were given DEN (40 mg each in 0 5 ml of 10% aqueous ethanol) by stomach tube and were kept for about 30 min away from their young in order to avoid direct transfer of DEN to the young on being licked by the mothers. At 2, 4, 6 and 49 h after the mother rats had been returned to their offspring, 5 of the young rats were killed by decapitation, the stomachs were dissected out and their content (consisting of clotted milk) removed, pooled and kept in glass bottles in deep freeze (at 20°C) until it could be analysed. Five of the offspring of the third mother rat, kept as controls, were killed when 7 days old, corresponding to the age of the experimental ones killed at 49 h after the beginning of the experiment. The milk removed from their stomachs was tested as negative control, in a similar way to that of the experimental sucklings. The milk samples were extracted with I ml of dichloromethane (DCM) and 5 Id of this DCM extract was injected without further clean up on to a Philips model R gas chromatograph (GC) linked to an AEI MS902 mass spectrometer. The GC was equipped with a 2-4 m x 2 mm ID GC column containing 15% carbowax 20M in series with a 5-4 m x 2 mm ID column containing 500 carbowax 20M, the stationary phase being supported on 80-100 mesh AW Chromosorb W on both columns. The GC oven temperature was 145°C. In order to separate the helium carrier gas from the GC eluant before entering into the mass spectrometer, a membrane separator (Gough and Webb, 1972) was used and a solvent venting device (Gough and Webb, 1973) was incorporated in the GC to prevent pressure surges in the mass spectrometer. The presence of DEN in the extracts was established by parent ion monitoring (Gough and Webb, 1972) The limit of detection for DEN was 1 mg/l. There was no evidence for the presence of free volatile metabolic derivatives of DEN. DISCUSSION It is of interest that significant concentrations of unchanged free DEN were found in the milk of suckling rats within a few hours after their mothers were given DEN (130 mg/kg) by stomach tube. Exact measurements of the amounts of DEN ingested with milk by the youing are difficult to obtain. The young were left with their mothers until shortly before administering the DEN, hence the stomachs of the young would contain some " clean " milk which would suppress the DEN concentration during the first few hours after administration. Diffusion of DEN from the stomach and digestion would also affect the accuracy of the data. However, on the basis that a suckling rat of 6-10 g body weight would receive about 1 ml of milk within the time that DEN was present in the mother's milk, a dose of about 20 ,tg of DEN would be received by the offspring. In a parallel experiment in which the mothers were given several doses of DEN (each 130 mg/kg), the suckling rats were allowed to survive. They grew and developed in apparent good health until tumours developed later in life (Schoental, 1974b). On the basis of the present observations, the formation of the tumours in the offspring can be attributed, at least in part, to the repeated ingestion of unchanged DEN during lactation. We thank Dr J. M. Barnes for the gift of diethylnitrosamine which was used in these experiments. One of us (R.S.) is indebted to Professor E. Cotchin for hospitality in his Department.

v3-fos

2020-12-10T09:04:17.311Z

1974-03-01T00:00:00.000Z

237231848

s2

Comparison of Macroscopic Examination, Routine Gram Stains, and Routine Subcultures in the Initial Detection of Positive Blood Cultures Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO2. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO2) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture. Blood was cultured in two vacuum bottles containing Columbia broth with sodium polyanethol sulfonate and CO2. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in C02) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture. There are many methods recommended for the routine-culture and examination of blood samples. There is agreement that blood cultures should be observed at least daily for macroscopic growth, but suggestions as to the need for routine Gram stains and blind subcultures vary from author to author. We are not aware of any published report comparing the efficacy of these procedures in the initial detection of positive blood cultures. Therefore, a comparative study was carried out to assess the value of the three approaches to detection of initial microbial growth in blood cultures. MATERIALS AND METHODS Blood cultures were obtained from patients in the University of Minnesota hospitals (approximately 800 beds) and were processed in the Diagnostic Microbiology Laboratory, which receives about 700 blood cultures per month. Blood was cultured in two vacuum bottles containing 100 ml of Columbia broth with 0.03% sodium polyanethol sulfonate and 10% CO2 (B-D Division of BioQuest). The blood was drawn by physicians, and the amount inoculated into each bottle varied from a few drops to approximately 10 ml. When the bottles were received in the laboratory, filtered air was admitted to one bottle by using a blood collection set (B-D Division of BioQuest); the collection set was removed from the bottle before incubation. The other bottle was considered to be anaerobic. Penicillinase (Difco) was added when indicated. The blood cultures were incubated at 35 C for 7 days or until growth was noted. Cultures from patients with suspected bacte-rial endocarditis or brucellosis were held for 2 to 3 weeks. Cultures were examined macroscopically for growth in the morning and afternoon on the 1st day of incubation and in the morning of each day thereafter. Cultures that appeared positive were Gram stained immediately, and subcultures were made according to the types of organisms seen. Gram stains were performed on all bottles that appeared macroscopically negative on the 1st, 4th, and 7th day of incubation. Blind subcultures were also made on the 1st and 4th days to a sheep blood agar plate (incubated anaerobically) and to a chocolate agar plate (incubated in C02). Subculture plates were held for 2 days before they were discarded as negative. Each procedure was performed in the routine laboratory by a total of 13 microbiology technologists on a rotation basis. RESULTS The method of first detection of growth is shown in Table 1. There were a total of 7,357 blood cultures examined over a period of 10.5 months, and 1,127 were positive. Of these, 734 Table 2 shows the day on which cultures were noted to be positive by three methods of detection. Forty-seven percent of those first detected by macroscopic examination were found on the 1st day. Of those first detected by Gram stain, 49% were found on the 1st day, 28% were found on the 4th day, and 23% were found on the 7th day. Of the positive cultures first detected by subculture, 76% were detected on the 1st day and 24% were detected on the 4th day. One hundred twenty-five positive cultures were not apparent macroscopically on the 1st day, and 106 positive cultures were not detected by Gram stain on the 1st day, nor were 33 positives detected by Gram stain on the 4th day. Of the 1,127 positive blood cultures, 467 (41.4%) were detected on the 1st day either macroscopically or by Gram stain. The numbers and types of organisms isolated, along with the mean times for detection (by all methods) are shown in Table 3. Of all the organisms isolated, Haemophilus influenzae, H. parainfluenzae, Moraxella sp., and Neisseria gonorrhoeae were detected first only by subculture. These organisms were never detected first by macroscopic examination or Gram stain, although approximately one-half of the Haemophilus cultures appeared macroscopically positive subsequent to subculture. Of the Pseudomonas aeruginosa isolated, only one-third were detected first macroscopically, one-third were detected first by Gram stain, and the remaining one-third were detected first only by subculture. Anaerobic organisms were almost always de- tected either by macroscopic examination or Gram stain. Only four strains of Bacteroides were first detected on the anaerobic subculture plate. The organisms detected first by the 7th-day Gram stain included Propionibacterium acnes, Candida, Corynebacterium, Peptococcus, Pseudomonas, Staphylococcus epidermidis, and Torulopsis glabrata, although some strains of these bacteria were also detected by the other methods. DISCUSSION The data presented indicate that, for optimal speed in detection and identification of organisms from positive blood cultures, both routine Gram stains and blind subcultures should be performed in addition to daily visual inspection of cultures. If routine Gram stains and subcultures had not been performed, the detection of a positive blood culture or presumptive identification of the organism would have been delayed by at least 1 day in 35% of the cultures. If, in PER addition to macroscopic inspection, only Gram stains were done, there would have been a delay in 12% of the cultures. If only subcultures had been performed, 23% of the positive reports would have been delayed at least 1 day. One might make the point that results of subcultures themselves were delayed by 1 day and that the culture in some cases may have been positive macroscopically the next day; however, even though this may be true, at the time of reading the subcultures a more definite identification of the organism could be given to the physician rather than just its Gram stain morphology. Subcultures were especially important in the more rapid detection of Haemophilus, since these organisms were all detected first only by this means. Both Gramstains and subcultures were also valuable in the more rapid detection of Pseudomonas, as two-thirds of those isolated were detected first only by Gram stain or subculture. Our experience with Pseudomonas bears out the study by Slotnick and Sacks (3), who stated that the use of visible growth or Gram stains alone are not sufficient to detect the presence of Pseudomonas in blood culture media. Although there is no question about the importance of a Gram stain to detect positive blood cultures on the 1st day, the value of Gram stains on the 4th day in relation to the amount of work involved and the clinical importance might be questioned. In this study, approximately 6% of the positives were first detected by Gram stains on the 4th day. Individual judg-ments would have to be made as to whether detection of the positive on the 4th day would be that much more important than detection by subculture the following day. The blood cultures in this study were incubated for a maximum of 7 days, except in cases of suspected brucellosis or endocarditis. This incubation period was based on the results of previous unpublished studies in our laboratory which demonstrated the rarity of isolation of clinically significant organisms after 1 week of incubation. Effersoe (1) has also shown that incubation for longer than 7 days is not necessary, especially if "control" Gram stain and subcultures are performed. It was not the intent of this study to assess the overall rapidity of organism detection. However, the information in Table 3 does allow for comparison with other recently published studies (2, 4) on this subject. On the basis of these comparisons, we feel that the spacing of the procedures evaluated in our study are appropriate and practical for the clinical laboratory.

v3-fos

2020-12-10T09:04:12.723Z

1974-04-01T00:00:00.000Z

237232826

s2

Relationship of Cellular Fatty Acid Composition to Survival of Lactobacillus bulgaricus in Liquid Nitrogen Concentrated cultures of Lactobacillus bulgaricus were prepared by resuspending cells grown in semisynthetic media in sterile 10% non-fat milk solids. The concentrated cultures were frozen in liquid nitrogen for 24 h. The cell suspensions exhibited decreased viability after storage, and the amount of death varied among the different strains tested. Storage stability of all strains examined was improved by supplementing the growth medium with sodium oleate. Radioisotopes were used to study the fate of sodium oleate with L. bulgaricus NCS1. [1-14C]sodium oleate was incorporated solely into the lipid portion of the cells, including both neutral and polar lipids. The fatty acid composition of L. bulgaricus NCS1, NCS2, NCS3, and NCS4 grown with and without sodium oleate was studied. The major fatty acids of strains NCS1, NCS2, and NCS3 grown without sodium oleate were dodecanoic, tetradecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids. In addition to these, strain NCS4 contained C19 cyclopropane fatty acid. The major fatty acids of all strains grown with sodium oleate were tetradecanoic, hexadecanoic, hexadecenoic, octadecenoic, and C19 cyclopropane fatty acids. All strains grown in broth containing sodium oleate contained larger amounts of octadecenoic and C19 cyclopropane fatty acid, and less saturated fatty acids than when grown without sodium oleate. Statistical analyses indicated that C19 cyclopropane fatty acid was most closely related to stability of the lactobacilli in liquid nitrogen. A negative regression line that was significant at P < 0.001 was obtained when the cellular content of this fatty acid was plotted against death. Previous work from this laboratory (25) has shown that cells of Lactobacillus bulgaricus grown in media containing Tween 80 (polyoxyethylene sorbitan monooleate) were more resistant to freezing than those grown without it. Although such an effect had not been previously reported for the lactobacilli, it has been well documented that non-ionic detergents containing oleic acid, and free oleic and cis-vaccenic acids are important in the metabolism of lactobacilli (22,29,30). Either oleic or cis-vaccenic acids can replace the requirement for biotin by the lactobacilli (4,10,29,30). Tween 80, which is a non-toxic form of oleic acid, can also replace this requirement (3, 29, :0). In addition, these acids are intimately involved in the control of the fatty acid synthesis in Lactobacillus plantarum (1,9,28). Oleic and cis-vaccenic acids are incorporated intact into the lipids of lactobacilli or converted to a C1, cyclopropane fatty acid (17,21). C,, I Paper no. 4145 of the Journal Series of the North Carolina State Univ. Agricultural Experiment Station, Raleigh, N. C. cyclopropane fatty acids are formed from oleic or cis-vaccenic acid by the addition of a methylene bridge carbon across the double bond. S-adenosylmethionine serves as the donor of the bridge carbon for this conversion (22). Although C1, cyclopropane fatty acid(s) are found in the lipids of many different types of bacteria, their exact physiological function is unknown. It is believed to have a role in maintaining cell membrane flexibility (16). This study was initiated to determine the mechanism(s) whereby Tween 80 imparted freezing stability to L. bulgaricus. MATERIALS AND METHODS Cultures. The strains of L. bulgaricus selected for this study are used commercially in the manufacture of yogurt and Italian cheese. They were propagated and stored as described previously (25). A medium containing 2% Tryptone (Difco), 1% yeast extract (BBL), 2% lactose, and 0.2% Tween 20 (Nutritional Biochemicals Corp.) was used as the control medium to study the effects of sodium oleate on the lactobacilli. A sterile solution of sodium oleate 7:38 was added to the medium to give a final concentration of 100 Mg/ml. The sodium oleate solution and control were sterilized by autoclaving 15 Preparation of concentrated cell suspension. Each strain of L. bulgaricus was grown in the control broth and was used to inoculate the test growth media using a 2% inoculum. The cultures were grown statically to the stationary phase (15 h) in the test media at 37 C. They were harvested, concentrated, and stored in liquid nitrogen, and viability was measured as described previously (25). Incorporation of [1_14C]sodium oleate. Cells from 200 ml of broth containing [1-4C Isodium oleate and from 200 ml of broth containing unlabeled sodium oleate were washed three times with cold distilled water at a ratio of approximately 250 mg of cells (dry weight) to 20 ml of water. The cells were recovered between washings by centrifugation at 0 C and 17,300 x g for 10 min. After the final wash the cells from each broth were suspended in 10 ml distilled water, and the resulting suspensions were combined and stored in a freezer until analyzed. The spent medium, washings, and cells were analyzed for 14C content by using a Packard model 574 Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.) to ascertain whether or not sodium oleate was incorporated into (or adsorbed onto) the cells during growth. The counting fluid contained Triton X-100, 333 ml; toluene, 167 ml; 1,4-bis[2(5-phenyloxazolyl)]-benzene, 0.1 g; and 2,5 diphenyloxazole, 5.5 g. Ten ml of the counting fluid was added to each sample, and the final volume was adjusted to 20 ml with toluene. Whole cells were kept in suspension with the aid of Thixotropic Gel Powder (Packard Instrument Co., Inc.). Counts were corrected for background and quenching. Free lipids. Free lipids were extracted from washed whole cells by the method of Bligh and Dyer (2). The cells were extracted three times at 4 C. One part chloroform and 2 parts methanol were mixed with 0.8 parts of an aqueous cell suspension. The first extraction was for 1 h and 40 min, and the second and third were for 30 min each. After each allotted extraction time, 1 part chloroform and 1 part distilled water were added. The mixture was shaken and set aside for 20 min to allow the phases to separate. Between extractions the cells were recovered by centrifugation at 0 C and 17,300 x g for 10 min. The three extracts were pooled and condensed for analyses. Bound lipids. Cell suspensions from which free lipids had been extracted were hydrolyzed for 1.5 h in 1 N HCl at 121 C. The hydrolysates were extracted for 10 min by using the Bligh and Dyer procedure (2). Whole cells (not previously extracted) were also hydrolyzed and extracted for 10 min to obtain information for comparing total and bound lipids. Silicic acid column chromatography. Free lipids were separated by silicic acid chromatography into neutral and polar lipid fractions by using procedures outlined by Dittmer and Wells (6). Gravimetric analysis. Lipid fractions were evaporated to dryness by using a rotary flash evaporator at 42 C. They were redissolved in chloroform and quantitatively transferred to a tared evaporating flask. The chloroform was removed by evaporation, and the flask was dried and placed in a desiccator for at least 30 min before weighing. Gas-liquid chromatography. The fatty acid methyl esters were prepared from the lipid fractions by the method of Metcalfe et al. (20) using boron trifluoride (BF,)-methanol reagent (Fisher Scientific Co.). The methyl esters were separated on a Packard 800 series gas chromatograph (Packard Instrument Co., Inc.) equipped with a flame ionization detector. A stainless-steel column packed with EGSSX (10%) on Chromosorb Q (100/120 mesh, Applied Science Co., State College, Pa.) was employed. The oven temperature was held at 140 C for 3 min after sample injection and then increased linearly at 4 C per min until the final temperature of 190 C was reached. The injector and detector temperatures were 230 and 205 C, respectively. Nitrogen served as the carrier gas with a flow rate of 20 ml/min at 22 C. The fatty acid methyl esters were tentatively identified by comparing their retention times with known standards. The peak areas were determined by triangulation, and the total area was used to determine the relative percent of each fatty acid present. Gas chromatographic detector response was linear over the range studied. Statistical analysis. The regression of percent death on percent fatty acid content was determined by using methods outlined by Snedecor and Cochran (26). RESULTS Relationship of sodium oleate to storage stability. Previous work in this laboratory (25) demonstrated that cells of L. bulgaricus grown in broth containing Tween 80 were more resistant to freezing in liquid nitrogen than cells grown in broth without Tween 80. When most of the free oleic acid associated with Tween 80 was removed by silicic acid column chromatography, the detergent lost part of the ability to impart freezing resistance to cells grown in its presence. Sodium oleate added to a broth composed of Tryptone, yeast extract, and lactose was toxic to the growth of L. bulgaricus. However, Tween 20 (polyoxyethylene sorbitan monolaurate) was added to the medium to detoxify the fatty acid (15,30). Cells grown in the control broth without sodium oleate exhibited susceptibility to freezing similar to that observed in a previous report (25). An improvement in the storage stability was noted for each strain when grown in the presence of sodium oleate ( Table 1). The cultures grown without sodium oleate varied in their resistance to freezing. Strain NCS4 was most resistant to freezing; strain NCS1 was most sensitive and exhibited the greatest response to sodium oleate. Although the level of sodium oleate utilized in these experiments improved the stability of strains NCS2 and NCS3, it did not impart to the cells sufficient resistance to survive the freezing completely. Previous studies in our laboratories (25) indicated that strains of L. bulgaricus varied with respect to the optimum level of Tween 80 required to produce cells that were resistant to freezing. Presumably a similar situation exists with respect to the optimum level of sodium oleate. Incorporation of [l-14C]sodium oleate. Radioactive tracer studies demonstrated that sodium oleate was incorporated into the lipid portion of L. bulgaricus NCS1 ( Table 2). Analysis of whole cells revealed that 100.8% of the radioactivity associated with the cells was incorporated as lipid material of which 28.5% was bound lipid and 72.3% was free lipid. The free lipid was further separated by silicic acid col- umn chromatography into neutral and polar lipid fractions. There was approximately 2.5 times more 14C associated with the polar lipids than with the neutral lipids. Gravimetric analysis of the lipids revealed that the percentage by weight of each fraction was similar to the percent distribution of radioactivity in each fraction. There was little quantitative difference in lipid content between cells grown with and without sodium oleate. The total amount of lipid for cells grown with and without sodium oleate was 4.2 and 4.9%, respectively. Fatty acid analysis of lipids from hydrolyzed whole cells. Lipids from L. bulgaricus NCS1, NCS2, NCS3, and NCS4 grown with and without sodium oleate were evaluated for fatty acid composition. Chromatograms of fatty acid methyl esters prepared from lipids extracted from hydrolyzed cells of strain NCS1 grown with and without sodium oleate are presented in Fig. 1. Similar chromatograms were obtained for strains NCS2, NCS3, and NCS4. Quantitative evaluations comparing percentage compositions based on peak areas for all four strains are presented in Table 3. The primary fatty acids in lipids from hydrolyzed whole cells grown in sodium oleate were tetradecanoic, hexadecanoic, hexadecenoic, octadecenoic, and C1,, cyclopropane fatty acids. The predominant fatty acids of strains NCS1, NCS2, and NCS3 grown without sodium oleate were dodecanoic, tetradecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids. Stain NCS4 grown without sodium oleate contained considerably more C19 cyclopropane fatty acid than the other three strains. Cells grown in sodium oleate in all cases contained larger percentages of octadecenoic and C19 cyclopropane fatty acids than cells grown without sodium oleate. Cells Fatty acid composition of lipid fractions from L. bulgaricus NCSl. Results from gas chromatographic analyses of lipid fractions from cells of strains NCS1 grown with and without sodium oleate are presented in Table 4. The same fatty acids were observed in all fractions. The major differences appeared to be the presence of greater amounts of octadecenoic and C19, cyclopropane fatty acids along with lesser amounts of the saturated fatty acids in cells grown in sodium oleate than in cells grown in the control medium. More C1,, cyclopropane fatty acid was present in the polar fractions than in the neutral fraction. The cells grown in sodium oleate had more octadecenoic acid in the neutral than in the polar fraction; the reverse was true for cells grown in the control broth. Relationship of fatty acid composition to death resulting from freezing. The amount of death that resulted from freezing was closely associated with the cellular content of C,1 cyclopropane fatty acid. Figure 2 shows the percent death plotted against the cellular content of Car cyclopropane fatty acid for the lactobacillus cultures. The line had a negative regression that was significant at P < 0.001. Similar comparisons involving dodecanoic and tetradecanoic acids revealed positive regression lines that were significant at P < 0.001 and P < 0.005, respectively. Octadecanoic acid also had a positive regression line that was significant at P < 0.025. C1, cyclopropane fatty acid content exhibited the smallest standard deviation from the regression line of all the individual fatty acids. The regression coefficient of percent death on percent total saturated fatty acids was significant at P < 0.001. cyclopropane fatty acid content of L. bulgaricus. DISCUSSION Results from this and a previous study (25) suggested that sodium oleate was the active portion of Tween 80 responsible for producing cells that were stable to freezing in liquid nitrogen. The incorporation of [1-4C ]sodium oleate into the lipid fraction of cells of certain lactobacilli has been reported (17,21). The amount of labeled sodium oleate incorporated into the lipid fractions of L. bulgaricus NCS1 was similar to the percent of each fraction on a weight basis, which suggested that the incorporation was random and not selective for a specific lipid fraction. The total amount of cellular lipids and the ratio of polar to neutral lipid were similar to those of other lactobacilli (12,13). Since cyclopropane fatty acids are usually associated with phospholipids of bacteria (5,11) it was not surprising that the polar lipids of L. bulgaricus NCS1 contained more C,19 cyclopropane fatty acid than the neutral lipids. The phospholipid fraction thus appears to be important in protecting L. bulgaricus during freezing. The data indicate that the oleate was incorporated into the lipids intact or was converted to C19 cyclopropane fatty acid. Such a conversion of octadecenoic or cis-vaccenic acid to C19 cyclopropane fatty acid has been demonstrated in other lactobacilli (17,21 Generally, the major fatty acids of all strains were similar to those reported by Veerkamp (27) for L. bulgaricus. Alteration of the growth medium resulted in changes in the relative percentages of the individual fatty acids present in the cells. Similar effects of growth medium composition on the fatty acid content have been reported for other bacterial cells (14,27). The increases in octadecenoic or C19 cyclopropane fatty acid, accompanied by decreases in saturated fatty acids that were observed when L. bulgaricus cells were grown in the presence of sodium oleate, can be explained by metabolic control mechanisms involving these fatty acids (1,9,28). The lipids of gram-positive microorganisms, which are found predominately in the cell membrane (13), are important in maintaining membrane structure. The primary site of damage to certain cells during freezing is the cell membrane (19). Thus, the fatty acid composition might be involved in maintaining cell membrane integrity during freezing. Kodicek (16) suggested that cyclopropane fatty acids prevent close packing of lipids in cell membranes, making them more elastic and flexible during exposure to adverse environmental conditions. Jungkind and Wood (Abstr. Annu. Meet. Amer. Soc. Microbiol., p. 143, 1972) reported that strains of Streptococcus faecalis deficient in cyclopropane fatty acids were more sensitive to deoxycholate, NaCl, sodium lactate at pH 4.0, and incubation at 47 C than the parent strain that contained more of these acids. However, work on liposomes prepared from structural lipids of Escherichia coli suggested that the formation of cyclopropane acids from unsaturated fatty acids does not alter their physiochemical properties (8). The results from the present study to support the flexible membrane theory proposed by Kodicek (16) and the work of Jungkind and Wood (Abstr. Annu Meet. Amer. Soc. Microbiol., p. 143, 1972). A relationship has been shown between the amount of unsaturated and saturated fatty acids and fragility to the cell membranes of Mycoplasma laidlawii (23,24). Gier et al. (7) demonstrated that an increase in double bonds in the fatty acids of liposomes increased their permeability. Plant mitochondria that were sensitive to chilling had a higher content of saturated fatty acids than chill-resistant plant mitochondria (18). Chill-sensitive mitochondria were apparently injured due to an inflexibility of the membrane at low temperatures. A similar phenomenon may exist with regard to L. bulgaricus, because cell death appears to be related to a decrease in saturated fatty acid content. Both unsaturated and cyclopropane fatty acids are believed to have important roles in cell membrane structure (5). However, in the case of L. bulgaricus C1, cyclopropane fatty acid appears to be the one most closely related to the resistance of cells to freezing. The exact mechanism of protection is not known, but death is closely related to both the amount of saturated and cyclopropane fatty acids. Perhaps the cause of increased protection is due to a favorable balance of C19 cyclopropane and saturated fatty acids. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant ES-61 from the Division of Environmental Health Sciences.

v3-fos

2020-12-10T09:04:20.868Z

1974-09-01T00:00:00.000Z

237231263

s2

Thermoradiation Inactivation of Naturally Occurring Bacterial Spores in Soil Samples of soil collected from the Kennedy Space Center near the spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilzation techniques. The inactivation kinetics of the naturally occurring spores in soil were investigated by using dry heat and ionizing radiation, first separately and then simultaneously. Dry-heat inactivation kinetics of spores was determined at 105 and 125 C; radiation inactivation kinetics was determined for dose rates of 660 and 76 krads/h at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C, with a dose rate of 76 krads/h. Combined treatment was found to be highly synergistic, requiring greatly reduced radiation doses to accomplish sterilization of the population. For some time, it has been recognized that the heat stability of bacterial spores may be altered considerably by the manipulation of cultural conditions during sporulation (10). Recent studies of the dry-heat inactivation kinetics of spore populations in soil have shown that the heterogeneous soil populations exhibited higher dry-heat resistance levels than subcultured survivors obtained at corresponding intervals on the survivor curves (2); subsequent to this study, a naturally occurring spore population in soil collected at the Kennedy Space Center, Florida, was found to be exceptionally resistant to dry heat at 125 C (1). Since the organisms indigenous to local soils could comprise part of the bacterial flora of industrial manufacturing and assembly areas, it was recognized that these highly resistant spore populations were possible contaminants of spacecraft surfaces (12,13,14,19). This report describes efforts to develop a more effective sterilization procedure applicable to labile articles, such as extraterrestrial life-detection spacecraft. MATERIALS AND METHODS Preparation of soil specimen. Soil samples were taken in three different locations at the Kennedy Space Center, Florida, and were mixed. The composite sample was spread in a thin layer on sterile paper, covered, and allowed to dry for 48 h at room temperature. This dry soil (2,771 g) was then processed dry through a stainless-steel sieve series (W. S. Tayler Co., Cleveland, Ohio) down to a 0.124-mm screen size to remove rocks, shell particles, and plant particles. A series of rinses in 95% ethyl alcohol (6) and then a final sieving with a 0.043-mm screen resulted in 4,900 ml of soil-spore suspension in 95% ethyl alcohol. Serial dilutions in sterile deionized water were plated with Trypticase soy agar (BBL), supplemented with 0.1% soluble starch and 0.2% yeast extract, and were incubated aerobically for 1 week at 35 C (3). The viable concentration was 2.7 x 105 organisms per ml, and the approximate weight of soil in the suspension was 0.03 g/ml. The soil suspension described was used as the inoculum for all the experiments, and the suspension was maintained at 4 C during storage. Prior to use, the soil-spore suspension was insonated (9, 11) for 2 min in an ultrasonic bath (TURCO 750-W output with a cavitation intensity of 1.75 W/cm2) to break up clumps within the ethyl alcohol suspension, and it was continuously agitated with a stirring bar during inoculation to prevent settling. The samples were prepared by applying 0.1 ml of the suspension onto the surface of 0.038-mm thick, biological-grade aluminum foil disks, 32 mm in diameter. The samples were then allowed to air dry until the ethanol evaporated. When dry, the inoculated disks were assembled on aluminum strips (38 by 200 by 0.51 mm); four sample disks were placed on each strip, a single clean foil disk was placed over each sample, and then another aluminum strip was placed on top and held firm with wire clamps. This assembly of the disks clamped between two strips permitted considerable handling and suspending of the assembly in a vertical position without loss or damage to the sample disks. The assembled sample strips were then placed in a desiccator over Drierite (W. A. Hammond Drierite Co.) in a vacuum for 15 h prior to exposure to the sterilization environment. All .of the inoculation and assembly operations were performed in a class-100 laminar airflow clean room (7,21,22). Exposure methods. The thermal environment was provided by a recirculating air temperature chamber (Delta Design model 1060 w/type Ill controller) having a volume of 0.016 mi, with a rail arrangement in the door to hold the aluminum strips; temperature was controlled and recorded to an accuracy of +0.2 C. The radiation environment was provided by Sandia Laboratories Gamma Irradiation Facility which contained remote handling equipment to introduce and remove the source, including visual, physical, and electrical access with necessary safety controls. The 60Co source was introduced in a corner of the cell (2.13 by 2.44 by 2.59 m), and the dose rates ranged from 106 to 4 x 103 rads/h depending on the location of the sample within the cell. Moisture content of the air in the temperature chamber was controlled by a system (8) which passed pressurized air through a saturator in a warm water bath and then through a condenser coil and water trap in a cold-water bath. The air was allowed to expand into a coil in the warm-water bath prior to entry into the temperature chamber. Adjustments of the air pressure and the temperature of the cold-water bath provided a controlled relative humidity for the experiments of 30 + 1% measured at 25 C. Relative humidity measurements of the air were made at the input to the temperature chamber with a dew-point indicator, and continuous measurements were provided by lithium chloride sensors and a strip chart recorder. The rate of airflow into the temperature chamber was controlled to 0.236 liter per s, and the air pressure in the chamber was regulated at approximately 23.2 kg/iM2 by adjustment of a bleeder valve on the chamber. For each radiation experiment, the temperature chamber was placed in the Gamma Irradiation Facility cell at the appropriate distance from the 60Co source for the desired dose rate, and the chamber was positioned so that the sample strips assembled with the foil disks were vertical and the faces of the strips were perpendicular to the direction of the gamma rays. The temperature chamber controller, temperature recorder, and humidity control system were located outside the cell with the necessary cable connections passing through the cell wall. A block diagram of the equipment setup is shown in Fig. 1. Silver phosphate or cobalt glass dosimeters, depend. ing on the dose range, were placed on selected sample strips to verify the computed dose rates. Recovery methods. Each sample strip, when removed from the temperature chamber, was wrapped in sterile aluminum foil and returned to the class-100 clean room facility for recovery operations. A 20to 30-min time period was required to transport the samples from the remote Gamma Irradiation Facility area. Each of the samples from the strips was placed in a separate 50-ml beaker containing 10 ml of sterile 0.1% Tween 80 (BBL Polysorbate 80) in distilled water. The samples were then insonated for 2 min to remove the organisms from the foil disks. Care was exercised in placing the foil disks into the beakers, to assure separation of the inoculated disk from the cover disk and to complete wetting and submersion of both disks. The insonation was accomplished with the beakers immersed in the ultrasonic water bath to a level just above the recovery fluid level in the beaker. Occasional agitation of the beakers kept the disks separated and prevented the disks from cold welding. Appropriate 10-fold serial dilutions of the recovery fluid for each sample were made in sterile deionized water and plated in duplicate with supplemented Trypticase soy agar. Prior to pour-plating, petri dishes were prepared by pouring a thin layer of agar medium to underlay the specimen. After pour plates of the dilutions were solidified, sterile agar medium was carefully overlaid on each plate to retard spreading bacterial growth. Plates were counted after a 1-week incubation at 35 C, and each data point represents the mean value from four replicate sample foil disks. RESULTS Experimentation with the naturally occurring spores in soil was directed primarily toward the response to dry-heat treatment, radiation treatment, and the combination of dry heat and gamma radiation (thermoradiation) (20). The results of the first base-line experiment to determine dry-heat resistance of the organisms in soil at 125 C are shown in Fig. 2. The viable population, beginning at 2 x 104 organisms per disk (zero heat treatment), underwent a rapid reduction to about 102 organisms per disk during the first treatment period. The initial drop, found to be characteristic of naturally occurring organisms in soil (2), was followed by a second logarithmic phase of destruction. The second phase represents a very heatresistant subpopulation of approximately 1% of the original sample. Except for radiation inactivation, this biphasic order of death was noted in all dry-heat and thermoradiation experimentation. The second phase on the logarithmic part of the survivor curve was used as a basis of comparison for various treatments. By using the method of least squares, the data were fit with a straight line so that slopes or D values (time at temperature or the radiation dose required to reduce the viable population by 90%) might be compared. The 95% confidence intervals are also shown. The D,2,c value of the resistant subpopulation was 29 h (Fig. 2). This value is roughly 50 to 100 times the D125c value for Bacillus subtilis var. niger spores (4), an organism commonly used as a biological indicator for dry-heat sterilization cycles. PRESSURE The radiation resistance of the naturally occurring spores in soil was then determined. Samples were exposed at room temperature (25 C) to gamma radiation from the 0°Co source. Temperature and moisture-conditioned air was supplied to the sample chambers at a flow rate -which provided one air change per minute. The results of irradiation are shown in Fig. 3. In this figure, the data from dry-heat inactivation at 125 C (Fig. 2) were repeated to simplify comparisons of the inactivation rates with various treatments. The second curve shows results of radiation at room temperature with a dose of 54 krads/h, and the D value was found to be 205 krads (3.8 h). Although the dose rate was 76 krads/h, the samples were actually exposed to the source for only 45 min out of each hour of treatment. The 15 min of down time was required to remove the samples from the Gamma Irradiation Facility cell. The third curve on Fig. 3 is the result of simultaneously combining dry heat and radiation. The thermoradiation D value was found to be approximately 1 h as compared to 3.8 h for radiation alone or 29 h for heat alone. of the synergism obtained, the singular effects of heat and radiation, when added, would reduce the population by 1 log after 3 h of-treatment. Thermoradiation, for the same time at temperature and total dose, reduced the population by 3 logs. If the slopes of these examples are examined, the singular effects of heat and radiation, considered in an additive sense, would result in a D value of 3.37 h. The thermoradiation D value, 1 h, is less than one-third that of the additive effects. This level of synergism means that sterilization of this spore population could be accomplished in one-third of the time at temperature with onethird of the normal dose. Additional tions were found to be 57 min at 125 C, 60 min at 120 C, 77 min at 115 C, 89 min at 110 C, and 95 min at 105 C (Fig. 4). The interesting aspect of this series of experiments was the manner in which the shape of the inactivation curve changed. As the temperature was lowered, the sharp initial drop usually experienced at 125 C was lessened, requiring a longer exposure to reach the second phase of inactivation. To illustrate the temperature-dose relationship, the D values presented in Fig. 4 were plotted as a function of temperature in Fig. 5. For example, at 105 C with a 95-min D value, the radiation dose per log population reduction would be 121 krads per log. As the temperature was elevated (constant dose rate), the D value dropped to 57 min with a total dose of 72 krads per log population reduction, or roughly 60% of the radiation required at the lower temperature. DISCUSSION Naturally occurring bacterial spores in soil present a very realistic and yet difficult contamination problem when dealing with a labile article, such as a spacecraft destined to undergo a terminal sterilization cycle prior to launch. Although many microorganisms are resistant to either heat or radiation, the naturally occurring bacterial spores in soil are resistant to both. As such, the spores in soil have been a realistic adjunct to past studies of heat and/or radiation resistance based primarily on the standard test spore of Bacillus subtilis var. niger. The synergistic behavior of dry heat and ionizing radiation on the spores in soil was found to be in agreement with observations of the inactivation kinetics of other bacterial spores, bacteriophages, proteins, viruses, and yeasts (5,15,17,18); however, the synergism exhibited with the naturally occurring organisms appeared to be more pronounced than with Bacillus subtilis var. niger tests (a factor of 3.25 versus 2.6, respectively 416]). To persons or organizations dealing with the sterilization of labile articles, thermoradiation offers several advantages over conventional sterilization cycles. When actual microbial populations contaminating a particular article are defined (these are naturally occurring populations directly from environments associated with the article), laboratory tests can easily be designed to identify the most effective regions of synergism. By manipulating either or both of the synergistic components, sterilization cycles can be adjusted to optimize treatment efficacy and minimize the degradation of the article being sterilized. Thermoradiation in many instances also has the distinct advantage of being less sensitive to environmental parameters than either heat or radiation when applied separately (21). ;kl

v3-fos

2018-04-03T05:09:23.982Z

1974-03-01T00:00:00.000Z

41662283

s2

Effect of sodium ascorbate and sodium nitrite on toxin formation of Clostridium botulinum in wieners. Toxin production by Clostridium botulinum was inhibited by sodium nitrite levels above 50 mug/g of wiener. Sodium ascorbate at levels of 105 and 655 mug/g of product did not decrease the effectiveness of the sodium nitrite inhibition, nor did sodium ascorbate potentiate it. The results indicate that the use of sodium ascorbate in vacuum-packaged wieners does not appreciably alter the inhibition of C. botulinum toxin formation by sodium nitrite. Previous studies in our laboratory demonstrated the effectiveness of sodium nitrite (nitrite) in preventing the formation of Clostridium botulinum toxin in wieners that were temperature abused (1). In many cured meat items, current industry practice includes the addition of sodium ascorbate (ascorbate) to accelerate the formation and improve the stability of cured meat pigments. Little information is available, however, regarding possible changes incurred by ascorbate on the effectiveness of nitrite in controlling toxin formation in these products. Conceivably, ascorbate could enhance the growth of C. botulinum by decreasing the redox potential. Also, it could reduce the effectiveness of nitrite inhibition by reacting with the nitrite and thus lowering its concentration. On the other hand, the possibility exists that ascorbate may potentiate the inhibition by nitrite. To gain information regarding these possible effects of ascorbate, wieners were prepared as follows. The wiener premix (without nitrite or ascorbate) was prepared according to the following formula: pork, 39.82%; beef, 34.11%; water, 3.20%; ice, 17.14%; salt, 2.52%; dried corn syrup solids, 1.80%; dextrose, 1.02%; and spice, 0.39%. Eighteen batches of product were made with six levels of nitrite (0, 15, 30, 50, 100, and 150 Ag/g) and three levels of ascorbate (0, 105, and 655 Mg/g). Each batch of product was inoculated with a heat-shocked preparation of C. botulinum spores. The inoculum was adjusted so that approximately 1,000 spores per g of raw premix were added. Spore levels in the premix and thermally processed product were estimated by decimal dilution and inoculation of thioglycolate broth, followed by toxicity testing (intraperitoneal inoculation of mice) of the incubated cultures. A 10-tube most-probablenumber procedure was used. The spore preparation consisted of approximately equal numbers of the following strains: type A-33A, 73A, 62A, 109A, and 3A; type B-53B, 213B, 113B, 169B, and Lamanna. The total viable counts (30 C, 40 h) were made by using APT agar (Difco). As a result of thermal processing of the wieners, an average decrease of less than 1 log unit occurred in C. botulinum spore count. The thermally processed product was vacuum packaged and incubated at 28 C. Samples were tested after 0, 7, 14, 21, 28, and 56 days for pH value and total viable count, and after 7, 14, 21, 28, and 56 days for toxicity. All general methods used in this study were reported previously (1). The average composition of the thermally processed product was: moisture, 53.2%; protein, 11.2%; fat, 28.9%; salt, 2.7%; and water, 8.7%. The effect of nitrite and ascorbate on the incidence of toxic samples during incubation of the wieners is shown in Table 1. Samples containing 50 ,ug of nitrite per g of product, or less, began to show toxicity after 7 days of incubation. The incidence of toxic samples increased significantly after 14 days and remained high throughout the incubation period. Wieners containing 100 and 150 gg of nitrite per g of product failed to develop toxicity, with the exception of one sample containing 100 ,ug of nitrite per g of product. In the latter instance, toxicity was evident only after 56 days of incubation. The total number of toxic samples in each series containing nitrite was not altered appreciably as the level of ascorbate was varied (Fig. 1). The incidence of toxicity declined sharply above the level of 50 jig of nitrite per g of product. number of toxic samples/five samples During the incubation period, total viable counts and pH values showed no appreciable variation among the 18 variables. The pH values dropped steadily after thermal processing through the incubation period, with the exception that at zero level of ascorbate, all nitrite levels showed a slight increase in pH at the final 56-day sampling. The total counts generally decreased in the 56-day-old samples. Apparently, acid production was not a factor in preventing toxin formation under the conditions of the experiment. From the results obtained in this study, it can be concluded that ascorbate did not affect the efficacy of inhibition of toxin production by nitrite. Thus, the inclusion of ascorbate in the formulation of wieners does not detract from the desired inhibitory effects of nitrite on the production of botulinal toxin.

v3-fos

2020-12-10T09:04:22.924Z

1974-09-01T00:00:00.000Z

237233193

s2

Malformin in Aspergillus niger-Infected Onion Bulbs (Allium cepa) Malformin was identified, by its biological activity and chromatography, in acetone extracts of the outer scales of onion bulbs infected with Aspergillus niger. Malformin was not detected in tissue underlying the infected areas or in the central portions of the bulbs, nor was malformein liberated from extracts or extracted tissues after reduction with zinc in acetic acid. This is the first report of naturally occurring malformin. Images Black mold is a storage disease caused by Aspergillus niger on both colored and white cultivars of onions (Allium cepa). Although signs of the pathogen are most obvious on the outer scales, the disease is not confined to the exterior portion of the bulb (12). We recently obtained a supply of black molded onions and examined them for the presence of malformins, a small family of cyclic pentapeptides produced by A. niger (1,11) and several other members of the A. niger group of Aspergillaceae (4). In addition to causing serious disturbances in the growth of higher plants (2,6), malformin is antibiotic to a variety of bacteria (9). We report the first example of the natural occurrence of malformin. MATERIALS AND METHODS Source and preparation of samples. Onion bulbs (A. cepa cv. Spartan Banner) were submitted to the Plant Disease Diagnostic Laboratory (Botany and Plant Pathology, Purdue University) in February 1974 for disease identification. There were irregular masses of black powdery spores generally restricted to two or three outermost scales. Internal scales lacking fungal sporulation appeared normal. Outer scales of severely infected onions were slightly desiccated and easily separated from inner scales. The onions were grown in northern Indiana on muck soil during the summer of 1973. Because of dry soil conditions, onion foliage dried prematurely during mid-August, and foliar fungicide sprays, normally applied weekly until harvest, were terminated 2 to 3 weeks prior to harvest in late August. Bulbs were field-cured before harvest and placed in conventional storage. Disease was not apparent at harvest. Onions were removed from storage in early February and processed by normal topping, sorting, and packaging. Onions showing disease were culled during sorting and either de-' Journal Paper no. 5514 of the Purdue Agricultural Experiment Station. stroyed or delivered to our laboratory for diagnosis. Grower losses were negligible. Three portions of the bulbs were extracted and analyzed for the presence of malformin. The outer, visibly infected scales were separated from healthy appearing tissue. After removal of infected scales, the bulbs were washed in tap water to remove surface contaminating conidia. A layer one scale in thickness was removed and termed the underlying tissue. The remainder of the bulb is referred to as the central area. ExtractioW Infected outer scales (638 g fresh weight), underlying scales (1,140 g), and central areas (1,280 g) were macerated and steeped separately for 24 to 48 h in acetone (2 liters). The extraction was repeated three times with fresh acetone. The extracts were combined, filtered through cheese cloth, and evaporated to dryness on a steam bath, and portions of the residues were examined for the presence of malformin by bioassay. Bioassay. Bean seedling malformations and corn root curvatures were used to assess weighed portions of the acetone-soluble compounds for malformin (3). Fractions (1.0 g) of the residue were dissolved in water (20 ml), diluted with water containing Tween 20 (0.1%), and applied to the apical bud of Phaseolus vulgaris cv. Harvester, and the seedlings were observed for malformations after 10 days in the greenhouse. Similar dilutions, without Tween 20, were assayed by the root curvature method in which seeds of Zea mays WF9 x 38-11 were germinated for 3 days at 28 C on filter paper, moistened with 3.0 ml of test solution, in petri dishes. The amount of malformin present was estimated from standard curves prepared from root curvature bioassays using authentic malformin A isolated from culture filtrates of A. niger strain 58-883 (10). Chromatography. A portion (4.0 g) of the acetone-soluble residue was dissolved in water (150 ml) containing an excess of NaHCO, (pH 8.0) and extracted four times with ethyl ether (150 ml). The ether, in which malformin is soluble, was evaporated in a hood, and the residue was dissolved in ethyl acetate (30 ml). Strips (2.5 by 40 cm) of Whatman no. 362 3 paper were streaked with small volumes (0.1 ml) of the solution, developed in a variety of solvents by descending chromatography, dried, and cut into 2.0-cm sections beginning 1.0 cm behind the origin. Each section was heated in water (20 ml) for 5 min on a steam table, and the solution was examined for malformin by the root curvature method. To locate malformin, similar chromatograms were streaked with fractions of the test solution containing [4C]malformin A (ca. 800 to 900 dpm) prepared by biosynthesis as described by Ciarlante and Curtis (Proc. 8th. Int. Conf. Plant Growth Substances, Tokyo, 1973, in press). These strips were developed, dried, sectioned, and analyzed for "4C by liquid scintillation counting. RESULTS Identification of pathogen. Five fungal isolates were obtained by streaking spores from onion tissues onto potato dextrose agar. Cultures transferred to Czapek agar were all identified as A. niger as described by Raper and Fennell (7). Bioassay. Bean seedlings treated with the acetone-soluble residue from the central and underlying tissues of infected onions at three concentrations (20, 2, 0.2 mg/ml) grew normally. Similar preparations from infected exterior scales induced severe malformations on 100% of the seedlings (20 mg/ml), moderate malformations on 70% (2 mg/ml), and slight malformations on 7% of the seedlings (0.2 mg/ ml). The malformations were identical to those induced by authentic malformin (Fig. 1). No evidence for the presence of malformin in the acetone-soluble residue from the central and underlying tissues was obtained by the root curvature bioassay (Table 1), but a typical malformin dosage-response relationship was obtained with extracts of the infected outer scales. These root curvatures were identical to those induced by authentic malformin (Fig. 2). By comparison with standard curves obtained with malformin, we estimated that infected tissues contained the malformin equivalent of 3 to 9 mg/kg (fresh weight). Malformin was not detected in extracts from any portion of diseasefree bulbs. The apparent absence of malformin-like activity in extracts of the central and underlying tissues is not proof for the absence of malformin in either the tissues or the extracts. Malformin reacts with thiol compounds to form 1:1 addition products (5), and when ["4C]malformin is supplied to P. vulgaris a substantial portion is bound to a cell wall fraction and can not be extracted with organic solvents (D. Ciarlante and R. Curtis, in press). However, much of the c Taken as equivalent to the response of corn roots to the optimal concentration of malformin (0.1 ,g/ml) as determined from standard curves prepared from assays using authentic malformin A. Curvatures on less than 15% of roots not considered significant. bound malformin is released as [14C]malformein (dithiol malformin) after reduction with zinc in boiling acetic acid. Consequently, acetone extract residues and the tissues remaining after extraction were reduced, filtered, evaportated to dryness, and extracted with ether, and the ether-soluble fraction was examined for malformein by the root curvature assay. No evidence for the presence of malformein in the central and underlying tissue was obtained. Furthermore, after reduction the biological activity of acetone-soluble residues from infected scales decreased by approximately 90%, which also occurs when malformin is similarly converted to malformein. Chromatography. The chromatographic behavior of the active substance extracted from infected onion scales was identical with that of [14C]malformin A ( Table 2). No other biologically active compounds were detected on the chromatograms, and we concluded that a malformin was present in A. niger-infected onion bulbs. DISCUSSION When compared with authentic malformin, the similar solubility properties, biological activity, chromatographic behavior, and partial loss of activity after reduction indicate that the active substance extracted from A. nigerinfected onion bulbs is a member of the malformin group. No attempt was made to determine which of the malformins was present, and the various types cannot be differentiated by paper chromatography. The presence of malformin in only the infected, external scales indicates little or no diffusion to the inner portions of the bulb. Reaction of malformin with thiol groups in the tissues may preclude or hinder movement to other portions. Little information is available concerning the toxicity of malformin to animals. The compound is quite toxic to mice (mean lethal dose = 0.72 mg/kg, intraperitoneally) and is cytostatic in vitro to P-815 mastocytoma cells (mean effective dose = 0.059 yg/ml) (H. P. Sigg, Sandoz Ltd., personal communication). Among aspergilli grown on wheat and soybeans and fed to mice and chicks, A. niger, A. ficuum, and A. phoenicis were toxic and/or growth stunting (8). Although not implicated in these studies, malformins are produced by each of these species (4). The presence of malformin in A. nigerinfected onions warrants an awareness for this compound in other foods molded by this ubiquitous species.

v3-fos

2020-12-10T09:04:12.332Z

1974-06-01T00:00:00.000Z

237231535

s2

Combined Effects of Water Activity, Solute, and Temperature on the Growth of Vibrio parahaemolyticus Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (aw) of 0.992 (±0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower aw, respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce aw to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of aw with glycerol was less inhibitory to growth than similar aw reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar aw levels lower than 0.992. Minimal aw for growth at 15, 21, 29, and 36 ± 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced aw was generally most tolerable at 29 C, whereas higher minimal aw for growth was required at 15 C. Solutes added to TSBS to achieve reduction in aw, minimal aw for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal aw for growth of V. parahaemolyticus depends upon the solute used to control aw. which corresponded to a water activity (a,) of 0.992 ( 0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower aw, respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a, to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a, with glycerol was less inhibitory to growth than similar a. reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a, levels lower than 0.992. Minimal a, for growth at 15, 21, 29, and 36 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a, was generally most tolerable at 29 C, whereas higher minimal a, for growth was required at 15 C. Solutes added to TSBS to achieve reduction in aw, minimal a, for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a, for growth of V. parahaemolyticus depends upon the solute used to control aw. The relationships between available water content and the potential for spoilage of foods by microorganisms have been of interest for many years. Various minimal, optimal, and maximal moisture levels, usually expressed in terms of water activity (aj), have been reviewed (17,20). Much of the available data on a, requirements for bacteria have resulted from studies on foodborne pathogens such as salmonellae (3,4,6), staphylococci (5,15,16,19), and Clostridium spp. (1,7,10,18). In light of the recent recognition of Vibrio parahaemolyticus as a cause of foodborne disease outbreaks in the United States (2) and the lack of definitive information regarding the organism's tolerance to reduced a, levels, several experiments were designed to determine the growth response of V. parahaemolyticus over a wide range of a,. In the present study, the optimal a, in a medium containing added NaCl was established. Effects from minor changes in optimal a, levels resulting from the addition of several solutes to growth media on lag times of V. parahaemolyticus are discussed. Minimal a, levels for growth at 15,21,29, and 36 C were achieved by the addition of solutes to a basal medium. MATERIALS Media. The basal medium used for all investigations was tryptic soy broth (TSB, Difco), which contains 0.5% NaCl when prepared according to the manufacturer's directions. For studies involving growth of the organism at elevated NaCl levels, quantities of NaCl were added to known volumes of TSB, and the percentage of NaCl was expressed on the basis of grams of NaCl per grams of final NaCl-TSB mixture, assuming the density of TSB to be 1.0. Each of the remaining solutes studied (KCI, glucose, sucrose, glycerol, and propylene glycol) were added individually to TSB containing 2.93% NaCl (TSBS) in progressively increasing amounts to achieve a. values above and below which V. parahaemolyticus would grow. Percentages of these solutes, which are referred to throughout this report, were calculated as grams of solute per grams of final mixture (solute plus TSBS), again assuming the density of TSBS to be near 1.0. All growth media were adjusted to pH 7.8 by adding 2 N NaOH, dispensed into either 13 by 100 mm or 16 by 150 mm screw-cap tubes and sterilized at 121 C for 15 min. Random measurement of pH after sterilization revealed changes of no greater than +0.2. Growth studies. Since V. parahaemolyticus is facultatively halophilic, an initial experiment was designed to determine the optimal NaCl concentration at which the organism would grow. Strain M5250J-2 was cultured in TSBS at 29 C on a gyratory shaker (150 rpm) for 16 h. The culture was diluted 100-fold in distilled water containing 0.1% peptone (Difco) and 3.0% NaCl; 1 ml of the diluted culture was then inoculated into 150-ml portions of fresh TSB containing concentrations of NaCl ranging to 7.87%. The 500-ml Erlenmeyer flasks containing the cultures were returned to the shaker at 29 C. Samples were withdrawn at selected times and appropriate dilutions were made in the peptone-NaCl diluent prior to surface-plating on TSBS containing 1.5% agar (TSBSA) and on thiosulfate citrate bile salts sucrose agar (TCBS). Counts were made after 12 h (TSBSA) and 24 h (TCBS) of incubation at 36 C, and generation times were calculated for the organism cultured in each of the TSB media containing added NaCl. The above experiment showed strain M5250J-2 to have the fastest generation time in TSB containing 2.93% NaCl (TSBS). Addition of various quantities of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol to TSBS was made to achieve only slight reductions in a, levels. The effects of these a. reductions on lag times of strain M5250J-2 were then examined by measuring absorbance of the growing cultures at 620 nm after inoculation with a loop of 16-h TSBS culture. Growth temperature for the inoculum and the test cultures was 36 C. Amounts of solutes added, their weight percents, and resulting a. are summarized in Table 1. Minimal a, for growth of each of four strains of V. parahaemolyticus at 15, 21, 29, and 36 C were determined. Each strain was cultured in TSBS for 16 h at 29 C and standard loop inocula were transferred to 10-ml portions of TSBS containing individually added quantities of test solutes. Each test was performed in quintuplicate. In all cases, sufficient solute was added to achieve several a, levels both above and below that required for growth. Caps were tightened on the tubes (16 by 150 mm), and the inoculated media were incubated for 20 days in walk-in incubators adjusted to 15,21,29, and 36 ± 0.2 C. Obvious turbidity during the 20-day period was recorded as positive growth, and tubes were discarded. After 20 days all remaining tubes were examined for number of viable cells by plating on TCBS and for number of total cells by using a Petroff-Hauser counting chamber. Tubes were recorded as negative if viable cell population and direct microscope counts per milliliter were both less than the original number of cells per milliliter of culture at the time of inoculation. Direct counts were necessary to determine whether cell division was followed by death of significant numbers of cells during the 20-day incubation period. Determination of aw. Equilibrium relative humidity measurements were made at 29 C with Hygrosensor elements (no. 4-4822; Hygrodynamics, Inc., Silver Spring, Md.) mounted in lids of 8-oz jars. Hygrometer sensors were attached to a Hygrometer Indicator (model no. 4-4900; Hygrodynamics, Inc.) and an equilibration time of not less than 8 h was allowed before measurements were recorded from 25-ml portions of each test solution. Sensory calibrations were made against saturated KNO.. Mean values were determined from triplicate readings for several concentrations of each solute and sorption isotherms were plotted (Fig. 1). All aw levels reported in this paper were taken from isotherm curves. Accuracy of the measuring system is considered to be no better than ±0.005 aw. However, for purposes of comparison, as will become evident upon examination of results, data are presented at the 40.001 aw level. RESULTS AND DISCUSSION Inhibitory effects of aw levels higher and lower than optimum on the growth and metabolism of several bacterial species have been reported (3,5,6,16,17). Extreme sensitivity to relatively minor variations in osmotic and ionic conditions in growth media have been most dramatic with V. costicolus (8) and V. metchnikovi (13). Reduction in aw from 0.999 to 0.995 resulted in a fivefold increase in the rate of growth of V. metchnikovi. Further reduction in aw generally resulted in decreased rates of growth, depending upon the solute added to control aw and the nutrient composition of the growth medium. Several reports indicate conflicting ranges and optima for percentage of NaCl tolerance for V. parahaemolyticus (11,21,22, reviewed by 14; C. R. Lazarus and J. A. Koburger, Abst. Southeastern Branch Amer. Soc. Microbiol., 51st and 52nd Ann. Mtg., p. 19,1973). It is difficult to compare these data because methods for preparing "percent NaCl" levels in media were not detailed and aw levels were not reported. Initial experiments were therefore designed to determine the optimal NaCl concentration (and corresponding aw) for growth of V. parahaemolyticus by using TSB as a basal medium. Results are shown in Fig. 2. As aw was decreased from 0.998 to 0.992 (0.5 and 2.93% NaCl, respectively), the generation time of V. Fig. 3; levels were calculated from sorption isotherms shown in Fig. 1. parahaemolyticus M5250J-2 decreased from 24.4 to 16.4 min. Increased NaCl levels above 2.93% prolonged generation times. Whether aw manipulation by the addition of other electrolytes or nonelectrolytes alone or in combination to TSB would have resulted in different aw optima and faster growth rates was not determined. The 16.4-min generation time was considered to result from nearly optimal culture conditions, and therefore TSBS (TSB containing 2.93% NaCl) was used as a basal medium in subsequent studies involving the effects of added solutes on lag phase extension and minimal aw tolerance of the four test strains. Realizing that only slight differences in aw achieved from the addition of NaCl to the growth medium resulted in substantial differences in generation times of strain M5250J-2, it was decided to measure the effects of low amounts of added KCl, glucose, sucrose, glycerol, and propylene glycol, in addition to NaCl, on lag times of the organism. Data are plotted in Fig. 3. Grams of solute added per 100 ml of TSB or TSBS, calculated solute concentrations, and corresponding aw levels are summarized in Table 1. Some trends can be observed in these data. NaCl studies show that 0.992 a, results in the shortest lag time, fastest growth rate, and highest total biomass production. Departure from 0.992 a, resulted in longer lag times with slower growth rates and depressed cell production. These data tend to confirm those derived from generation time studies. Addition of solutes to TSBS (0.992 a.) reduced a. in all cases, but the magnitude of change in growth parameters was varied, depending upon the solute added. Glycerol had the least inhibitory effect on V. parahaemolyticus of all solutes tested. Addition of glycerol to achieve a 0.961 a, Table 1 for summary of media preparation. extended the lag phase only by 4 h compared to greater lag phase prolongation at even higher a. levels for the other test solutes. Inhibitory effects of NaCl and KCl were similar at comparable reduction in a, levels, whereas propylene glycol, glucose, and sucrose, in that general order, were most effective in delaying logarithmic growth and depressing cell production by strain M5250J-2. The glycerol data are in agreement with those reported for Staphylococcus aureus survival (15) and enterotoxin production (19) and growth of Salmonella oranienburg (4), Bacillus cereus (9), and Clostridium perfringens (10), wherein glycerol was found to be less inhibitory than other test solutes. Marshall et al. (12), on the other hand, reported that at aw levels between 0.96 and 0.90 the inhibition of S. aureus growth rate was about 10% greater in glycerol than in NaCl. Glucose and propylene glycol proved to be most bacteriostatic to V. parahaemolyticus when compared to the other solutes at similar aw levels. Total inhibition of growth of V. metchnikovi in nutrient broth containing glucose in amounts to achieve aw less than 0.997 has been noted (13). These authors reported no growth of the organism in brain heart infusion broth containing glucose. Glucose appears, therefore, to exhibit a toxic effect on V. metchnikovi and V. parahaemolyticus. By-products arising from chemical reactions between glucose and medium constituents during sterilization may account for these inhibitors. The reasons for V. parahaemolyticus sensitivity to propylene glycol cannot be explained. Table 2 shows the minimum a, for growth of V. parahaemolyticus in TSB and TSBS adjusted to reduced a, levels by the addition of various solutes. Calculated concentrations of added solutes are also listed. The four test strains responded similarly in their minimal a, levels at various temperatures. In most cases the a, levels listed in Table 2 are representative of at least three of the four strains. Therefore, only one minimal a, is listed for each solutetemperature combination. In general, solutes which were more inhibitory with respect to prolonging lag phase of growth were also more effective in completely inhibiting growth. Glycerol was least effective in controlling growth, followed by NaCl and KCl which were approximately equal, and then sucrose, glucose, and propylene glycol. The 29 C incubation temperature proved to be most satisfactory for the organism's tolerance to low aw, whereas 15 C adversely effected the response to aw stress. V. metchnikovi was reported to have lower tolerance to glycerol than to NaCl when aw adjustment was made in quarter-strength brain heart 1078 I E 30 so u z .r (12). Although the source of broth was not stated by the authors, if a Difco or BBL product was used, the initial concentration of NaCl was 0.5%. In view of the relatively high ionic strength required in growth media by Vibrio spp., the reversal in apparent tolerance of V. metchnikovi and V. parahaemolyticus to NaCl and glycerol might partially be explained on the basis of electrolyte concentration of the basal media used in the two experiments. The TSBS basal medium used to establish solute tolerances in the present study may have provided sufficient electrolyte to satisfy V. parahaemolyticus requirements. Therefore, addition of a nonelectrolyte such as glycerol to TSBS may have resulted in minimal a, levels which measured tolerance to the nonelectrolyte instead of tolerance to a combination of stresses induced from low electrolyte and high nonelectrolyte concentrations concurrently. The relationship between limiting a, levels for growth of microorganisms and the solute added to achieve those levels is unclear. Several authors (7,17) have stated that biological response to a, by some organisms is independent of the types of solutes used to reduce the aw. Other reports have shown that nutrient availability (3), pH (1), oxygen level (6,16), and moisture content (15), in addition to the test solute, effect a microorganism's ability to grow at limiting aw. It appears that the limiting aw for growth of the four V. parahaemolyticus strains examined in this study depends upon the solutes used to attain these lower limits. Notwithstanding the possibility of nutrient dilution effects inherent in the methods employed to prepare the test media, the differences in physico-chemical properties of the solutes apparently have a significant influence on the ability of V. parahaemolyticus to tolerate suboptimal aw levels. Further experiments are required to determine the response of V. parahaemolyticus to low aw levels achieved by the addition of other halogen salts to growth media. Studies involving the combined effects of various electrolytes and non-electrolytes on tolerance of the organism at reduced aw would also provide valuable information on growth characteristics of this facultative halophile. ACKNOWLEDGMENT The technical assistance of B. Vaughn is gratefully acknowledged.

v3-fos

2018-04-03T03:05:09.937Z

1974-11-01T00:00:00.000Z

347485

s2

Fermentation of Feedlot Waste Filtrate by Fungi and Streptomycetes The soluble and dispersed nitrogen and carbon components in the filtrate fraction of cattle feedlot waste are a potential nutrient source from which single-cell protein could be produced for animal feeds. The ability of more than 200 fungi and streptomycet es to grow in this liquid was determined; these included isolates from the waste and associated sources, as well as organisms maintained in the Culture Collection of the Agricultural Research Service in Peoria, Ill. Utilization of waste nutrients was measured by changes in nitrogen content and chemical oxygen demand. Only 20% of the organisms were able to grow appreciably in the filtrate. Of these, dry-weight yields varied from 0.6 to 2.7 g of mycelium per liter; from 21 to 50% of the nitrogen in the filtrates was used during growth, whereas chemical oxygen demand levels diminished from 4 to 60%. In general, streptomycetes isolated from the feedlot used nutrients from the filtrates better than fungi did. Addition of readily available carbon sources such as glucose or whey significantly increased (as much as sixfold) cell yields of selected organisms and promoted better utilization of nitrogen (from two- to threefold); the effect on chemical oxygen demand varied (0 to 33% increase). The soluble and dispersed nitrogen and carbon components in the filtrate fraction of cattle feedlot waste are a potential nutrient source from which single-cell protein could be produced for animal feeds. The ability of more than 200 fungi and streptomycet es to grow in this liquid was determined; these included isolates from the waste and associated sources, as well as organisms maintained in the Culture Collection of the Agricultural Research Service in Peoria, Ill. Utilization of waste nutrients was measured by changes in nitrogen content and chemical oxygen demand. Only 20% of the organisms were able to grow appreciably in the filtrate. Of these, dry-weight yields varied from 0.6 to 2.7 g of mycelium per liter; from 21 to 50% of the nitrogen in the filtrates was used during growth, whereas chemical oxygen demand levels diminished from 4 to 60%. In general, streptomycetes isolated from the feedlot used nutrients from the filtrates better than fungi did. Addition of readily available carbon sources such as glucose or whey significantly increased (as much as sixfold) cell yields of selected organisms and promoted better utilization of nitrogen (from two-to threefold); the effect on chemical oxygen demand varied (0 to 33% increase). Livestock production centers must cope with huge quantities of waste which offer a pollution hazard and a disposal problem (6,7,13,15). Biological treatment by oxidation ditches and lagoons to stabilize the waste before land disposal is the most common system used (6,9). We have enumerated and identified the microbial groups in feedlot waste (FLW; 8, 12), but not the ability of individual aerobic organisms to grow on feedlot pollutants. Organisms grown on waste pollutants might provide a source of microbial protein for animal feed while decreasing the pollution potential of the material. In a survey of isolates from waste and others from our Culture Collection, we sought filamentous organisms that could reduce pollutants and filter easily for cell recovery. More than 200 fungi and streptomycetes were studied for their ability to use nitrogen and organic material in the waste, the latter being measured by chemical oxygen demand (COD). The production of cell mass and the effect of adding glucose and dairy whey to waste filtrates also were investigated. (This paper was presented at the 1974 Annual Meeting of the American Society for Microbiology in Chicago, Ill.) MATERIALS AND METHODS Source of microorganisms. Samples were taken from pens of a cattle feedlot located near Peoria, Ill. (12). Fungi were isolated from plates of Mycophil medium (pH 7.0; Bioquest, Div. of Becton, Dickinson and Co., Cockeysville, Md.) to which has been added 0.2 mg of dihydrostreptomycin sulfate and 330 U of penicillin G per ml. A salts-starch agar (11) amended with cycloheximide (0.5 mg/ml) was used to isolate streptomycetes. Preparation of liquid waste for fermentation. Feedlot manure (21 to 40% solids) was diluted with distilled water to a solids content of 15%. After it was mixed to break up lumps, 15% (wt/vol) diatomaceous earth was added to aid separation. Filtrate obtained by suction filtration through filter paper (Whatman 54) served as substrate for the fermentation studies. The pH ranged between 6.0 and 6.8. The filtrate presented a qualitatively predictable substrate without the particles which would make equivalent samples difficult to obtain. Survey of organisms. Initially the ability of organisms to grow in FLW filtrate was evaluated in two ways. (i) Streptomycetes isolated from feedlot sites and fungi from the Agricultural Research Service Culture Collection were first grown on agar prepared with FLW filtrate as the sole nutrient source. Those which visibly grew well were further tested in liquid fermentations. (ii) Fungi isolated from feedlot sites, together with streptomycetes and fungi selected from (i), were grown in sterile FLW filtrate in single, shaken test tubes (10 ml in tubes 25 by 150 mm). Inoculum was provided by 1-week-old plate and slant cultures. Tubes were shaken on a rotary shaker (200 rpm, 5-cm displacement) at 28 C. After 7 days, fermentation samples were brought back to volume with distilled water, and the mycelium was recovered by vacuum filtration through Whatman 54 paper. Cell masses were dried at 103 C overnight. Filtrate was analyzed for nitrogen (2), COD (1), and total carbohydrate (4). Flask fermentations. Organisms selected from the preliminary survey were further evaluated in duplicate flask fermentations. Flasks containing FLW filtrate (50 ml in a 300-ml Erlenmeyer flask) were inoculated with 2% (vol/vol) washed and blended mycelium. The mycelial inoculum was grown in a medium of 2% glucose, 0.1% peptone, 0.1% yeast extract, and 2% malt extract. Flasks were incubated on a rotary shaker at 28 C; the contents were analyzed for nitrogen and COD as were the tube cultures. RESULTS Survey. Streptomycetes isolated from FLW were streaked on agar plates made with waste filtrates. Of 59 cultures, 35 grew well and were transferred to FLW filtrate in tubes. Eight isolates produced more than 2 g of cell mass per liter in the liquid. Filtrates of these were lower in nitrogen and COD content than initial levels by 37 to 50% and 53 to 60%, respectively. Mycelium weight ranged from 2.0 to 2.4 g/liter (Table 1). Only 17 of 170 fungi from the Agricultural Research Service Culture Collection, streaked on waste filtrate-agar, grew well; the test organisms represented 14 different genera. The 17 fungi were inoculated into tubes of FLW filtrate. Nitrogen and COD levels were diminished below initial values by 31 to 58% and 4 to 52%, respectively. Cell mass ranged from 0.7 to 1.9 g/liter. Isolates of fungi from feedlots were grown on FLW filtrates supplemented with 0.6% glucose ( Table 2). They produced more cell mass and lowered COD and nitrogen levels further than fungi grown in waste liquid without added glucose. Nutrient additions. Trichoderma viride Persoon ex S. F. Gray NRRL 3652 and Fusarium aquaeductuum (Radlkofer and Rabenhorst pro parte) Lagerheim NRRL 2503 grew poorly on FLW filtrate in shaken flasks and were ineffective in reducing COD and nitrogen ( Table 3). Addition of glucose increased cell mass and decreased nitrogen and COD levels more. Fusarium oxysporum grew twice as well as T. viride and F. aquaeductuum and was 2 to 3 times as effective in diminishing pollution potential. F. oxysporum, an isolate from FLW, reduced COD levels by one-half in 1 week of fermentation ( Table 4). Addition of glucose yielded more cell mass and greater use of nitrogen from the waste; however, residual COD was not lowered. Common nutrients that might be limiting were added to FLW filtrate; these mixtures were fermented for 1 week with F. oxysporum. Levels of COD were diminished by half in all flasks (Table 5). Nitrogen content was lowered by 37% with FLW filtrate without additives. Glucose alone and in combination with peptone and phosphate diminished nitrogen and COD levels by 44 and 47%, respectively, and produced more mycelium than in the control flasks. Addition of ammonium ion diminished nitrogen levels by 28% as compared with 37% for controls, but phosphate supplement allowed Filtrates obtained from heated FLW, as compared with unheated material (Table 5), contained more nitrogen (78%) and organic mate-rial (40%, measured as COD). F. oxysporum, grown 1 week on filtrate from heated FLW, took up one-third more nitrogen and one-tenth less COD substances than in unheated liquid. Mycelium production was the same with both filtrates. When a sterile mixture of dairy and FLW on May 8, 2020 by guest http://aem.asm.org/ h Additions are final concentrations in flasks (wt/vol). Sterile system, autoclaved before inoculation; unsterile not autoclaved. Heated system was prepared by heating raw FLW at 100 C for 1 h before filtration; unheated used standard preparation. liquids (FLW filtrate-whey, 2:1, vol/vol) in flasks was inoculated with F. oxysporum, 10.3 g of cell mass per liter was made in 1 week. Initial COD and nitrogen levels were lowered by 85 and 73%, respectively (Table 5). F. oxysporum inoculated into the mixed culture of unsterile dairy liquid and filtrate from FLW overgrew other organisms. After 7 days of growth, nitrogen and COD materials were diminished 62 and 96%, respectively (Table 5). Sterile mixtures, similarly fermented by this organism, had an equivalent amount of nitrogen but less organic material. Dairy whey was fractionated by dialysis in an Amicon apparatus and combined with waste filtrate in ratios of 1:2 (Table 6). F. oxysporum produced 6.4-fold greater cell mass on this mixture (10.3 g/liter) than on FLW filtrate alone (1.6 g/liter). Although nitrogen uptake was improved, comparable final COD values indicate that admixture with whey did not increase utilization of FLW filtrates. Dialysates of whey gave similar results. Thus, increased yields of mycelium from combined waste are attributable to usable nutrients in whey. Amendment of FLW filtrate with 1.7% lactose gave two-thirds the cell mass and showed decreases of nitrogen and COD material comparable to that for combined waste liquids. DISCUSSION As expected, a large proportion of streptomycetes isolated from FLW grew well as streaks on FLW filtrate-agar. Such growth may reflect survival and adaptation in a limiting environment. However, limited microbial growth in FLW in situ was indicated by the relatively constant number of organisms found in waste at a feedlot regardless of season (12). Streptomycetes which were isolated from the feedlot reduced pollutants in waste filtrates with modest yields of cells (Table 1). Fungi which were similarly isolated and grown on FLW filtrate with glucose also reduced nitrogen and COD levels, but formed more mycelium ( Table 2). Fungi that were not adapted to FLW nor supplemented with glucose reduced pollut- ants less well and produced less cell mass (Table 1). FLW filtrate contains 3 mg of carbohydrate and 0.5 mg of N per ml. Assuming that carbohydrate was the major nonprotein carbonaceous material in FLW, the calculated carbon-tonitrogen ratio of FLW filtrate is 2.4. This ratio was compared with 6.6 and 7.6, which values are based on the elemental composition of mycelium (10). Nitrogen levels of Aspergillus niger mycelium have been shown to be a function of initial nitrogen content of medium (14). Foster (5), reporting elemental data by Porges, gave a carbon-to-nitrogen ratio of 18 for fungi. A suitable carbohydrate source is often crucial for growth of molds (3). Consequently, carbohydrate supplements were supplied to microorganisms isolated from a feed lot and to others selected from the Agricultural Research Service Culture Collection. Selected fungi exhibited varied ability to take up pollutants from waste filtrates as a function of glucose amendent. Although T. viride, grown in FLW filtrate with glucose, reduced COD levels below those found without supplement, utilization of COD by F. aquaeductuum and F. oxysporum was either unaffected or impaired by glucose addition. Less uptake of organic material with increasing glucose content suggested that diminished utilization occurred because of a sparing action. In contrast, uptake of nitrogen was invariably increased by addition of glucose to the waste. Addition to FLW filtrate of nutrients common to microbial media did not greatly affect uptake of nitrogen or COD substances ( Table 5). Liberation of waste nutrients by heating FLW (before filtration) affected subsequent pollutant utilization even less. Evidently, F. oxysporum is so acclimated to waste substrates that common supplements give little benefit. Two dissimilar wastes that complemented deficiencies in nutrient composition were combined. For example, fermentation of dairy whey in combination with FLW filtrate resulted in a high uptake of nitrogenous and organic materials (Table 6). It is likely that dairy whey complemented feedlot filtrate by providing lactose because waste was deficient in total carbohydrates. Liquids resulting from fermentation of supplemented waste filtrate are not suitable for release to surface waters because of residual nitrogen and COD levels. However, stabilized fermentation liquids could be used for flushing feedlot surfaces and for irrigation. In conditions of protein shortages, the fungal mycelium of those fermentations might prove useful in animal feeds.

v3-fos

2018-04-03T02:32:38.262Z

1974-01-01T00:00:00.000Z

33958987

s2

Morphological observations of Diplodia maydis on synthetic and natural substrates as revealed by scanning electron microscopy. Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion. Diplodia maydis (Schw.) Lev is one of the major pathogens causing stalk rot (4) of corn (Zea mays L), a complex disease resulting in an annual loss of more than 109 bushels of corn in the corn-growing areas of the world (8). Many studies concerning resistance or susceptibility of corn to D. maydis have been reported (see 4,8), but only a few studies have been reported on the in vitro growth of D. maydis (1,2,6,10,11), and none of these at the ultrastructure level. The paucity of information concerning the gross morphology of D. maydis led us to examine (via scanning electron microscopy) the ultrastructural morphology of D. maydis grown on laboratory media and natural substrates to lay ' Present address: Center for Electron Microscopy, Southern Illinois University, Carbondale, Ill. 62901. 'Present address: University of Delaware, 104 Hullihen Hall, Newark, Del. 19711. the groundwork for further studies of host-parasite interaction. Growth conditions. Cultures were maintained at 25 C on sterile oats inoculated with D. maydis and on potato dextrose agar plates. The inoculum for the liquid media and PDA plates was prepared by transferring a 5-mm plug of D. maydis grown for 4 days on PDA. Oats were inoculated with a few infected oat kernels from maintenance cultures of D. maydis. Spores were obtained from maintenance oat cultures by adding 10 ml of sterile distilled water into an inoculated oat flask, allowing spores to swell for 10 to 15 min with occasional gentle agitation of the flask, and decanting the suspended spores. For purposes of comparison, D. maydis was grown at 25 C in MR2 medium on a rotary shaker, a reciprocal shaker, and under stationary conditions. In other studies D. maydis was grown for 5 days at 25 C in CPK, MR1, or MF2 media. Corn kernels and corn husk tissue from field-grown plants infected with D. maydis were provided by A. L. Hooker. Fixation and specimen preparation methods for scanning electron microscopy. To determine a suitable fixation procedure for D. maydis mycelia and spores, the glutaraldehyde and Parducz fixation methods were tested. In glutaraldehyde fixation, mycelia and spores were immersed for 1 h in 0.1% s-collidine-buffered glutaraldehyde (Electron Microscopy Sciences), followed by three washes in buffered s-collidine and a subsequent 1-h fixation in 1% s-collidine-buffered glutaraldehyde, followed by 10 washes in s-collidine buffer. In Parducz fixation (9), mycelia and spores were immersed for 10 min in 6 parts of 2% OsO4 to 1 part of saturated HgCl2, followed by 10 washes in a buffered solution. Both fixatives were used alone as well as in conjunction with each other. Freeze-dried (Edwards Pearse tissue dryer) preparations were compared with air-dried preparations on all samples examined. Mycelia grown on PDA were compared by using freeze-drying, critical-point drying (Bomar 900EX or Denton criticalpoint drying apparatus), and air-drying. Scanning electron microscopy. Fixed and dried D. maydis mycelia, infected corn kernels, corn tissue, and infected oat kernels were attached to Cambridge specimen stubs by using double sticky tape slightly melted with acetone. Spores were pipetted onto specimen stubs by the method of Murphy and Campbell (7). Samples were rendered electrically conducting by evaporating a thin 40:60 palladium gold alloy coating onto the surface in a vacuum evaporator while the samples were simultaneously tilted and rotated to insure even coating. The specimens were examined in a Cambridge Stereoscan Mark 11A scanning electron microscope operated at 20 kV. RESULTS Fixation results. Glutaraldehyde fixation followed by Parducz fixation and freeze-drying gave the same morphological preservation as Fig. ld, illustrating well-preserved spores). Glutaraldehyde fixation followed by freeze-drying resulted in mycelia and spore collapse. All air-dried samples and the samples unfixed and freeze-dried resulted in collapse of mycelia and spores as well as a matting of the mycelia and slime material. Freeze-drying versus critical-point drying. In comparing preparation methods, mycelia were preserved equally well with fixation and freeze-drying ( Fig. 2a and b) and fixation and critical-point drying ( Fig. 2c and d). The advantages and disadvantages of each method are discussed by Boyde and Vesely (3) but, considering equal preservation, critical-point drying is of greater advantage because the drying time (45 min) is considerably less than that for freeze-drying (4 h). Rotary versus reciprocal shaker cultures versus stationary cultures. D. maydis mycelia obtained from rotary shaker and from reciprocal shaker cultures were well preserved and had a similar overall appearance ( Fig. 3a and b). D. maydis grown in stationary cultures, however, was found to be surrounded by slime-like material. The copious precipitate on the mycelia surfaces is illustrated in Fig. 4a and b, and a coalescence of the slime material is shown in Fig. 4c. Coalescence of slime-like material does not appear to be an artifact of fixation since unfixed controls showed the same type of coalescence. Comparisons on different growth media. (i) MR2 medium. Figure 3a illustrates the mycelial organization in a mycelial pellet. Fixed and air-dried samples collapsed, and mycelia appeared to be matted. This is even more evident in samples which were air-dried without fixation. Figure 4a illustrates the overall mycelial appearance after growth for 4 days in stationary cultures. Copious particulate matter is found on the surface of mycelia grown in stationary cultures (Fig. 4b); also apparent is the coalescence of slime-like material over the mycelia, as indicated by an arrow in Fig. 4c. This might be expected, as copious quantities of slime-like material are elaborated during the growth of D. maydis in liquid media (1,2,6). The greatest amount of the precipitate is found on the mycelia grown under stationary conditions. Figure 5 shows a finely laced net randomly appearing throughout the mycelia with small bead-like structures interspersed along the net. The amount of netting interlaced in the mycelia was well correlated with the amount of slimelike material elaborated by the fungus during growth. The fungus grown in MR2 medium elaborated the least amount of slime-like material when compared with growth in MR1, MF2, and CPK media. (ii) MF2 medium. The mycelial pellet has a slightly different overall appearance when grown in MF2 medium in that it showed less regularity than found in MR2 medium. Typically, a film of secreted material overlying and stretched between the mycelia is apparent (Fig. 6a). Compared with cultures grown in MR2 medium, there is a greater abundance of beadlike structures interlaced throughout the mycelia (Fig. 6b and c), and larger thickened node-like structures are observed. The mycelia from cultures grown in MF2 and MR2 media both have a smooth surface with no discernible characteristic structures. (iii) CPK medium. When grown in cpk medium, the mycelial pellet has a honeycomblike appearance. In juxtaposition, one can see the honeycomb netting and smooth-surfaced mycelia (Fig. 7a); in certain areas, the honeycomb-like appearance takes on a more or less regular pattern (Fig. 7b). Mycelia were of two types: those covered with particulate material and those with a smooth surface, relatively free of other material. The bead-like structures and the coalescing of these structures noted in cultures grown in MR2 and MF2 media are also apparent in cultures grown in CPK medium ( Fig. 8a and b). The thickened node-like structures observed in MF2-grown cultures were not observed in CPKgrown cultures. (iv) MR1 medium. After 6 weeks of growth under stationary conditions in MR1 medium, the mycelial pellets were engulfed in a gel-like matrix; sporulation had occurred and the formation of bead-like structures and a coalescence of slime material were also noted. The mycelium appeared to be composed of a continuous series of bulges ( Fig. 9) suggestive of intercalary spores, although these are not reported to occur in this organism. (v) PDA medium. After 5 days of growth on PDA plates, coalescing of slime material on the smooth-surfaced mycelium, thickened nodes, and bead-like structures was observed. However, nodes and bead-like structures were not as abundant as in cultures grown in liquid media. This may be due to the relatively dry growth conditions in agar plate culture. D. maydis spores are ovate, most commonly two-celled and slightly curved with rounded to tapered ends. Figure 10a illustrates the most commonly observed spore shape where a septum between the cells can be noted. Air-drying of D. maydis spores mimics the condition in which the spores are found in naturally infected tissues. Air-dried spores are generally flask shaped (Fig. 10c) or collapsed (Fig. 10b). The average size of noncollapsed spores was 25 by 4 ,um. Size calculations are only approximate due to the variable specimen to beam angles inherent in the scanning electron microscope. (vi) D. maydis on oat substrate. On oats, D. maydis produces thousands of spores in small, black, flask-shaped pycnidia (Fig. 11a). Although the pycnidia may be globose, flask shaped, or irregular in shape, a typical pycnidium is illustrated in Fig. lib and c. Pycnidia are filled with spores (Fig. lie), and an opening (Fig. 11d) in the pycnidium for the dissemina-tion of spores can be seen. Inside the pycnidium, a fine-laced netting is found interspersed between the spores (Fig. 12a). A few of the bead-like structures noted in cultures grown in liquid media are also present (Fig. 12a). The interior of some of the pycnidia appears filled with spores engulfed in a thick gel-like matrix (Fig. 12b). Possibly the laced netting thickens to a gel-like matrix, giving some advantage in preservation under dry conditions. Interpycnidial mycelia are illustrated in Fig. 12c. A thin film overlying and stretching between the mycelia was observed (Fig. 12d), as well as a more extensive coalescing of slime material (Fig. 12e). Node-like structures were also observed in D. maydis-infected oats (Fig. 12f). Mycelia exhibited a smooth surface, and spores were two celled, rounded to tapered, and many were flask shaped due to natural collapse upon drying. Although samples were somewhat naturally dried during normal growth conditions, fixation was found to be necessary, as samples that were not fixed were found to have an artifactual cracked appearance both in the pycnidium and on spore surfaces. (vii) D. maydis on corn husks. The appearance of D. maydis on corn husks was found to be similar to that grown on oats. Small pycnidium dotted the surface ( Fig. 13a and b). Although often random in distribution (Fig. 13a), pycnidia sometimes appear to be closely associated with vascular bundles (Fig. 13b). Pycnidia were filled typically with two-celled, flask-shaped spores (Fig. 13c). The mycelial surface was smooth (Fig. 13d), although it was collapsed because of the naturally dry growth condition. Coalescing of slime-like material was noted interspersed between the mycelia. Less slimelike material covered the spores on the pycnidial surface compared to that found in infected oat tissue. Neither thickened nodes nor bead-like structures were noted in the fungus obtained from infected corn husks. (viii) D. maydis on corn kernels. D. maydisinfected corn kernels were examined from various locations in Illinois (Carbondale, Cornell, Morris, Lincoln, and Varne). Figure 14a illustrates a typical overall view of D. maydis-infected kernel. Pycnidia were not as compact (Fig. 14b) as that found on oats or corn husks. Thickened nodal areas were found in all samples examined. Many of the nodes tended to be round rather than ovate (Fig. 14c); however, the latter-shaped nodes were also present ( 14d), as indicated by an arrow. Mycelia were smooth surfaced without evidence of precipitated material on the surface. Spores were collapsed due to natural drying conditions during growth. DISCUSSION Glutaraldehyde fixation followed by Pardupz fixation and critical-point or freeze-drying resulted in the same morphological preservation as Parducz fixation alone followed by criticalpoint or freeze-drying. Mycelia and spores collapsed if treated with glutaraldehyde fixation followed by freeze-drying or air-drying, or prepared by freeze-drying or air-drying without fixation. From these results, we decided to routinely use the double-fixation procedure for field studies (glutaraldehyde fixation in the field followed by Parducz fixation and criticalpoint or freeze-drying after returning to the laboratory) and eliminating the glutaraldehyde fixation when sampling D. maydis from laboratory cultures. The collapse in mycelia, spores, and pycnidia observed in the air-dried field collections (husks and kernels of corn) are believed to be real and not induced by a fixation procedure. Major variations in fungal morphology were not observed due to culture conditions (rotary, reciprocal, or stationary liquid culture) or media, although the greatest amount of precipitate was observed on mycelia from stationary liquid culture. Apparently, the slime-like material is dispersed by movement in rotary and reciprocal shaker culture. Fine-lace netting with beads was found in all synthetic cultures. Minor differences appeared in the amounts of slimelike material observed and in the presence or absence of thickened nodes. Also apparent in MR1 medium was cell-like bulging of the mycelia. These cell-like bulges are suggestive of intercalary spores; however, these are not reported to occur in this organism, and no crosswalls (indicative of intercalary spore formation) are found in thin-section studies (Murphy, unpublished data). From this study, it appears that the degree of coalescence of slime-like material progresses as follows: the formation of bead-like structures (Fig. 6b); progressive accumulation of these precipitate on mycelia at a higher magnification. Marker represents I um. x5,000. (c) Note coalescence of slime material. Marker represents 10 gm. x 180. All cultures were grown in MR2 medium at 25 C. Im. FIG. 9. D. maydis grown in MR1 medium, 6-week stationary culture, Parducz fixed and freeze-dried. The mycelium appears to be composed of a continuous series of bulges. Marker represents I 'sm. x5.100. structures ( Fig. 8a and b); formation of thickened film of secreted material overlying and stretched between the mycelia (Fig. 6a); and finally a coalescence of the slime-like material (Fig. 4a). We believe that the same type of progression results in matting and coalescence of mycelia in the pycnidium. It should be realized that these suggestions are made from studying hundreds of micrographs, and the coalescence sequence is only briefly represented in the five micrographs listed. The slime-like material appears to be different from the material precipitated on the mycelia. However, it may be that the two arise from the same metabolic pathways since both seem to occur simultaneously. Why the great variation in amounts of slimelike material observed with the culture media tested is not known. It may be that the slimelike substance produced by D. maydis plays a role in the prevention of desiccation under normal conditions. One variation that did occur in the mycelial organization in the cultures was the formation of a honeycomb netting of mycelia in CPK medium. Why thickened nodes were observed in MF2 and PDA media and were not found in MR2, CPK, and MR1 media is also not known. Again, in thin-section studies (Murphy, unpublished data), cross-walls are not found. Perhaps the nodes represent storage material in the hyphae and occur only when certain metabolic products are in excess, depending on the food source. The amount of slime secreted by the fungus does not seem to be correlated with the presence or absence of thickened nodes. When comparing morphology of D. maydis on artificial versus natural substrates, the biggest difference appears to be in the formation of pycnidia. On natural substrates, these occurred along vascular bundles of the corn husk and along the oat and corn kernels. The tightness of the pycnidium was least on corn kernels and greatest on infected oats. The presence of finelace netting with beads on infected oats but not on corn kernels or husks, and the lack of thickened nodes in fungal samples from infected corn husks but not corn kernels or oats may indicate significant nutritional and environmental differences resulting in physiological differences in spores of the pathogen. Similarly, there may be differences in these characteristics when host-pathogen responses with a wide variety of cultivars and pathogen isolates are studied. Regardless of these possibilities, from our study we suggest that there should be little morphological differences expected in the pathogen mycelia, spores, and pycnidia. Although scolecospores have been reported (5) Marker represents 1 Aim. x2,300. 249 VOL. 27, 1974 on May 5, 2020 by guest http://aem.asm.org/ Downloaded from strains of Diplodia, and including D. maydis, none were observed in the material studied. From this study, we have enough morphological data about this pathogen to identify it if observed in corn stalk tissue infected with D. maydis. With the scanning electron microscopy preparation methods now available, and with knowledge about the morphological differences in various synthetic and natural environments of the pathogen, several other investigations of D. maydis can be undertaken. Of particular interest now is the documentation and clarification of the stalk rot process as D. maydis penetrates its natural host and spreads from cell to cell within the stalk, over kernels, and through the husk tissue. Included in this is the need to study the digestion process and changes in morphology when production of cellulolytic enzymatic enzymes are stimulated and their release occurs (1,2).

v3-fos

2020-12-10T09:04:16.767Z

1974-04-01T00:00:00.000Z

237229760

s2

Stomach Fermentation in East African Colobus Monkeys in Their Natural State The microbial fermentation in the stomachs of two monkeys, Colobus polykomos, collected in Kenya, was studied. The gas accumulated within the stomach contained H2 but no CH4. Volatile fatty acid concentrations were high, but accumulated acid prevented determination of the fermentation rate in untreated, incubated stomach contents. Upon addition of bicarbonate, a very rapid rate could be demonstrated. Some D- and L-lactate were in the stomach contents. Starchy seeds or fruits rather than leaves appeared to have been consumed. Microscopically, the most prominent microorganisms seen were large, very refringent cocci, possibly Sarcina ventriculi, and various smaller cocci and rods. The 28 cultured strains of bacteria included 14 Staphylococcus, 2 Streptococcus, 10 Propionibacterium, and 2 Peptostreptococcus. The culture count constituted 10 to 20% of the direct count. No protozoa or cellulolytic bacteria were found. An active microbial fermentation in the stomach of leaf-eating monkeys has been inferred (14) from the large amount of digesta in the stomach, from production of methane, and from the relatively high concentration of volatile fatty acids in the stomach contents (8). Measurements of fermentation in the langur monkey, Presbytis cristatus (3), and in Procolobus and Presbytis (14) indicate rates comparable to those reported for stomach contents of domestic (6) and wild ruminants (12). A brief field trip in Kenya in September 1969 afforded an opportunity to obtain two individuals of Colobus polykomos living on the north slope of Mt. Kenya and to culture the stomach contents and measure the rate of fermentation. MATERIALS AND METHODS Animals. Two male specimens were collected immediately adjacent to the Landrover containing the equipment used in the study. The first animal, no. 263, was in the Timao Forest on the north slope of Mt. Kenya at an elevation of about 3,200 m; the second, no. 264, was on the Burguret River on the lower edge of the timberline at an elevation of about 2,000 m. 'Present address: Institut fur Lebensmittelhygiene, Freie Universitat Berlin, 1 Berlin 33, West Germany. 2Present address: Veteringrische Anatomische Institut, 6300 Giessen, West Germany. ' Present address: Department of Animal Physiology, University of Nairobi, Nairobi, Kenya. Each was weighed and dissected as rapidly as possible. For monkey 263, samples of the gas in the stomach were removed 27 min after the animal was killed. For monkey 264 the gas was sampled at 21 min, and the pH was sampled after 26 min. The contents of the glandular portion of the stomach of 263 were almost white in color and were finely comminuted. The contents of the saccular portion were about as well comminuted but had a slightly green color, presumably due to ingested leaves. Leaves appeared to be a minor component of the diet. The stomach contents of 264 contained almost no green material. A number of large whole seeds (about 1 cm in diameter) were observed in 263. They were somewhat more abundant in the glandular than in the saccular portion of the stomach. Material from the saccular stomach of each animal was used for the culture and rate experiments. A sample of the contents from the saccular stomach of each animal was fixed with approximately 1 volume of acid-free Formalin. Direct microscopic counts of the bacteria in these samples were made with a Petroff-Hauser counting chamber and corrected for the dilution by the Formalin. Some of the contents of the glandular portion of the stomach of colobus 264 was Formalin fixed and later examined microscopically. Field equipment. Transportation by air and in the field necessitated miniaturization of equipment and materials. A special autoclave was made by turning a stainless-steel cylinder to dimensions of 44 by 160 mm and fitting it with an adjustable safety valve. An easily portable supply of carbon dioxide was contained in a small cylinder (50 by 265 mm) provided with a needle valve outlet. The valve was threaded into a 15-mm opening at one end of the cylinder. To replenish the gas supply, the needle valve assembly was unscrewed and the cylinder was filled with powdered dry ice. The assembly was then screwed tightly into the cylinder, the valve was opened, and carbon dioxide was allowed to escape for 5 or 10 min. This washed out other gases. The CO2 was used untreated from the cylinder; facilities for scrubbing traces of oxygen were not feasible in the field. Small Pyrex culture tubes (10 by 75 mm) were provided with special butyl rubber stoppers (Jenkyns Rubber Co., London, England) (8 mm in diameter at the small end and 11 mm at the large end, 20 mm long). A small hole was drilled in the center of the upper surface, extending two-thirds through the length of the stopper to permit easier penetration by a syringe needle. Prior freezing in dry ice facilitated boring the hole with an ordinary drill press. The tubes were gassed out with carbon dioxide, stoppered, and sterilized before being taken into the field. Monoject 1-ml sterile disposable syringes, graduated in 0.01-ml divisions, fitted to 1-inch (about 2.5 cm), 20-gauge disposable needles (Becton Dickinson and Co.), were used for quantitative dilutions and for subcultures. In the latter case, the inoculum in only the dead space of the syringe and needle was transferred. Disposable 10-ml syringes were used for filling the tubes and for measuring out media. Media. Media used for initial cultivation included sheep rumen fluid cellulose broth, sheep rumen fluid without added carbohydrate, sheep rumen fluid cellulose agar (SF), colobus stomach fluid agar (CF), and colobus stomach fluid cellulose agar (CC). The methods of anaerobiosis were essentially as described earlier (10), except that the later modification using syringes and needles (11) was substituted for the earlier dilutions by pipettes and the gassing with glass capillaries. CF medium was prepared in the field. Some of the stomach contents were diluted with an equal volume of water, mixed, and filtered by squeezing the mixture through several layers of cheese cloth. The filtrate was used as two-thirds of the final medium. The remainder was a mixture of equal parts of mineral solutions A (0.6% NaCl, 0.3% KH2PO4, 0.15% (NH4)2SO4, 0.06% CaCl2, and 0.06% MgSO4) and B (0.3% K2HPO4). Ionagar (Colab laboratories, Inc., Glenwood, Ill.) at a 0.7% concentration was melted in the mineral mixture before addition of the stomach fluid. Resazurin (0.0001% final concentration) was included in all media. The medium, total volume of 18 ml, was placed in a 30-ml Pyrex Kjeldahl flask with a shortened neck, constricted to take the small stopper used on the culture tubes. After being autoclaved, the medium was cooled to 50 C, and the required amounts of 10% NaHCO, and 2.5% cysteine hydrochloride were injected and mixed to give final concentrations of 0.5 and 0.025%, respectively. The melted medium was tubed in 1.8-ml amounts and kept in a bucket of water at 50C until inoculated. The temperature of water baths was maintained by addition of boiling water as needed. CC medium was similarly prepared except that a pebble-milled suspension of 2% filter paper cellulose composed one-third of the medium, undiluted colobus stomach fluid composed one-third, and equal parts of mineral solutions A and B constituted the remainder. The sheep rumen fluid broth (one-third each water, sheep rumen fluid filtered through cotton, and mineral solution AB), sheep rumen fluid agar (same as broth but agar added), and SF (2% pebble-milled cellulose suspension substituted for the water) had been prepared in the culture tubes at Cambridge, England, by using rumen fluid from a sheep fed on grass. No additional substrate was added to the rumen fluid agar medium since previous experience with the rumen indicated that the colony count obtained without added substrate was about as high as with substrate and, though the colonies were smaller, longevity was greater, presumably because of decreased metabolite production. The bicarbonate and cysteine were added to the tube after the medium was melted and ready to be inoculated in the field. The colobus stomach contents were the consistency of a thick paste but were sufficiently liquid that, after mixing, a 1-ml sample could be sucked up into the calibrated portion of a 5-ml measuring pipette broken off squarely at the 5-ml calibration mark and provided with a rubber mouth tube. The sample was transferred to a tube containing sheep rumen fluid broth, the tube was gassed out, and the contents were thoroughly mixed. A 0.2-ml portion was then injected into a second rumen fluid dilution tube and further serially diluted through seven more tubes. From these, 0.1 ml was inoculated into each tube of the various culture series. After removing the inocula from the tubes of the original sheep rumen fluid dilution series, 0.2 ml of 2% cellulose suspension was injected into each tube to test for cellulose digestion that might occur in liquid but not in agar cultures. After inoculation, the tubes from the first monkey Were rolled in ice water, labeled, and incubated in the investigator's pocket. They were taken to the guest house overnight, where they were placed in a vacuum bottle containing water at 45 C. This had cooled by morning, when the temperature was adjusted to 39 C. The cultures from monkey 264 were placed in the vacuum bottle immediately after the agar had solidified, and that same day were transported back to the laboratory at Muguga, where all tubes were incubated at 39 C. After 8 days, the incubated tubes were examined and the colonies were counted in the higher dilutions showing growth. Individual colonies selected as representative of those observed were subcultured to a similar medium and also to rumen fluid glucose agar. In the higher dilutions containing fewer than 12 colonies, all colonies were subcultured. Subcultures were made at Muguga with the intent of shipping them immediately to California for further study, but permission to import was not obtained in time and it was necessary to take the cultures to New Zealand, where they were maintained until they could be sent to California. Between the initial subculture and the final characterization, a number of the strains were lost. Zero time rate measurement. A 40-g sample of the fresh stomach contents was placed anaerobically in a rubber-stoppered container and immersed in the water bath at 39 C. At 0, 15, 30, and 60 min for colobus 263 and at 0, 21, and 41 min for 264, a subsample of the incubated material was removed to a previously weighed plastic tube containing 1 ml of 10 N NaOH. The subsample was mixed with the alkali, and its weight was determined by difference. The alkaline samples were shipped to Davis by air freight and were analyzed immediately for volatile fatty acids. Nonvolatile acids in these samples were determined in 1971 after adding water to make up that lost by evaporation during storage. Gas analyses. A sample of the gas in the stomach was taken by syringe before the stomach wall was opened. The short sampling needle was replaced with a 20-gauge, 6-in (about 15 cm) needle, and the gas was transferred to the base of a dry culture tube (16 by 150 mm), drawn out to a capillary in its central portion. Some of the gas sample was used to flush out the base of the tube, and the narrow portion was then quickly sealed with a propane torch to enclose the sample in an all-glass container. The'samples were sent to Davis and analyzed with a Perkin-Elmer model 154B gas chromatograph, using a silica gel column and N2 as carrier gas. Analysis of fermentation products and substrates. The pH of the stomach contents was tested with paper having a range of 5.5 to 7.0. Volatile fatty acids in the stomach contents and in the products of the metabolism of pure cultures were determined on a 700 F and M gas chromatograph with a flame ionization detector and a 6-ft (about 1.9 m) column filled with FFAP on Chromosorb W. Carrier gas was helium at a flow rate of 40 ml/min. Column temperature was 125 C, injection port was 210 C, and detector was 290 C. Membrane-filtered (0.22 uim pore size; Millipore Corp.) acidified supernatant fluid was injected directly into the column, or, in analyses of pure cultures, the volatile fatty acids were first distilled at low temperature in a closed vacuum system with the receiving tube dipped in ice water. Qualitative examination for nonvolatile acids was performed by spotting acidified stomach contents on a thin-layer chromatographic plate of ECTEOLA cellulose 300 (Macherey, Nagel and Co.), separating the acids by development with ethanol-water-NH4OH (16:3: 1) and detecting with a spray of 0.04% bromothymol blue adjusted to pH 8.0. For quantitative measurements, samples were first made alkaline and evaporated to dryness; they were then acidified and the volatile fatty acids were separated by vacuum distillation. The residue was extracted with three 5-ml volumes of freshly distilled anhydrous peroxide-free ether. Water (0.2 ml) was added, and the acids were titrated. The ether was evaporated off at room temperature, and water was added to a total volume of 1 ml. L(+)-Lactate in this material was determined with the Lactostat Kit (Sigma Chemical Co.). This involved enzymatic oxidation to pyruvate and reduced nicotinamide adenine dinucleotide, the latter being determined spectrophotometrically. D(-)-Lactate also was determined from enzymatic nicotinamide adenine dinucleotide reduction by using D(-)-lactic dehydrogenase (Boeh-ringer Co., Mannheim). In both reactions, the pyruvate formed was removed by a pyruvate-glutamate transaminase reaction Total nitrogen was measured by the Kjeldahl method. The anthrone method was used for carbohydrates. To determine the starch content of the Formalinfixed stomach contents, a 75-mg sample was dried to constant weight and suspended in 25 ml of water; 1 ml of human saliva was added, and a 10-ml sample was immediately cooled in an ice bath and centrifuged at 4 C. The remainder was incubated for 24 h at 37 C, and a 10-ml sample was similarly centrifuged. The increase in anthrone values of each supernatant after saliva treatment was used as a rough measure of the starch content. Total carbohydrate was estimated by taking 0.1 ml of the above suspension and analyzing it by the anthrone method. For analyses of the fermentation products of the pure cultures, the strain was inoculated into either peptone beef extract broth (PB) or, for some requiring additional nutrients, into dilute rumen fluid broth. PB contained 4.5 g of peptone (Difco), 2.25 g of beef extract, 0.45 ml of 0.1% resazurin, 210 ml of deionized water, 75 ml of mineral solution A, 75 ml of mineral solution B, 90 mg of cysteine hydrochloride, 11.25 ml of 4% glucose solution, and 33.75 ml of 10% NaHCOs solution. The rumen fluid medium differed in containing only 0.135 g of peptone and 0.135 g of beef extract, with 45 ml of rumen fluid clarified by centrifugation replacing 45 ml of water. To assure identical CO2 content in all tubes, the medium (except for the glucose and bicarbonate) was prepared in a round-bottom, 1-liter flask, equilibrated at room temperature with 100% CO2, sealed, and sterilized. The sterilized medium was cooled to room temperature and then opened, with a gassing needle to exclude air from the flask but without bubbling the CO2 through the medium. The sterile glucose and bicarbonate solutions were then added aseptically and anaerobically and mixed, and 9 ml of the medium was transferred anaerobically tp the sterile culture tubes, each containing 1 ml of water. Air was continuously excluded also from the culture tube without bubbling the contents, and the tubes were closed with a recessed butyl rubber stopper. They were inoculated with 0.2 ml of culture, and some were refrigerated as a control. The same media without glucose were inoculated and incubated as a control for fermentation products formed from substrates other than glucose. After completion of growth at 37 C, the cultures were allowed to come to room temperature and the gas produced was measured. A 1-inch, 21-guage needle, attached to a 20-ml syringe well lubricated with water, was inserted through the stopper, and the contents of tube and syringe were equilibrated by vigorous shaking. The amount of excess gas in the syringe was recorded. Without removing the first syringe, 1 ml of normal HCl was injected and, after equilibration, the total gas volume was again read. The second volume minus the first gave a measure of the residual bicarbonate in the tube. The difference between the second volumes in the incubated and in the refrigerated tubes gave the volume of gas pro-duced in metabolism, aside from that released from bicarbonate. The difference in residual bicarbonate gave a measure of the amount of acid produced during metabolism. The differences in gas volumes for duplicate cultures differed usually by less than 2%. One milliliter of 2 N NaOH was then injected, still without removing the initial syringe, and the CO2 was absorbed. Ten milliliters of N2 was injected to maintain a gas pressure above atmospheric. This third volume was read, the gas in the syringe was injected back into the tube, and the syringe was withdrawn. The difference between the gas absorbed in the incubated and refrigerated cultures gave the amount of CO2 produced or used. The gas in the tube was analyzed for H2 by injecting 0.5 ml into a Perkin-Elmer 154B gas chromatograph with a silica gel column and N2 as carrier gas. Peak heights were compared with a standard. Fermentation rate measurements. A volume of mineral solution containing 0.5% NaHCO, was added to an equal volume of stomach contents in each of three wide-mouth bottles closed with a rubber stopper perforated by a 2-inch (about 5 cm) needle attached to a water-lubricated 10-ml glass syringe (16). An initial interval was allowed for temperature equilibration to 39 C and to check the uniformity in the rate of gas production in the three bottles. Strong acid was then added to one of the bottles as a control to release all CO2 from the bicarbonate. Differences in residual bicarbonate gave a measure of the difference in acid production. The total gas released during the fermentation minus that due to acid production gave a measure of the amount of gas produced in metabolism. Before each reading, the contents of each bottle were vigorously shaken by hand to equilibrate the CO, between liquid and gas phases. The amounts of gas evolved were corrected to standard conditions. A temperature of 39 C and vapor pressure of 52 mm of Hg were assumed, though the actual temperature and vapor pressure were somewhat lower than this because the measuring syringe was not immersed in the water bath. Barometric pressures of 522 and 564 mm of Hg (=6.95 x 10' and 7.5 x 10' N/in) were assumed for the experiments with colobus 263 and 264, respectively. The pressure increase due to the weight of the syringe barrel, 10 g/cm2 (=9.806 x 102 N/M2), amounted to less than 1.5% of the total pressure and was neglected. All of these errors made the reported rates of gas production slightly less than the actual. Determination of guanosine-cytidine percentages. Strains identified as Staphylococcus were treated with lysostaphin (Schwartz/Mann, Orangeburg, N.Y.) according to Klesius and Schuhardt (13), and the base composition of the recovered deoxyribonucleic acid was estimated from its buoyant density in a cesium chloride gradient by using a Spinco model E analytical centrifuge. Tests on Staphylococcus aureus. For production of enterotoxin, strains were grown in sac cultures according to the method of Donnelly et al. (7). The amount of enterotoxin formed was measured according to the microslide method of Untermann (17). Stan-dard disks were used to measure antibiotic sensitivity. For arsenic and cadmium sensitivity measurements, disks were impregnanted with 250 gg of Na2HAsO4 7H20 or 3.1 ug of Cd(N02)2 4H20. RESULTS Measurements in the field and many of those from the laboratory are shown in Table 1. The results of analyses of zero time rate samples are shown in Table 2. These were from gas chromatographic analyses completed in the fall of 1969 just after the materials were shipped to Davis. The zero time rate experiments were started 41 min after colobus 263 was sacrificed and 35 min after colobus 264 was sacrificed. The Formalin-fixed stomach contents, analyzed in 1971, contained roughly the same kinds and quantities of fermentation products. The Formalin had been specially purified for fixation of tissues and was free of acids. In 1971, the zero time rate samples were examined also for nonvolatile acids. The thinlayer chromatography plates did not show any succinic acid but did show a little lactic acid. For neither one did the relative size of the spots indicate that their concentration increased during the incubation period. The amounts of lactic acid are shown in Table 2, including the results of analyses for D-and L-lactate on the Formalin-fixed stomach contents. Fermentation rate. In the experiment with stomach material from colobus 263, three bottles were incubated, each containing 42 ml of colobus stomach contents (saccular portion) mixed with 42 ml of balanced salt solution containing 0.5% NaHCO,. The samples were incubated at 39 C, and the gas production was measured to determine whether the rates in the three vessels were comparable. After 15 min of incubation, excess acid was added to one vessel, releasing 4.3 ml (corrected) of gas from bicarbonate. The other vessels, incubated an additional 10 min before acid was added, also showed only 4.3 ml of gas liberated by the acid. The amount of bicarbonate remaining after equilibration was apparently insufficient, and the experiment did not measure the fermentation acids formed. The rates of gas production in the three vessels were 31, 27, and 25 ,umol per h per g of fresh stomach contents, respectively. In the run the next morning with colobus 264, only 32 g of stomach contents was used, and the bicarbonate solution was increased to 50 ml. Figure 1 shows the gas evolution in the three vessels and also the amount of residual bicarbonate in each when acid was added. The rates of gas production in bottles 1, 2, and 3 were 148, 194, and 195 Mimol per g per h, respectively. Comparison of bottles 2 and 3 between 17 and 23 min (Fig. 1) shows a difference of 7.1 ml (corrected) of CO2 released from the residual bicarbonate, indicating that 316 ,gmol of the gas evolved during this part of the fermentation was CO, released from bicarbonate by the fermentation acids. The total gas produced and released by acid in bottle 2 during this period was 408 umol, giving 92 Mmol as the amount of CO2 and H2 produced in the fermentation and 316 ,mol as the fermentation acids. In calibration experiments done previously in California with bovine rumen contents plus mineral solution AB containing 0.5% NaHCO,, incubated in an atmosphere of CO2 (giving a pH of about 6.7), the amount of CO2 released by added lactic acid was equivalent to only about 70% of the amount expected if eq of acid liberates 1 mol of CO2. Correction for a similar lack of stoichiometry in the colobus 264 experiments gave a ratio of fermentation acid to gas of about 5. For bottles 1 and 3 between 17 and 22 min, 132 ,mol of CO2 was released by fermentation acids, and 118 Amol of CO2 and H2 was produced in metabolism. With the 0.7 correction, the ratio of acid to gas was 1.6. The low ratio can be explained in part by the longer incubation of sample 1, with consequent greater depletion of bicarbonate. The experimental error was fairly large in these determinations because of the short incubation time. Nature of the bacteria. The Formalin-fixed stomach contents were examined at Muguga with an oil immersion phase microscope. The microscopic appearances of the two animals were remarkably similar. Large cocci arranged in diplo and tetrad forms were morphologically the most distinctive bacteria observed. Some of them were very refringent, bright in appearance under dark-phase illumination, and regularly arranged in twos or tetrads. A few spirochetes were seen. Photomicrographs of the Formalinfixed material gave the appearance shown in Fig. 2. In colobus 264, the large refringent cocci were relatively more abundant in the contents from the glandular region of the stomach as compared with the forestomach material. The former was not collected from colobus 263. Culture counts for colobus 263 on the CF series and on the sheep rumen fluid series showed 2.2 x 109 and 7.7 x 109 colony-forming units per ml of fresh forestomach contents, respectively. For colobus 264, the counts were the same for both of these media, namely 2.6 x 10g. Direct counts on the Formalin-fixed material were 3.8 x 1010 and 2.6 x 10'0/ml for colobus 263 and 264, respectively. None of the culture series containing cellulose showed any cellulose digestion even after 7 weeks of incubation. Two large, non-cellulolytic colonies in tubes 5 and 6 of the CC medium were subcultured as strains 1-6cc-1 and 1-7cc-1. In this strain designation, the first number indicates the first, 263, or second, 264, monkey. The second number is the dilution tube from which the colony was picked, with the letters indicating the medium (cf for colobus fluid agar, cc for colobus fluid cellulose agar, and sf for sheep rumen fluid agar). The third number is that of the colony picked, the letter following being used to designate the strain when more than one was isolated from that colony. Ten colonies were subcultured from the eighth dilution tube of the CF agar series inoculated from animal 263, and five were subcultured from the ninth dilution. Ten colonies were subcultured from the eighth dilution tube of the CF series inoculated from 264, five were subcultured from the eighth dilution of the sheep rumen fluid agar series, and nine were subcultured from the ninth dilution. Three first subcultures and some subsequent subcultures failed to grow even though some colobus fluid was included in the medium. Of the remaining picked colonies, 27 survived further vicissitudes of shipment and various mishaps. From one of them two different bacteria were isolated, giving a total of 28 strains that were studied. Sixteen of the 28 strains characterized were euryoxic, of which 14 formed catalase. These 14 were gram-positive cocci, in single, diplo, tetrad, or irregular chain arrangement, and fermented glucose without producing visible gas bubbles. The diameter of the cells varied from 0.6 to 1.2 ,m, and in some strains was quite variable. Nine of the strains formed white colonies, of which four strains liquefied gelatin; four of them were light yellow and did not liquefy gelatin. One strain was golden yellow and fermented mannitol, whereas the others did not. It reduced nitrate but did not liquefy gelatin. The golden strain (2-8sf-2) was identified as S. aureus. It was identical to S. aureus ATCC 14458 in morphology and in production of coagulase, proteinase, lipase, nuclease, and phosphatase. ATCC 14458 gave only delta-hemolysis on sheep, cow, rabbit, and human blood, whereas strain 2-8sf-2 showed a hemolysin for all bloods tested and was beta-hemolytic on sheep and cow blood. Strain 2-8sf-2 also differed in being sensitive to arsenic and cadmium, streptomycin, tetracycline, and penicillin. It was negative for production of enterotoxins A, B, C, and D ( Table 3). The phage type was not typable at routine test dilutions, but at 1,000 x concentration the phage type was 42A/52/42D/29/55/79 (4). The other 13 euryoxic catalase-positive strains (Table 4) also showed the characteristics of the genus Staphylococcus and were presumably S. epidermidis (1). For several representative strains the guanosine-cytidine percentages were determined (Table 5). Lactic acid was the chief fermentation product of the three tested strains of staphylococcus grown anaerobically on glucose ( Table 6). The recovered lactate accounted for 75 to 88% of the glucose provided. The lactate was chiefly the L-form for two of the strains. The third strain formed almost equal amounts of L-and D-lac-tate. The acetate in the experimental culture was no greater than in the uninoculated control. The calculated yield of cells was low for a homolactic fermentation, only 11.2, 13.2, and 10.6 gg of cells per Amol of glucose for the three cultures, respectively. The yield of cells was estimated from the nitrogen content of the sediment of the culture, because it was necessary to add CaCO, to obtain good growth. In an experiment without CaCO,, the nitrogen content of the cells of strain 2-9sf-4 was 12.6% of the dry weight. They were nonmotile and without capsules. They produced large, circular, entire, thin, lenticular, deep colonies and opaque, smooth, white colonies on the surface of the agar in the roll tubes. Three tested strains (1-8cf-2b, 1-8cf-10, and 2-8cf-4) were catalase positive and produced acetic and propionic acids in the ratios expected for Propionibacterium. The production of H2 varied (Table 7). A slight amount of H2 was produced by all three strains when the medium contained 0.5% peptone and 0.3% beef extract, and only in strain 1-8cf-10 was the H2 production increased by glucose. One strain produced a slight amount of H2 from rumen fluid. All three strains grew on the glucose-free peptone, and the amount of fermentation products was not greatly increased by addition of glucose ( Table 8). The eight strains were assigned to the genus Propionibacterium. The other four anaerobic strains were not as easy to classify. Cells of strain 1-8cf-9 ranged in shape from cocci to rods, 0.5 Am in diameter, single, in V's or close clusters. The Gram reaction was not clearly negative or positive. The strain produced 9 Amol of acetic acid per ml when grown on PB, and this amount was not increased if glucose was added. No propionic acid was formed on the PB medium, but if glucose was added, 1 gmol of propionic acid per ml was formed. The strain did not produce H2 at any time and is probably closely related to Propionibacterium. Strain 2-8sf-1 contained gram-positive rods to cocci, single, in pairs or chains, 0.75 to 1.25 Am in diameter and 1.25 to 2.0 Am long. They were nonmotile and without capsules, and formed acetic and propionic acids in the ratio expected for Propionibacterium, but they also produced a great deal of H2 (Table 7). In its fermentation products, the strain appeared to be intermediate between Propionibacterium and Veillonella. A slight amount of H2 was formed on PB or rumen fluid medium alone, but addition of glucose caused copious H2 production. There was very little growth on rumen fluid medium alone but a fair amount with glucose added. Even with added glucose, the optical density on rumen fluid medium was not as great as that on PB, which was about half of that on PB plus glucose ( Table 7). Traces of n-butyric and isovaleric acids were formed, the amounts being slightly increased by glucose. Strain 1-9sf-2 was non-saccharoclastic and formed some acetate and H2 from the PB medium without glucose. It grew very poorly on the few media tested. Morphologically it consisted of chains of cocci and is probably referable to the genus Peptostreptococcus. No propionic or butyric acid was formed. Strain 2-9sf-1 resembled 1-9sf-2 in morphology and culture characteristics. DISCUSSION The results from the zero time rate experiments demonstrate that within 30 min after death of the animal, the fermentation was inhibited by the acids formed, in agreement with the observations of Kuhn (14). Rapid acid production was indicated by the in vitro fermentation rate experiments. With colobus 263, the bicarbonate added was insufficient to bring the pH into the range (about pH 7.0) at which the acids produced could be measured by release of CO2 from bicarbonate. In this experiment, 2,520 Amol of NaHCO. (60 ,omol/ml) were added. Addition of excess acid at 34, 44, and 45 min of incubation of the three a Optical density (OD) was measured at a wave length of 600 nm in a cuvette with a 1-cm light path. The acid production in the live monkey stomachs must have been considerably greater than in ruminants collected in nature and similarly studied (12). In the ruminant studies, only 30 Mimol of NaHCO, per ml of rumen contents was sufficient to maintain an excess of bicarbonate during the in vitro fermentation rate measurements made at about the same time after death of the animal. With colobus 264, 99 ,gmol of NaHCO, per ml of stomach contents was added, and in this case 48, 714, and 536 Mmol of gas were released by acid added after 17, 23, and 24 min of incubation, respectively, as compared with the 3,168 ,gmol total of added bicarbonate. With colobus 264, the total gas released, both during the run and by acid added later, amounted to 2,076, 2,634, and 2,813 ,umol for the three samples, respectively. The more rapid development of acidity as compared with the rumen could be due to a greater availability of substrate in the colobus, to poorer buffering, or to poorer absorption. The stomach contents appeared to be chiefly finely comminuted, starch-like white material and gave an intense black color when tested with iodine, though the percentage digestible by saliva was less than 5% ( Table 1). The fermentation gas production rate for the first animal, 28 gmol per h per g (wet weight), is of the same order of magnitude as the values of 38 and 12 reported by Kuhn (14) for Presbytis cristatus and Procolobus badius, respectively. Values of 63 to 79 umol per h per g reported by Bauchop and Martucci (3) for Presbytis cristatus are considerably greater but are less than the average rate of 179 ,umol per h per g for colobus 264. This high rate represents the potential for a very rapid fermentation when sufficient bicarbonate is added to maintain a favorable pH. It is doubtful that the high rate for colobus 264 obtained in the animal. More likely, after feeding, acid is produced more rapidly than it can be absorbed, and the resulting acidity slows the fermentation. The slower rate observed with colobus 263 is unexplained, but may be due to a longer interval between the last feeding and the time of sampling. It seems unlikely after death that mixing of the acidic contents of the glandular stomach contents with the forestomach contents could account for the high acidity of the latter, since the contents of both compartments were relatively dry. The percentage of dry matter in the saccular stomach contents of colobus 264 (Table 1) was much higher (31.7%) than in colobus 263 (18.7%), and the percentage nitrogen was much lower. Colobus 264, collected at 8:32 a.m., may have completed a morning feed on dried seeds and not yet consumed water. The ratios of the concentrations of the various fermentation acids in the zero time rate samples and in the Formalin-preserved material ( Table 2) are similar to those typical of rumen samples. For colobus 264, the total concentrations of acids is about as high as the highest values encountered in the rumen. There is more lactate in the colobus stomach than is usually found in forage-fed ruminants but comparable to those on a high concentrate ration (2). The absence of methane from the gas in the colobus stomach contrasts with its occurrence in the langur (3) and in the ruminant. The stomach fermentation of the colobus differs also from that in a marsupial, the quokka (Setonix brachyura) (Moir and Hungate, unpublished data), which formed significant quantities of both hydrogen and methane. Another difference in the colobus fermentation from that in ruminants is the absence of cellulolytic bacteria. No bacterial colonies surrounded by zones cleared of cellulose were observed in any of the cellulose agar cultures containing rumen fluid or colobus fluid, and none of the liquid cultures showed any disappearance of cellulose. These negative results might be explained in lower dilutions as due to too great an acidity introduced with the inoculum, but this could not explain the negative results in the higher dilutions. The stomach contents seemed to contain very little plant fibrous material, suggesting that "leaf eating" may not apply to Colobus polykomos. Acidity may be a factor preventing the development of both methanogenic and cellulolytic bacteria in the colobus stomach. Acidity may also explain the absence of protozoa, though Kuhn (14) postulated that it was the consistency of the contents which prevented protozoal growth. Purser and Moir (15) found that the rumen protozoa could not survive continuous exposure to acidities much below pH 6. Another difference from the forage-fed ruminant is the greater number of euryoxic cultures as compared with anaerobes. Inability to absorb traces of 02 from the CO2 used in the field experiments might be a factor in the increased proportion of aerobes and in the low ratio of culture count to direct count. But the absolute number of cultured aerobes per milliliter is much higher than for the rumen. The culture counts were 20 and 10% of the direct counts for the two monkeys, respectively. This is a slightly lower ratio than is obtained in careful culture experiments on rumen contents from forage-fed cattle, but is of the same order of magnitude. A higher ratio of culture count to direct count is usually obtained for ruminants on high concentrate rations. Although propionibacteria are occasionally abundant in the rumen (9), they are not usually a prominent element, nor are staphylococci commonly found. The morphology of the cultured bacteria is consistent with the morphology of some of those seen by direct microscope examination. The pure culture strain 1-8cf-4, a staphylococcus, showed tetrads of cocci very similar to the tetrads of smaller cocci in Fig. 2. None of the cultured strains resembled the very large refringent cocci. The appearance of these cells is similar to that of Sarcina ventriculi, and, in view of their greater abundance in the contents of the more acidic glandular stomach contents, it seems possible that this species is a normal inhabitant of the colobus stomach. The observed high refringence might be due to the cellulose produced by this species (5). It seemed to be more marked as the cells increased in size. The relative abundance of staphylococci in the colobus might be the result of grooming with the teeth for fleas and other external parasites, a process which could conceivably provide a continuous inoculum, proliferating further under the conditions in the stomach. Thorough examination failed to demonstrate any enterotoxin production by the isolated Staphylococcus aureus (Table 3). This suggests that the staphylococcus strains in the colobus stomach may have been selected for host digestive compatibility and may be a normal component of the stomach microflora, maintaining themselves without ingested inocula. The concentration of fermentation products affords some index to their rate of absorption, since absorption of volatile fatty acids is a function of the concentration gradient between stomach contents and blood. The concentration of volatile fatty acids in the stomach of colobus 263 ranged between 107 and 125 ,mol per ml of stomach contents and between 212 and 434 for colobus 264 ( Table 2). The values obtained from analyses of the Formalin-fixed material are roughly the same, if the dilution by the Formalin is taken into account. ACKNOWLEDGMENTS We are much indebted to many who assisted during the course of these studies. Thanks are due to the Kenya Game Department for permission to collect the animals and espe-

v3-fos

2020-12-10T09:04:22.502Z

1974-10-01T00:00:00.000Z

237232198

s2

Microbial Response to Drought in a Texas Highplains Shortgrass Prairie The population of the microbial flora of a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) prairie near Amarillo, Texas, was studied during 1971 after a severe drought. Bacteria, fungi, and algae were estimated by plate count and terminal dilution procedures. Rates of grass and paper decomposition were determined. The microbial flora of soil associated with bovine-grazed grass did not differ significantly from the flora associated with ungrazed grass, either qualitatively or quantitatively. During drought, a greater number of fungi were found in soil associated with prickly pear than in that associated with blue gramma grass. The microbial biomass decreased one full log between the surface and a depth of 50 cm, and the percentage of anaerobes increased with depth. The maximum numbers of fungi and algae detected were 8 × 105 and 6 × 104/g respectively. A linear relationship existed between the microbial biomass and soil moisture. The maximum number of aerobic, heterotrophic bacteria detected was 1.5 × 108 viable cells per g of soil. The population of the microbial flora of a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) prairie near Amarillo, Texas, was studied during 1971 after a severe drought. Bacteria, fungi, and algae were estimated by plate count and terminal dilution procedures. Rates of grass and paper decomposition were determined. The microbial flora of soil associated with bovine-grazed grass did not differ significantly from the flora associated with ungrazed grass, either qualitatively or quantitatively. During drought, a greater number of fungi were found in soil associated with prickly pear than in that associated with blue gramma grass. The microbial biomass decreased one full log between the surface and a depth of 50 cm, and the percentage of anaerobes increased with depth. The maximum numbers of fungi and algae detected were 8 x 105 and 6 x 104/g respectively. A linear relationship existed between the microbial biomass and soil moisture. The maximum number of aerobic, heterotrophic bacteria detected was 1.5 x 108 viable cells per g of soil. Clark and Paul (4) have reviewed the sparse literature on the microbial ecology of native grasslands. The biomass in the top 30 cm of a Canadian grassland is estimated by Babiuk and Paul (2), by direct microscope counts with fluorescein isothiocyanate, to be 30 to 76 g/m2 on a dry weight basis. Aerobic soil extract dilution plates showed 4 x 107 bacteria/g in the 0-to 10-cm layer (during April) with a linear decrease of total number with depth. This study provided a taxonomic and physiological activity profile of the microbial population of the soil during the 1971 growing season after a year of severe drought in a mixed blue gramma grass (Bouteloua gracilis H. B. K.) and prickly pear (Opuntia polyacantha Haw.) grassland. The contribution of prickly pear to the total ground cover varied from 3 to 11%. The significance of the cactus is due to its tendency to grow in thick clones. During the dry season, it may at times appear to be the predominant ground cover, because of greatly reduced amounts of grass. Minor amounts of buffalograss (Buchloe dactyloides Nutt.), sand dropseed (Sporobolus cryptandrus Torr.), and purple three-awn (Aristida purpurea Nutt.) prevail throughout the undisturbed grassland. There were specific questions of interest in this study. (i) Are there qualitative or quantitative differences between the microbial flora of soil associated with either bovine-grazed grass or ungrazed grass? (ii) Do the microbial flora associ-ated with grass ground cover differ from that of prickly pear? (iii) Is there any change in total population or type with increasing depth? (iv) Is the microbial biomass linked with available soil moisture? MATERIALS AND METHODS Site description. At Texas Tech University Research Farm (Amarillo, Texas), a moderately grazed site of 32 hectares and a 14-hectare site which had been ungrazed for 5 years were selected, approximately 6.8 km apart at an elevation of 1,180 m. The weather is extremely variable, characterized by strong prevailing winds, dry winters, and sharp temperature changes (9,12). Precipitation of only 14 cm was received at the site during 1970; the average is 56 cm. During 1971, the following amounts of precipitation were received: in April, 1.5 cm; May, 1.3 cm; July, 8.8 cm; August, 9.8 cm; September, 6.3 cm; and October, 3.5 cm. The soil at the sites is classified as Pullman silty loam (6). Each of the sites was subdivided into two replicate sites. Each replicate was further subdivided into 200 quadrats (2.74 by 2.74 m), which were arranged in two parallel rectangles (5.49 by 137.2 m) with an access alley inbetween. Bacterial population studies. Two cores (2.5 by 50 cm) were taken from each of six plots, chosen at random from each replicate of both the grazed and ungrazed sites at each sampling period, for a total of 48 cores. The cores were obtained with a hollow soil sample tube hydraulically pressed into place. Two complete additional sets of cores were obtained when the sites were subdivided for comparison of prickly pear and grass ground cover. Each core was divided 700 into five zones, or depths, from 0 to 5 cm, 5 to 10 cm, 10 to 20 cm, 20 to 30 cm, and 30 to 50 cm. The sections from each core were placed separately in sterile plastic bags, sealed, and immediately stored in an ice chest. All 12 samples of each zone from each replicate were blended together in 90 ml of sterile, physiological saline for 3 min, and appropriate serial dilutions were prepared. Pour plates were prepared with five replicates at each of at least three dilutions. Plate counts were made from the maximal colony development after incubation at 30 C. Aerobic heterotrophs were assayed (15) with standard methods agar (SMA; tryptone glucose yeast agar from BBL). Anaerobes were assayed with anaerobic agar (BBL) and with incubation under prepurified nitrogen; the methylene blue confirmed anaerobiosis. Aerobic and-anaerobic sporeformers were assayed by heat-shocking the appropriate dilutions at 85 C for 20 min before plating. Fungi were cultivated in rose bengal streptomycin agar (7). The population of algae was estimated by the dilution-frequency method (1). The total populations of different nutritional types were also determined by the dilution-frequency method, with the basal salts media of Lockhead and Chase (8). Three additions were made to this set of media: (i) Phenol red broth base (BBL) was chosen to approximate the SMA; (ii) a 1.0 g amount of microcrystalline cellulose (Calbiochem, Los Angeles, Calif.) per 100 ml was added to one set of the basal medium; (iii) the chitin basal salts medium of Campbell and Williams (3) was used to determine chitinase activity. The most common colony types from each horizon were selected from the SMA and classified according to nutritional type as described previously (8). Decomposition. Rates of litter decomposition were evaluated with squares (8 by 8 cm) of Whatman no. 1 filter paper or 8-g samples of blue-stem hay (Andropogon gerardi Vitman) sewn lengthwise in 1-mm mesh nylon net bags. These bags were placed horizontally on the soil surface. Blue-stem hay was used throughout the U.S. grassland study as a standard litter material. Filter paper samples were placed both on the surface and at a depth of 5 cm in the soil. Five samples were used per replicate per treatment of each sample. The samples were incubated at 50 C, and dry weights were obtained. The total sample was ashed, and the excess ash weight was subtracted from the sample weight to correct for soil contamination of the sample. Respiration. The open end of round metal cans (12.6 cm in diameter by 17.0 cm high) were inserted 2 cm into the soil, and carbon dioxide was absorbed in 10.0 ml of 0.1 M potassium hydroxide, which was placed in open vials in the cans for an average period of 16 h. The cans were shaded with plastic covers, and five samples per replicate were taken at each period and compared to unexposed 10.0-ml samples of base. Trapped carbon dioxide was estimated by automatic titration with 0.05 M potassium biphthalate. Physical measurements. Pooled cores from each horizon were weighed to 0.1 g. Moisture content was determined gravimetrically on 10.0-g samples by drying at 105 C for 24 h. Ash content was determined from samples ashed at 600 C for 6 h. Statistical analysis. The total plate count data were analyzed for covariance with a prewritten computer program. Significant differences were identified by contrasts. The grazed-grass plate count data means were analyzed separately for covariance, and mean differences were then compared by the method of Tukey (14). RESULTS The results of viable cell counts of bacteria, fungi, and algae are presented in Fig. 1 through 4. Both the viable cell and/or spore counts in the 0-to 5-, 10-to 20, and 20to 30-cm zones roughly followed the 5to 10-or 30-to 50-cm zones, with appropriate corrections for the effect of depth. The computer-based analysis of all data sets for bacteria identified highly significant (1%) effects due to date, treatment, and zone depth. A marked difference (99% confidence) was found between the number of fungi associated with grass versus that associated with prickly pear ground cover. The effect of depth on the number of observed fungal colonyforming units was significant at the 99% confidence limit. To clarify the above results, the data obtained from the grazed grass site were analyzed separately. Highly significant effects were identified for the numbers of aerobic heterotrophs due to depth but not to the date. The means from both the 0-to 5-and 5to 10-cm zones differed significantly (95% confidence) from the means of the 10-to 20-, 20-to 30-, or 30-to 50-cm zones. The difference between the 0to 5and 5-to 10-cm zones was not significant. Analysis for covariance of the total population of aerobic heterotrophs to a soil depth of 50 cm identified a highly significant effect due to the date. Comparison of means showed that the total population of aerobic heterotrophs on 30 September differed significantly from the populations on 4 August, 29 June, and 8 June. The total population on 2 September differed significantly from those on 29 June and 8 June 1971. In the top two horizons, the 0-to 5-and 5-to 10-cm zones, the number of fungal colony-forming units associated with prickly pear cacti decreased nearly one-half log during the period from May until August. The number of fungi associated with grass ground cover was consistently smaller than the number associated with the prickly pear cacti (during the spring and early summer). During this same period, many of the cacti were heavily infested with Coccidae scale insects. The computer analysis of data from both sites placed a 99% confidence limit on this effect. There was a significant effect due to Microbial populations and moisture in the 5to 10-cm soil horizon during 1971. Symbols: A, ungrazed site (U), grass ground cover; 0, grazed site (G), grass ground cover; and 0, prickly pear ground cover (pp), results averaged from grazed and ungrazed sites. the date of sampling on the number of fungal colony-forming units at the grazed site. There was a highly significant fungal-colony population change associated with increasing depth. The number of fungal colony-forming units in the 0to 5-and 5to 10-cm soil depths differed significantly from the numbers associated with any other depth. It was not determined whether the fungal colony-forming units were due primarily to spores or to mycelial fragments. Limited data did not permit evaluation of algal population changes as the season progressed. The number of algae in this arid soil was greater than expected. The average populations per gram of wet soil in the 0to 5-cm horizon on the following dates were: on 29 June 1971, 9 x 103; 2 September 1971, 0.3 x 103; and 30 September 1971, 50 x 103. Highly significant decreases in the average populations of aerobic bacteria, actinomycetes, anaerobic bacteria, and the aerobic fungi ( Fig. 3) were observed with increasing depth. Because the latter data might be misleading because of the presence of many faculatively anaerobic organisms, dilutions were heated to kill the vegetative cells and plated in the appropriate medium. The number of aerobic sporeformers tended to decrease with depth, but the number of anaerobic or faculative sporeformers did not. Additional data will be required to firmly establish this relationship. Actinomycete-type colonies were counted on the same plates as the bacteria, but represented only 2% of the colonies in the top horizon and 4.5% in the bottom horizon. The medium was not ideal for growth of the actinomycetes, and these data provide only preliminary estimates. In some ways SMA was an unfortunate choice for the plating medium, since it is too rich to allow the growth of many of the soil bacteria. However, its choice by U. S. International Biological Program permits the comparison of results at several sites; the usual soil extract agar would vary with the soil used for its preparation. There are, in addition, several different formulas for soil extract agar. To evaluate partially the influence of SMA on the results, terminal dilution studies were conducted with several media (8). The dilutions were prepared from the same soil suspensions used for the plate count studies. Only slightly higher counts were obtained by using a basal salts medium supplemented with amino acids. Cellulose in the basal salts medium resulted in slightly lower counts. Apparently, the SMA allowed adequate growth of the bacteria from this soil. The most common colony types on SMA were selected and cloned from samples of each depth collected on 30 June 1971. These were compared by using basal salts medium plus various supplements, including soil extract (8). All but two of the bacteria were gram-positive rods and 12 of these were Bacillus species. Large variations existed in the nutritional requirements of these organisms; some required soil extract, amino acids or yeast extract for growth. Several cultures were inhibited by the addition of growth factors, yet all 18 isolates grew well in the presence of both soil extract and/or yeast extract. No clearly identifiable changes in nutritional requirements were associated with soil depth. The moisture content and bulk density of the soil increased with depth (Fig. 5). The ash content varied only slightly in the hundreds of samples examined and averaged (95.2 ± 4.0 g on a dry weight basis). The ash content increased slightly with depth. During May and June, soil samples collected at 0 to 5 cm below prickly pear had 0.5% more moisture than samples collected beneath grass. The biomass of platable, aerobic heterotrophs on the grazed site to a depth of 50 cm on 8 June 1971 is estimated at 3 x 1012 bacteria per square meter, or approximately 3.3 g/m2 (1.1 x 10-12 g/cell; 10). Analysis of covariance indicated that significant (a = 0.01) effects were associated with date, treatment, and depth, but not moisture. The difference between the number of bacteria associated with the grazed and ungrazed site was not significant at 1%, but it was significant at the 5% level for the 0-to 20-cm zone. There was a marked difference (a = 0.10) between the number of fungi associated with grass versus fungi associated with prickly pear ground cover. The negative relationship between soil moisture of individual horizons and their bacterial populations seemed questionable as populations increased after rains during July and August. A linear correlation was found to exist between the aerobic bacterial populations and total moisture per 50 cm of core (Fig. 4). This effect was identical to that for date of sampling. Decomposition rates were significantly different for surface versus subsurface placement and may have depended on moisture. During June, grass litter samples lost 17.0% in weight. Filter paper samples which were placed on the surface lost an average of 1.2% by weight, and subsurface samples lost an average of 15.2%. During August, the grass lost 23.1% of its weight, the surface filter paper lost 2.4%, and the subsurface filter paper 23.9%. Soil carbon dioxide evolution estimates (Table 1) indicate no significant differences between the two sites, but soil respiration increased as the season progressed. Relationship of the number of aerobic viable bacteria per gram of wet soil. The results are the averaged and normalized (for zone depth) data to a depth of 50 cm. Each dot represents the averaged and normalized datum for one replicate on a particular date, from either the grazed or ungrazed site. The least squares equation for the regression line is Y = 0.005 + (0.0015)(X). The 95% confidence interval on the slope is 0.00061 to 0.00239. DISCUSSION There were minor but consistent differences in microbial populations on the two sites, resulting in the significant covariant. These differences never exceeded one-half log and were of only minor importance. Sampling difficulties made it inadvisable to continue at the ungrazed site after September. Statistical analysis indicated a highly significant difference between the microbial flora associated with grass ground cover and that associated with prickly pear. Data in Fig. 1 showed that more fungi were associated with the prickly pear ground cover during the early summer (in the first two horizons), when the moisture content of the soil was lowest and cacti were diseased. Shade provided by cacti may have allowed the soil to retain a greater moisture content during early summer than soil under the sparse grass. Comparison of soil moisture values for grass and prickly pear indicates that the soil in the first horizon had 0.5% higher moisture content under the prickly pear. This differential was true during June, but decreased during the remainder of the season. The greater fungal population during the dry weather may have resulted both from stored moisture in the cacti and/or higher soil moisture under the cacti. The populations of both bacteria and fungi decreased significantly in number with depth (Fig. 3). This was verified by statistical analysis. Though the total number of aerobes and anaerobes decreased with depth, analysis of the data in Fig. 3 showed that the percentage of anaerobes doubled from the top to the bottom horizon. This is in agreement with the theory that oxygen must diffuse from the soil surface downward, and that the rate of diffusion might be slow in this very hard, unworked soil. The decrease in numbers of aerobic bacterial and fungal colony-forming units with depth would be expected. There is no direct evidence that oxygen is limiting at greater depths in this soil. Such effects are usually associated with watersaturated soils. The primary producers (grass, algae, and prickly pear cacti) were at the surface. Thus, nutrients produced by photosynthesis diffused downward and decreased with depth. The ash content of the soil increased slightly with depth, which may indicate a lower percentage of organic matter at greater depths, as was found at the Amarillo Experiment Station (5). The experimental plate count data provide no information about the autotrophic bacteria. Terminal dilution studies indicated no shift with depth in nutritional types. The same results were obtained from the nutritional groupings of organisms selected from SMA. The latter data is biased, because SMA favors heterotrophic metabolism. All of the pure cultures grew in basal salts medium, supplemented both with soil and/or yeast extracts. A large percentage of the aerobic heterotrophic bacteria were Bacillus species. Values of 20% have been 70"a given for other grasslands (4). Twelve out of 18 clones were Bacillus species, and in plate counts a preponderance of the colonies were Bacillus (Fig. 3). This may be due to lack of moisture or simply to spore survival despite insufficient water activities for vegetative cell growth. The estimate of' biomass (aerobic, heterotrophic bacteria) is a very low 3.3 g/m2 in June and 6.7 g/m2 in November. Babiuk and Paul (2) calculated that the active biomass based on plate counts was only '41 that of direct counts. However, they concluded that the plate count procedure may be a better estimate of metabolizing cells in the soil. Our results were in accord with a very dry soil and a maximum carbon dioxide evolution of 0.7 g per m2 per 24 h ( Table 1). Several workers obtained similar plate counts with a peak of 20 to 75 x 106/g of soil at the 0-to 5-cm depth at this and associated sites in 1970 (12). The maximum plate count in this study was 150 x 106/g of soil at the 0-to 5-cm depth. Reuss (11) reported that another worker, Doxtader, estimated the total dry weight biomass of' bacteria plus fungi to a depth of 30 cm at a grassland site in Colorado to vary from 51 to 82 g/m2. Babiuk and Paul obtained counts of 4 x 107 to 27 x 107 bacteria per gram in the 0-to 10-cm layer on a Canadian grassland soil, by using soil extract agar. These counts compare very favorably with the results of this study. Since our figures did not include the true anaerobes, actinomycetes, autotrophs, and the fungi, and also were based on the wet soil weight, the estimate of biomass was very low. If Babiuk and Paul's estimate (2) of the relationship between viable cell and direct count is applicable, the maximum biomass in our experiments may have been as much as 100 g/m2. On the basis of individual samples from each horizon, no correlation between moisture and the number of viable bacteria was demonstrated by statistical analysis. Yet, one would expect such a correlation if for no other reason than the dependency of grass on available moisture. Since the herbage is the primary producer in the system, the number of active bacteria should be linked to the growth of the grass or other plants. The data were reexamined. The sum of the total viable cell counts of aerobic heterotrophs for 0 to 50 cm was directly proportional to the sum of the available moisture (Fig. 4). Lack of moisture altered the grassland drastically. The soil at the Texas Tech University Research Farm had not been studied extensively. Results obtained at the Amarillo Experiment Station, which also has Pullman soil, indicated that the moisture tensions during this study were always in excess of 15 bar. The wilting point for grass on the Pullman soil is approximately 14 bar (5). The soil moisture was always lowest in the first 20 cm (Fig. 5), exactly where one would expect the greatest microbiological and plant activity. The top 5 cm was often dust, with much of the remaining soil brittle and dry to touch. The water activities may have been below the critical points for growth of some bacterial species. The parched, sparse spring and summer vegetation changed to a luxuriant grass cover in the fall, accompanied by increased microbial biomass. The decomposition studies provide an indication of the soil activity. Though cattle-grazing influence upon the microbial flora of this grassland was negligible, it must exist directly below their excrement. The influence of the ruminants on the decomposition of litter is not known, beyond their eating the decomposition samples. Jack rabbits ate tongue depressors marking the locations of buried samples during the spring' During June, surface grass samples lost an average of 17% in weight. The average litter biomass of the blue-gramma grass during June of 1970 (13) was estimated at 106 to 181 g/m2; thus, if the 17% decomposition is representative, then 18 to 31 g of litter decomposed per m2 during the month, or 0.6 to 1.0 g per m2 per day. The carbon dioxide evolution measured during this period was 0.26 g per m2 per 24 h. The two figures are not greatly disproportionate. Decomposition, respiration, and microbial biomass changes were all apparent responses to rainfall and available moisture. The effects of extreme drought were the controlling factors in this grassland ecosystem.

v3-fos

2020-12-10T09:04:12.765Z

1974-03-01T00:00:00.000Z

237234087

s2

Effect of Sodium Ascorbate and Sodium Nitrite on Toxin Formation of Clostridium botulinum in Wieners Toxin production by Clostridium botulinum was inhibited by sodium nitrite levels above 50 μg/g of wiener. Sodium ascorbate at levels of 105 and 655 μg/g of product did not decrease the effectiveness of the sodium nitrite inhibition, nor did sodium ascorbate potentiate it. The results indicate that the use of sodium ascorbate in vacuum-packaged wieners does not appreciably alter the inhibition of C. botulinum toxin formation by sodium nitrite. product did not decrease the effectiveness of the sodium nitrite inhibition, nor did sodium ascorbate potentiate it. The results indicate that the use of sodium ascorbate in vacuum-packaged wieners does not appreciably alter the inhibition of C. botulinum toxin formation by sodium nitrite. Previous studies in our laboratory demonstrated the effectiveness of sodium nitrite (nitrite) in preventing the formation of Clostridium botulinum toxin in wieners that were temperature abused (1). In many cured meat items, current industry practice includes the addition of sodium ascorbate (ascorbate) to accelerate the formation and improve the stability of cured meat pigments. Little information is available, however, regarding possible changes incurred by ascorbate on the effectiveness of nitrite in controlling toxin formation in these products. Conceivably, ascorbate could enhance the growth of C. botulinum by decreasing the redox potential. Also, it could reduce the effectiveness of nitrite inhibition by reacting with the nitrite and thus lowering its concentration. On the other hand, the possibility exists that ascorbate may potentiate the inhibition by nitrite. To gain information regarding these possible effects of ascorbate, wieners were prepared as follows. The wiener premix (without nitrite or ascorbate) was prepared according to the following formula: pork, 39.82%; beef, 34.11%; water, 3.20%; ice, 17.14%; salt, 2.52%; dried corn syrup solids, 1.80%; dextrose, 1.02%; and spice, 0.39%. Eighteen batches of product were made with six levels of nitrite (0, 15, 30, 50, 100, and 150 Ag/g) and three levels of ascorbate (0, 105, and 655 Mg/g). Each batch of product was inoculated with a heat-shocked preparation of C. botulinum spores. The inoculum was adjusted so that approximately 1,000 spores per g of raw premix were added. Spore levels in the premix and thermally processed product were estimated by decimal dilution and inoculation of thioglycolate broth, followed by toxicity testing (intraperitoneal inoculation of mice) of the incubated cultures. A 10-tube most-probablenumber procedure was used. The spore preparation consisted of approximately equal numbers of the following strains: type A-33A, 73A, 62A, 109A, and 3A; type B-53B, 213B, 113B, 169B, and Lamanna. The total viable counts (30 C, 40 h) were made by using APT agar (Difco). As a result of thermal processing of the wieners, an average decrease of less than 1 log unit occurred in C. botulinum spore count. The thermally processed product was vacuum packaged and incubated at 28 C. Samples were tested after 0, 7, 14, 21, 28, and 56 days for pH value and total viable count, and after 7, 14, 21, 28, and 56 days for toxicity. All general methods used in this study were reported previously (1). The average composition of the thermally processed product was: moisture, 53.2%; protein, 11.2%; fat, 28.9%; salt, 2.7%; and water, 8.7%. The effect of nitrite and ascorbate on the incidence of toxic samples during incubation of the wieners is shown in Table 1. Samples containing 50 ,ug of nitrite per g of product, or less, began to show toxicity after 7 days of incubation. The incidence of toxic samples increased significantly after 14 days and remained high throughout the incubation period. Wieners containing 100 and 150 gg of nitrite per g of product failed to develop toxicity, with the exception of one sample containing 100 ,ug of nitrite per g of product. In the latter instance, toxicity was evident only after 56 days of incubation. The total number of toxic samples in each series containing nitrite was not altered appreciably as the level of ascorbate was varied (Fig. 1). The incidence of toxicity declined sharply above the level of 50 jig of nitrite per g of product. number of toxic samples/five samples During the incubation period, total viable counts and pH values showed no appreciable variation among the 18 variables. The pH values dropped steadily after thermal processing through the incubation period, with the exception that at zero level of ascorbate, all nitrite levels showed a slight increase in pH at the final 56-day sampling. The total counts generally decreased in the 56-day-old samples. Apparently, acid production was not a factor in preventing toxin formation under the conditions of the experiment. From the results obtained in this study, it can be concluded that ascorbate did not affect the efficacy of inhibition of toxin production by nitrite. Thus, the inclusion of ascorbate in the formulation of wieners does not detract from the desired inhibitory effects of nitrite on the production of botulinal toxin.

v3-fos

2018-04-03T05:20:51.595Z

1974-06-01T00:00:00.000Z

20593479

s2

Rapid method for detection and enumeration of fecal coliforms in fresh chicken. A rapid method for enumerating fecal coliforms in foods was developed employing an agar pour-plate medium. After 7 h of incubation at 41.5 +/- 0.05 C, this medium effectively allows the growth of fecal coliforms only. This rapid method was compared with the Association of Official Analytical Chemists multiple-tube dilution method for Escherichia coli, by using 21 samples of fresh, cut-up chicken and a surface rinsing procedure for sample preparation. Verification of picked colonies was carried out in EC broth using parallel incubation temperatures of 45.5 and 44.5 +/- 0.05 C. Verifications for these temperatures averaged 79 and 98%, respectively. All positively verified isolates were E. coli types I and II, as were the negatives. Geometric means for the verified 7-h plate count were within 12% of the standard means for both EC broth incubation temperatures. A rapid method for enumerating fecal coliforms in foods was developed employing an agar pour-plate medium. After 7 h of incubation at 41.5 0.05 C, this medium effectively allows the growth of fecal coliforms only. This rapid method was compared with the Association of Official Analytical Chemists multiple-tube dilution method for Escherichia coli, by using 21 samples of fresh, cut-up chicken and a surface rinsing procedure for sample preparation. Verification of picked colonies was carried out in EC broth using parallel incubation temperatures of 45.5 and 44.5 0.05 C. Verifications for these temperatures averaged 79 and 98%, respectively. All positively verified isolates were E. coli types I and II, as were the negatives. Geometric means for the verified 7-h plate count were within 12% of the standard means for both EC broth incubation temperatures. Various methods that have accelerated bacteriological assessment of the sanitary quality of foods have been described (7,17). However, the Association of Official Analytical Chemists (A.O.A.C.) multiple-tube dilution method for Escherichia coli (6) has remained the accepted method for judging quality of many foods. Several attempts have been made to improve this laborious and time-consuming procedure (1,5,11), but thus far no really rapid method has been devised. The fecal coliform group of bacteria, comprised primarily of E. coli type I (13), defined according to criteria listed in Standard Methods for the Examination of Water and Wastewater (3) as those bacteria that produce gas in EC medium when incubated at 44.5 C for 24 h. The use of the fecal coliform group as an index of the sanitary quality of water stems from their identification as indicators of fecal contamination by man and other warm-blooded animals (13) and from the correlation of their densities with the presence of pathogens such as salmonellae (14). Geldreich Geldreich, Bacteriol. Proc., p. 25, 1969). The purpose of this study was to modify the rapid water fecal coliform procedure for use with foods and to evaluate it by comparing it with the A.O.A.C. standard method. MATERIALS AND METHODS Medium. The modified medium was formulated after intensive preliminary studies by using pure cultures of fecal and nonfecal coliforms suspended in buffer and in various food products. Ingredients and techniques were adjusted to give maximum recovery of fecal strains and still screen out nonfecals. The final formulation of the medium is as follows: proteose peptone no. 3, 5.0 g; yeast extract, 3.0 g; lactose, 10.0 g; NaCl, 7.5 g; sodium lauryl sulfate, 0.05 g; bromothymol blue, 0.3 g; agar, 15.0 g; glass-redistilled water, 1,000.0 ml. In preliminary studies, the use of glass-redistilled water was shown to give more consistent results. The sodium lauryl sulfate concentration is moderately inhibitory to noncoliforms, but selectivity is primarily effected by the incubation time-temperature combination. To insure complete dissolution, bromothymol blue is dissolved separately in a portion of the measured quantity of water which is then added to the rest of the ingredients in the solution. The pH is adjusted to 7.3 before the addition of agar. The medium is heated to boiling to dissolve the agar, dispensed into sterile bottles, and stored at 4 C, with a shelf life of at least 1 month. Enumeration is carried out in standard pour plates that are overlaid with the medium, heat-sealed in plastic pouches, and incubated submerged in a water bath for 7 h at 41.5 ± 0.05 C. After incubation, the plates are counted on a FRANCIS, PEELER, AND TWEDT Quebec colony counter, with fecal coliform colonies appearing yellow to orange, with yellow haloes against the bluish-green background color of the medium. Methods of sample preparation. After surveying various foods, we chose fresh, cut-up chicken as a representative food for evaluating our method, since fresh chicken containing suitable levels of fecal coliforms could be obtained consistently. For sample preparation, we modified the rinsing procedure recommended by the American Public Health Association (A.P.H.A.) (2). Our procedure consisted of rinsing the chicken part in a 1-gallon plastic food storage bag with Butterfield's A.P.H.A. sterile phosphate buffered diluent (4), and subsequently enumerating the fecal coli rinsed off the surface of the sample. A recovery efficiency study was performed to evaluate this bag-rinsing method. Packages of six drumsticks served as single samples; five were surfaceinoculated with a known level of an EC broth positive strain of type I E. coli recently isolated from fresh ground beef. The sixth drumstick was left uninoculated to serve as a base-level control. The inoculated pieces were rinsed with five ratios of rinse volume to sample weight: 2:1, 1:1, 1:1.5, 1:2, and 1:4. Since the results of the analysis showed recoveries for all ratios to be > 90%, the ratio of 1:4 was chosen because it produced the greatest concentration of organisms and thus the maximum sensitivity for our method. Comparative evaluation study. Twenty-one samples of fresh, cut-up chicken were enumerated for fecal coliforms by the rapid fecal coliform procedure and the A.O.A.C. standard method. The evaluation procedure, which enumerated fecal coliforms and E. coli by both methods, was carried out as outlined in Fig. 1. Rinse from each sample prepared as described above was used for both plating and most probable number (MPN) tube inoculation. Plating was done in duplicate, by using 101 (10 ml of rinse pipetted into three plates), 100, and 10-1 dilutions. All 10-fold dilutions were made by pipetting 11 ml into 99-ml dilution blanks. A 5-tube MPN series was set up by pipetting five 1-ml portions of each dilution into each of five tubes containing 9 ml each of lauryl sulfate tryptose (LST) broth. Dilutions made were 100 through 10-6. Counts are expressed per milliliter of rinse. Representative numbers of colonies were picked from 7-h plates into LST broth. All colonies were picked when the plate count was 10 or below; 10 colonies were picked from plates having a count greater than 10. Positive LST tubes, in addition to presumptive positives from the standard method MPN series, were confirmed in brilliant green lactose bile broth and EC broth. Since preliminary work had shown that the EC broth incubation temperature of 45.5 C failed to verify about 20% of the E. coli found in chicken, it was decided to include a parallel EC broth incubation temperature of 44.5 C for both the rapid and standard procedures. All positive EC tubes from both methods and also EC negatives from colonies picked from 7-h plates were subjected to the standard completed test. RESULTS The results of this study are shown in Tables 1 to 3. Table 1 shows counts for 21 individual samples of fresh chicken for both the rapid and standard procedures with verification in EC broth at both 44.5 and 45.5 C. A paired t test was computed to compare methods, with the first value of the rapid method duplicate counts being used for this purpose. Significant difference between methods could not be demonstrated at a = 0.01 for either temperature ( Table 2). The geometric mean difference between our method and the A.O.A.C. standard method, by using first value means, was 11.5% at 44.5 C verification and 5.2% at 45.5 C. By using the means for duplicate counts, they were 12.1 and 9.8%, respectively, for the two temperatures. Log variance for the rapid method duplicates was 0.00224 for 44.5 C and 0.00896 for 45.5 C. Overall percentage of verification of colonies picked from 7-h plates was 98% at the 44.5 C EC broth incubation temperature and 79% at 45.5 C (Table 2Y. At 44.5 C, 97% of the EC positives were E. coli type I, and 3% were type II; all of the EC negatives were type I. At 45.5 C, 98% of the EC positives were E. coli type I, and 2% were type II; 91% of the EC negatives were type I, and 9% were type II (Table 3). DISCUSSION When n = 21, the paired t test could only demonstrate significance of a difference between the rapid and standard methods greater than 40 to 50%. This result stems primarily from the great variability inherent in the MPN procedure, despite the fact that we made use of the five-tube technique rather than the usual three-tube. More than 200 samples would be required to detect a difference of 10%, and this effort is patently not feasible. However, given the nature of the MPN procedures, a 50% difference between methods was not considered unacceptable. Furthermore, since the geometric means of the two methods were no more than 12% apart, we felt that our method performed very well in comparison with the standard procedure. The log variance of 0.00224 for the 7-h count verified at 44.5 C was good. At 45.5 C, however, with a 19% drop in verification, the log variance increased to an unacceptably high value of 0.00896 (0.005 is considered an acceptable upper limit) (8). Preliminary studies employing the EC broth incubation temperature of 45.5 C specified by A.O.A.C. resulted in a verification of picked colonies averaging only about 80%. Nevertheless, all the EC-negative picked colonies proved to be E. coli types I and II. Consequently, in an effort to improve our percentage of verification and possibly demonstrate that 45.5 C is too high a temperature for fecal coliform verification, we included parallel EC broth verification at 44.5 C. Our data show the improvement effected by the 1 C reduction in EC broth incubation temperature-both a 19% increase in verification for the rapid method, with an improved log variance, and a substantial increase in the verified standard MPN count. Fishbein (9,10), working with pure cultures of E. coli and Aerobacter strains and also with frozen foods and nutmeats, concluded that 44.5 C was too low a temperature for EC broth incubation. Many Aerobacter strains and a significant number of non-E. coli strains isolated from the food products studied were verified as positive. To screen out these interfering organisms, an incu- bation temperature of 45.5 C was recommended. From our experience with fresh chicken, however, no organisms other than E. coli types I and II were encountered; thus, we feel that, with this product at least, 44.5 C is the preferred temperature for EC broth incubation. Some difficulties were encountered in counting 7-h method plates. These arose primarily when dealing with the 101 dilution. The pH of the rinse was found to vary from 6.0 to 6.9; samples having a rinse pH in the low end of the range caused an overall yellowing of the medium color. Such loss of medium contrast against yellowish fecal coliform colonies sometimes made these colonies hard to see. Also, debris in the rinse could either obscure fecal coliform colonies or be confused with them. Experience in counting helped to overcome these difficulties. We feel that the value and potential of this rapid method has been well demonstrated. It has value as a rapid screening procedure for the detection and enumeration of organisms indicative of fecal contamination of fresh chicken, and it has potential application to other foods. This method has been evaluated with other food products including fresh frozen and dried foods as well as dairy products. Data obtained have provided us with confidence that the procedure can be useful for a variety of foods. Continuing studies in this laboratory are directed toward evaluating the wider applicability of the rapid method for enumeration of total, as well as fecal, coliforms.

v3-fos

2020-12-10T09:04:23.017Z

1974-07-01T00:00:00.000Z

237235122

s2

Effect of Growth Rate and Nutrient Limitation on the Composition and Biomass Yield of Acinetobacter calcoaceticus Acinetobacter calcoaceticus was grown on ethanol in a chemostat as a model system for single-cell protein production. The substrate yield coefficient (Ys, grams of biomass/gram of ethanol), protein yield coefficient (Yp, grams of protein/gram of ethanol), and biomass composition were measured as a function of the specific growth rate. Nucleic acid, protein, Yp, and Ys all increased at higher growth rates. Although protein content increased only 14% (from 53 to 67%), Yp almost doubled over the same range of growth rates. The increase in Yp was due to the higher protein content of the biomass and to higher values of Ys. The higher values of Ys were attributed to maintenance metabolism, and the value of the maintenance coefficient was found to be 0.11 g of ethanol per g of cell per h. When A. calcoaceticus was cultivated under a phosphorus limitation protein content, Yp and Ys were lower than in carbon-limited cultures. It was concluded that a single-cell protein fermentation using A. calcoaceticus should be operated at a high growth rate under ethanol-limiting conditions in order to maximize both the protein content of the biomass and the amount of biomass and/or protein made from the substrate. was due to the higher protein content of the biomass and to higher values of Y8. The higher values of Y8 were attributed to maintenance metabolism, and the value of the maintenance coefficient was found to be 0.11 g of ethanol per g of cell per h. When A. calcoaceticus was cultivated under a phosphorus limitation protein content, Y,, and Y, were lower than in carbon-limited cultures. It was concluded that a single-cell protein fermentation using A. calcoaceticus should be operated at a high growth rate under ethanol-limiting conditions in order to maximize both the protein content of the biomass and the amount of biomass and/or protein made from the substrate. The composition of a microorganism is not invariant but is strongly dependent on its growth rate and environment. The chemostat is an ideal tool for studying changes in cell composition because both environment and growth rate can be controlled readily and accurately. By using chemostat cultures, it was found that there is a close correlation between specific growth rates (,u) and ribonucleic acid content of microorganisms (5,12,16,19,22). This variation is due primarily to the changes in ribosome content (6,10). Protein content also varies with growth rate, but unlike ribonucleic acid, the extent of variation usually is not great (10,21). The carbohydrate content usually does not change greatly when cells are grown at different rates under carbon-limiting conditions; however, chemostat cultures of nitrogenor sulfurlimited cells exhibit wide variations in carbohydrate content (10,21). The growth rate of a microorganism influences not only its composition but also the substrate yield coefficient (Y8; grams of biomass produced per gram of substrate consumed). Usually one of two types of relationships between ,g and Y8 are reported: (i) Y, increases as ,u increases, attaining a maximum value as the growth rate approaches /2max (13); or (ii) Y8 at-'Present address: Eli Lilly and Co., Dept. M539, Indianapolis, Ind. 46206. tains a maximum value at some intermediate growth rate (11). The first type of behavior has been attributed to the maintenance metabolism of the microorganism (13,14), which is a measure of the consumption of substrate by microorganisms for functions other than the production of new biomass. These functions include maintaining concentration gradients, providing energy for motility, resynthesizing unstable macromolecules, etc. The amount of substrate consumed for these purposes is a progressively smaller proportion of the total substrate consumption as the growth rate increases. Thus, more substrate goes to biomass production, causing Y, values to increase. This relationship can be expressed as: where (1/x) (ds/dt) is the specific rate of substrate consumption (grams of substrate consumed per gram of cell per hour), m is the maintenance coefficient (grams of substrate consumed for maintenance per gram of cell per hour), and Yg is the yield coefficient when m = 0 or whengu approaches infinity (14). By plotting (1/x) (ds/dt) versus A, m and Yg can be found as the intercept and the reciprocal of the slope, respectively. 58 The chemostat, in addition to its usefulness for studying changes in biomass yield and composition, is the preferred method of cultivation for the production of single-cell protein. In developing this process, it is important to establish how biomass composition can be varied, since the composition is a determinant of the nutritional and toxicological properties of the product. The protein content of single-cell protein also influences its market value, and the cost of production is strongly dependent on the protein yield coefficient, Y,, (grams of protein produced/gram of substrate consumed). Thus, for single-cell protein production, it is desirable to increase Yp, Y,, and protein content, three factors that may vary with the growth rate of the microorganism or the nutrient limitation under which it is grown. MATERIALS AND METHODS Organism. The organism used was Acinetobacter calcoaceticus strain 4736. Taxonomic characteristics and procedures for maintaining stock cultures were described previously (1,2). Fermentation. A. calcoaceticus was cultivated in a mineral salt medium containing ethanol as a sole carbon source. The medium, temperature, and pH for chemostat cultivation were described previously (1). For ethanol-limited cultures, the entering medium contained 6 g of ethanol per liter. Similar medium, temperature, and pH were used for phosphoruslimited cultures, except that HPO4 was deleted from the medium and added as a dilute solution via a second stream. The rate of PO4 addition was gradually reduced until about 0.05% ethanol accumulated in the culture broth. At low growth rates, the chemostat was operated for longer periods of time before biomass yields and composition were determined. At least four changes of medium were made at all growth rates to insure steady-state conditions. The dissolved oxygen tension in the fermentor was continuously monitored by a galvanic electrode and was maintained at greater than 20% of air saturation by varying agitation and aeration rate. At each steady state the concentrations of ethanol, acetate, and acetaldehyde in the culture broth were measured (1,2). In ethanol-limited cultures, the steady-state concentration of these metabolites was less than 0.05 g/liter. In phosphate-limited cultures, the ethanol concentration in the broth was measured by quantitative gas chromatography (1,2) and subtracted from the amount added to obtain the amount of ethanol consumed. Analytical methods. (i) Cell mass. Cell mass was determined by dry weight measurements. A portion (5.0 ml) of the cell suspension from the steady-state chemostat was transferred into a tared centrifuge tube. The biomass was sedimented by centrifugation at 12,000 x g for 15 min, and the supernatant fluid was discarded. The pellet was-resuspended in 5.0 ml 0.1 N HCl, sedimented, and washed with distilled water. The washed pellet was dried at 105 C, cooled in a desiccator over CaCl,, and weighed. The amount of precipitated salts in carbon-limited cultures was about 0.3 mg/ml, but there was very little precipitation in phosphorus-limited cultures. The HCl wash removed precipitated salts that sedimented with the biomass, compensating for the differences in the two types of medium. Control experiments indicated that the HCl and distilled water washes did not cause appreciable cell lysis. The dry weight measurements were performed in quadruplicate, and the averages are reported. (ii) Carbohydrate. For total carbohydrate measurements, 0.5 ml of the biomass suspension was added to 5.0 ml of anthrone reagent (7). The mixture was placed in a boiling-water bath for 10 min and cooled, and the absorption was read at 620 nm in a Gilford 2400 spectrophotometer (Gilford Instrument Laboratories, Oberlin, Ohio). A standard curve was prepared by using glucose, and the determinations were preformed in triplicate. (iii) Lipid. A cell suspension (1.0 liter) was removed from the chemostat, centrifuged, and resuspended in 200 ml of cold 1.0 N HCl. This suspension was incubated for 1 h to remove the low-molecularweight (cold acid-soluble) components of the biomass. The supematant fluid was discarded, and the pellet, containing high-molecular-weight components, was resuspended in 200 ml of 10 -I N HCl. A portion of this suspension was retained for protein and nucleic acid analyses, and the remainder (100 ml) was used to determine lipid content. For lipid analysis, the biomass was extracted first with 30 ml of acetone at room temperature. The material remaining after the acetone extraction was extracted with a mixture of 15 ml of ethanol and 5 ml of ether at 100 C for 5 min. The solvents were recovered by centrifugation, and the remaining biomass was refluxed for 1 h with a mixture of 5 ml of methanol and 15 ml of chloroform. The solvent extracts were combined, filtered, dried, and weighed. (iv) Nucleic acids. The biomass pellet remaining after removal of the cold acid-soluble (low-molecularweight) fraction was used for protein and nucleic acid analyses. The pellet from 10.0 ml of suspension was resuspended in 0.5 N perchloric acid and incubated at 90 C for 45 min. The suspension was cooled and the supernatant fluid was recovered after centrifugation. The supernatant was adjusted to pH 7.0 with KOH and the absorbance at 260 nm was determined in a Gilford spectrophotometer. An extinction coefficient of 0.0315 cm2/4g was used to convert absorbance to nucleic acid concentration (8). (v) Protein. The cell pellet remaining after the perchloric acid-catalyzed hydrolysis of nucleic acid was used for the protein analysis. The pellet was resuspended in 1.0 N KOH and incubated at 90 C for 45 min. The reaction mixture was cooled and the supernatant fluid was used for a biuret protein determination (18). A standard curve was prepared with bovine serum albumin. Protein content also was expressed as (%N x 6.25) -% nucleic acid. Total nitrogen was determined by a carbon, hydrogen, nitrogen analysis (Perkin-Elmer 240 CHN analyzer, Perkin Elmer Corp., Norwalk, VoL 28, 1974 ABBOTT, LASKIN, AND McCOY Conn.) of the biomass fraction that remained after removal of the low-molecular-weight fraction. Y, was calculated from the relationship Yp = (Y.) (% protein in biomass). RESULTS A. calcoaceticus was cultivated in a chemostat under ethanol-limiting conditions. A steady state was obtained at various growth rates, and the substrate yield coefficient, carbohydrate, lipid, nucleic acid, and protein content were measured. Substrate yield coefficient. YB of A. calcoaceticus was strongly dependent on the growth rate, particularly at low specific growth rates. The specific rate of substrate consumption, (-) (d) = y, was calculated at each growth rate and plotted as a function of t (Fig. 1). A straight line was obtained that intersected the ordinate at a positive value, indicating that the variation of Y8 with At was due to maintenance metabolism. The values of m and Yg calculated from Fig. 1 of the biomass remained essentially constant when the growth rate was varied (Fig. 2), whereas nucleic acid varied from about 7.5 to 12% (Fig. 3). There were no large differences in protein content when protein was measured by the biuret assay; however, the results obtained were highly variable and these variations would obfuscate all but large changes. In addition, when protein was determined by an amino acid analysis, it was found that the protein content was I much higher than that indicated by biuret assay. Closer agreement with the amino acid analysis and more consistent results were obtained by determining protein content from the nitrogen and nucleic acid content of the biomass (see Materials and Methods). The resultant data suggested that the protein content of A. calcoaceticus increased from about 53 to 67% of the biomass as the growth rate was increased from 0.1/h to 0.53/h (Fig. 2). Although the protein content of the microorganism varied by only 14%, the protein yield n .6- coefficent (Yr) almost doubled, increasing from 0.24 to 0.43 over the same range of growth rates (Fig. 4). This large variation was due to the additive effects of the higher values of Y., and the higher protein content of the microorganism. Phosphorus limitation. A. calcoaceticus was cultivated in a chemostat on ethanol under phosphorus-limiting conditions. A steady state was achieved at a specific growth rate of 0.5/h, and analyses were made of the biomass composition and the ethanol and phosphate yield coefficients. The steady state was maintained for 3 days, and daily measurements were made. Y8 was significantly lower under phosphoruslimiting conditions than under an ethanol limitation (Table 1). These yield coefficients were corrected for unused ethanol in the culture broths. The protein content appeared to be somewhat lower, whereas the total nucleic acid content was slightly higher (11.8 versus 14.2%) than in ethanol-limited cultures growing at a similar rate ( Table 1). As a result of the declines in protein content and Y,, Y,, was about onethird less in phosphorus-limited cultures. DISCUSSION Our data suggest that the protein content of A. calcoaceticus in an ethanol-limited chemostat increases when the growth rate increases. In contrast, others have reported that the protein content of microorganisms remains relatively constant or decreases when the growth (3,4,9,21). The protein content, as we measured it, was based on the total nitrogen content of the biomass. As a result, these protein values may be higher than the true protein content because of the presence of non-protein nitrogen other than nucleic acids (e.g., n-acetylglucosamine and muramic acid in cell walls). Nitrogen-based protein analyses also are dependent on the somewhat arbitrary extinction coefficient used for the total nucleic acid estimation. These sources of error influence primarily the magnitude of the protein content, but they should not hinder the determination of relative differences in protein values unless large changes in non-protein or non-nucleic acid nitrogen content occur. The identity of proteins responsible for the apparent increase in protein content of A. calcoaceticus at higher growth rates is not known. At least part of the increase may reflect the increase in enzyme level that presumably must occur to sustain high specific growth rates. Also, part of the increase may be due to ribosomal protein. Schaechter et al. (17) and Ecker and Schaechter (6) reported that the rate of protein synthesis per ribosome is independent of growth rate. Thus, larger numbers of ribosomes are needed to support higher growth rates. Although Sykes and Young (20) have shown that the rate of protein synthesis per ribosome increases with increases in growth rate, the rate of synthesis per ribosome was constant at growth rates above g = 0.5/h. Of particular significance to single-cell protein production is the variation of Y, with growth rate. This variation was due primarily to maintenance metabolism. At higher growth rates, the amount of substrate diverted to maintenance becomes a progressively smaller proportion of the total amount of substrate consumed. As a result, Y, increases at a greater rate than Y8 because both protein content and Y, are higher at higher growth rates. Earlier reports indicated that the protein content of a microorganism may be higher under a phosphorus limitation than under a carbon limitation (15,21). A similar increase in a single-cell protein fermentation would have a beneficial impact on process economics if concomitant decreases in Y, and Y, did not occur. The present study indicates that A. calcoaceticus contained slightly less protein in phosphorus-limited cultures, and this decrease was accompanied by substantial declines in Y, and Y,. In conclusion, a single-cell protein fermenta-tion using A. calcoaceticus should be operated under ethanol-limiting conditions at high growth rates in order to maximize the protein content of the product and the amount of protein and/or biomass produced from ethanol.

v3-fos

2018-04-03T00:33:36.749Z

1974-02-01T00:00:00.000Z

12865782

s2

Comparison of Fluorescent-Antibody Methods and Enrichment Serology for the Detection of Salmonella Four rapid methods for detection of Salmonella, (i) the conventional fluores-cent-antibody (FA) technique, (ii) a rapid direct FA technique, (iii) microcolony FA, and (iv) enrichment serology (ES), were compared with conventional cultural procedures. A total of 347 subsamples representing 16 different food prototypes, alleged to be naturally contaminated with Salmonella, were analyzed. From these samples, 52 were found to contain Salmonella by cultural methods. Conventional FA identified all 52 culturally positive samples, ES identified 51, microcolony FA identified 48, and the rapid FA method identified 34. The number of false-positive samples for each procedure was: ES-selenite, 7; tetrathionate, 8; rapid FA, 26; microcolony FA, 33; conventional FA-selenite, 27; tetrathionate, 26. Tetrathionate enrichment was found to be superior to selenite for Salmonella recovery from most foods, but the concurrent use of both media allowed maximum recovery. red suppression Smears a and 16 oculars. A oil fluorite was used for more detailed of to the presence of attached flagella. Approxi- 100 microscopic fields were scanned on each slide. One or more strongly fluorescing rods with discernable lumen in 10 or more fields was considered as positive. The demand for routine analysis for the detection of Salmonella in food and feeds has increased significantly in the last few years. As a result of this increased testing, research has been directed toward the development of faster and/or more sensitive methods to detect these organisms. The use of the fluorescent-antibody technique (FA) is not new, and recently two modified FA procedures for the detection of Salmonella have been described (5,12). In addition, an enrichment serology (ES) method (1,2,10) has been evaluated and reported for use as a quality control measure in bacteriological laboratories. The primary objective of this study was to compare five different methods for the detection of Salmonella in food and feeds. These methods were: (i) the conventional FA technique; (ii) a rapid direct technique described by Insalata et al. (5); (iii) the microcolony technique as described by Thomason (12); (iv) the ES technique as proposed by Sperber and Deibel (10); and (v) the standard cultural methods (13), with minor modifications. The procedure referred to as "conventional FA technique" is a modification of direct FA staining as described by numerous investigators (3,6,9). The experimental design used allowed a comparison of the time for pre-enrichment (7 versus 24 h) and an evaluation of the efficiency of tetrathionate and Selenite-F selective enrichments. MATERIALS AND METHODS Food and feed samples suspected of being naturally contaminated with Salmonella were obtained from various sources. The products, number of lots, and subsamples analyzed are presented in Table 1. Two sources of commercially available FA antisera were used throughout this work: FA Salmonella poly antiserum (11) (Difco, Detroit, Mich.) diluted 1:4 with sterile saline, and Salmonella Fluoro-Kit (4) (Clinical Sciences Inc., Whippany, N.J.) prepared as directed by the manufacturer. No differences between antisera were noted. Pre-enrichment. Samples (25 g) of each food or feed prototype were pre-enriched in 225 ml of FAS broth (Difco) which was tempered at 35 ± 2 C. Milk products were pre-enriched in brilliant green water as recommended in the Bacteriological Analytical Manual (13). Incubation was at 35 ± 2 C for 24 h, with the exception of the rapid FA technique for which the incubation time was 7 h (5). Incubation was without agitation. Selective enrichment. After incubation, the preenrichments were shaken and allowed to settle for about 5 min. In the rapid FA technique, 50 ml was transferred to 450 ml of prewarmed (35 ± 2 C) Selenite-F broth (BBL) and incubated for 16 to 18 h at 35 ± 2 C without agitation. For all other tests, 2-ml portions of the pre-enrichment broth were transferred to 18 ml of Selenite-F broth and 18 ml of tetrathionate broth, respectively, and incubated at the same temperature for 24 h. Elective enrichment. Selenite-F broth was agitated and allowed to settle for 5 min for the rapid FA technique. Two milliliters was then withdrawn from Cereal-spice mix a Number of samples confirmed positive. the top third of the culture, transferred to 18 ml of tempered FAS broth, and incubated for 5 h at 35 4 2 C. For conventional FA analysis, 1 drop from Selenite-F and 1 drop from tetrathionate broth were transferred separately into 3 ml of Trypticase soytryptose broth (TST) (7) and were incubated at 35 2 C for 3 h or until visible growth was obtained. This elective enrichment medium for conventional FA was suggested by Wallace H. Andrews, Jr., of the Food and Drug Administration. The use of an elective enrichment in FA analysis resulted in cleaner slides with less background debris. For the enrichment serology technique, 1 drop from each selective enrichment culture was added to M broth and incubated for 6 h at 35 2 C in a water bath. Serological analysis was performed as recommended by Sperber and Deibel (10). FA staining procedure. A loopful (2 mm) of the appropriate elective enrichment broth was placed onto the Fluoro-Kit slides, and they were stained by using the materials and methods specified by the supplier. If it is desired to observe nonfluorescent cells, FA Rhodamine counterstain (Difco) may be applied for 1 min after FA staining and then slides are rinsed for 30 s with distilled water. All nonfluorescent cells would be stained red. For the microcolony technique, a 2-mm loopful from tetrathionate enrichments was placed onto brilliant green agar plates in such a manner as to coincide with the prepared areas on the Fluoro-Kit slides. The plates were incubated for 3 h at 35 i 2 C. After this time, impression smears were made by placing the slides on the agar plates and exerting slight pressure. Slides were removed, allowed to airdry, and were then stained by the recommended method. Microscopy examination. Slides were examined on a Leitz Ortholux microscope equipped with incident-light fluorescence using exciting blue filters KP490 (FITC) and dichroic beam splitters with a K495 built-in suppression filter and 510 suppression filter slide. The light source was an Osram HBO-200 mercury arc burner equipped with a transmission heat filter (2 mm KGl) and a red suppression filter (4 mm BG38). Smears were examined by using a dry x40 objective and x 16 oculars. A x54 oil fluorite objective was used for more detailed examination of smears to confirm the presence of attached flagella. Approximately 100 microscopic fields were scanned on each slide. One or more strongly fluorescing rods with discernable lumen in 10 or more fields was considered as positive. Cultural methods. Selective enrichments of Selenite-F and tetrathionate broths were streaked on XLD agar (Difco) and hectoen enteric (HE; Difco) agar (8). XLD and HE agars were incubated at 35 4 2 C for 24 h. In our laboratory, we found XLD and HE to be as good or better than Salmonella-Shigella agar and brilliant green agar for the isolation of Salmonella. Typical colonies were picked to triple sugar iron agar and lysine iron agar, and cultures exhibiting a presumptive positive Salmonella characteristic were confirmed serologically. Subcultures in TST broth from Selenite-F and tetrathionate were incubated at 35 2 C for 7 h and then streaked on XLD and HE agar. The experimental procedures are illustrated schematically in Fig. 1. RESULTS Out of 347 subsamples, 52 were positive by the cultural method employed in this study. Five of the isolates were not recovered from selenite enrichment but were recovered from tetrathionate enrichment. One of these five cultures was isolated from tetrathionate only after the additional elective enrichment in TST broth. This isolate would have been missed by routine cultural methods. Another isolate did not grow in tetrathionate broth but was recovered from selenite broth ( Table 1). The conventional FA method correctly identified 48 positive samples from selenite enrichment and 52 from tetrathionate enrichments. The rapid FA method correctly identified 34 positive samples. Microcolony FA detected 48 of the 52 positive samples. The ES method gave 43 and 50 positive results from selenite and tetrathionate, respectively. Since the ES method calls for analysis of both selective enrichments, the method detected a total of 51 positives (Table 1). In the following discussion, a false-negative result is defined as a culturally positive sample which was negative for Salmonella by either FA or ES methods. A false-positive result is defined as a culturally negative sample which exhibited a presumptive positive test for Salmonella by either FA or ES methods. The rapid FA procedure gave 18 false-negative results compared to 4 false negatives for conventional FA from selenite enrichment. No false-negative results were obtained with the conventional FA technique using tetrathionate enrichment. Microcolony FA resulted in four false negatives. The enrichment serology method showed nine and two false negatives from selenite and tetrathionate, respectively. Since the method calls for analysis of both enrichments, the use of the ES technique resulted in only one false negative ( Table 2). False-positive results were found with all methods. However, the number was quite different for the various procedures. The lowest was ES, with seven and eight from selenite and tetrathionate, respectively. Conventional FA showed 27 and 26 from selenite and tetrathionate. The microcolony method resulted in 33 false positives, and rapid FA had 26 (Table 2). DISCUSSION Any screening procedure for Salmonella must be as sensitive as standard cultural methods. This means the procedure should not result in any false negatives. However, as a practical quality control method, it should also not produce excessive false-positive results, since these require lengthy and costly cultural confirmation. The rapid FA method resulted in 18 false negatives. This was probably due to the shortened pre-enrichment phase. When the same samples were analyzed by conventional FA using 24-h pre-enrichment followed by selenite selective enrichment, only four false negatives were obtained. Since the rapid FA technique relies on selenite enrichment, 5 of the 18 falsenegative results can be explained by the failure of the Salmonella in those samples to grow in selenite. The conventional FA procedure using tetrathionate selective enrichment resulted in no false negatives. In one sample Salmonella was not recovered from tetrathionate culturally, yet the conventional FA produced a positive test. This positive result was confirmed by the 326 irs. use of the elective TST enrichment, which is not part of the AOAC method. The number of Salmonella cells in the tetrathionate enrichment was too low to be detected on streak plates, but cells were found in FA smears. The false-positive rate found with conventional FA using the TST elective enrichment is comparable to that reported without the use of elective enrichment (5). In the microcolony technique, 24-h preenrichment was combined with tetrathionate enrichment, resulting in four false negatives. Some of these false negatives may have resulted from excessive growth on the brilliant green agar elective enrichment, creating a "quenching" of fluorescence on the slides. Reducing the incubation time of the brilliant green agar plates may overcome this problem. The microcolony technique did not offer any time saving over other methods. Microscopy examination of all FA smears was facilitated by the use of incident light illumination. Fluorescence was extremely bright and easily seen under x640 magnification. Since no dark-field condenser was needed, adjustment or oiling of the substage condenser was not necessary, thereby saving time and avoiding the possibility of inaccurate adjustment which would reduce brightness of fluorescence. The ES method using both enrichments gave only one false-negative result. The amount of growth in the elective M-broth enrichment was very scant in this instance and may have been the cause of this single failure. The need for a longer incubation time for M broth has been recently demonstrated (1). In the opinion of the authors the ES technique, using 6-h elective enrichment, is the preferred Salmonella screening method. Incubation of the elective enrichment must be extended in cases where culture turbidity is insufficient for serological testing. The ES method is simpler to perform, gives fewer false-positive results, requires less costly equipment, and requires significantly less technical expertise. The ES results found in this study are similar to those seen by Sperber and Deibel (10) and more recently by Boothroyd and Baird-Parker (1). Conventional FA would be the method of second choice. It has the advantage of detecting low numbers of Salmonella cells from elective enrichment broth. This sensitivity was demonstrated by the absence of false negatives when tetrathionate enrichment was used. However, due to numerous false positives, additional cultural confirmation would be required. The rapid FA procedure was found to be inadequate because incubation of the preenrichment broth for 6 h was insufficient. Fourteen additional Salmonella-positive samples were found when pre-enrichment incubation was extended to 24 h. If only one selective enrichment broth is to be used, then tetrathionate would be the broth of choice. However, in these studies limiting the enrichment to just tetrathionate would have missed two positive samples that were isolated from selenite broth. Studies are currently under way to evaluate the use of antibiotics and elevated temperatures for enhancement of Salmonella isolation.

v3-fos

2017-06-28T17:10:44.516Z

1974-04-15T00:00:00.000Z

6461182

s2

Breeding goals for beef cattle Specialised beef breeds can be used as : i. both sire and dam in commercial beef production ; 2. component of the dam in commercial beef herds ; 3. sire line for crossing on dairy or commercial beef cows. Evidence from crossbreeding trials suggest that the first role will be a declining one. Field data indicate that a beef x dairy cow may be the best commercial beef dam. The requirements of a beef sire line are analysed in economic terms, and the potential contribution of a sire line used for crossing on dairy cows is calculated. For fairly reasonable conditions, it is shown that this can double the economic value of the genetic merit from an average insemination in the dairy cow population. 3 . sire line for crossing on dairy or commercial beef cows. Evidence from crossbreeding trials suggest that the first role will be a declining one. Field data indicate that a beef x dairy cow may be the best commercial beef dam. The requirements of a beef sire line are analysed in economic terms, and the potential contribution of a sire line used for crossing on dairy cows is calculated. For fairly reasonable conditions, it is shown that this can double the economic value of the genetic merit from an average insemination in the dairy cow population. There are several quite distinct roles which specialised breeds of beef cattle can play, depending on the structure of the cattle population in which they are used. They can function as : I . Both sire and dam in commercial beef production. This is the situation in the great beef populations of the worldas in Argentina, the U. S. A., Australia and in parts of France. 2 . Component of the dam in commercial beef herds. This is generally the situation in Ireland and Britain, where the commercial beef cow is commonly a crossbred. 3 . Sire line for crossing on dairy cows. 4 . Sire line for crossing on commercial beef cows. Each of these roles calls for its own set of specifications in a beef breed. In order to examine the goals which a breed should set itself, it is necessary to consider which one or combination of these roles it is expected to perform. -USE OF BEEF BREEDS IN PURE FORM Only four countries in Western Europe have substantial numbers of specialised beef cows. Their approximate populations of beef and dairy cows are as follows. -. Two quite different patterns in the breed background of these beef cows are apparent. It is generally recognised in Britain and Ireland that commercial beef production from the beef breeds in their pure form is not economic. The traditional suckler cow is a beef X dairy cross. This practice has a sound base in genetic theory, in that the whole complex of reproductive and mothering traits that make for efficient beef dams are likely to show considerable heterosis. However, it takes a particular type of population structure to provide a steady flow of such replacement females for the beef herd. The Irish and British cow populations are so structured, in that each contains about 30 p. 100 of beef cows and 70 p. 100 of dairy cows. A proportion of the dairy cows are usually bred to beef bulls. So there is available throughout the population a steady supply of beef X dairy heifers. The movement of these heifers from dairy to beef herds is not difficult, because both kinds of herds are found in all grassland areas. Accidents of history and geography have dictated quite different structures in France and Italy and in the great beef producing countries overseas. In the U. S. A., for instance, the beef cow population is located mainly in the western states, far from the main dairy areas. In addition, there has been a tradition of highly specialised dairying, with beef crossing little practised. For these reasons, beef producers have had to breed their own replacement females. The result has been a beef industry based on pure Hereford and Angus cattlenot because this is technically the best way to use these breedsbut largely through force of circumstances. Recent experiments (GREGORY, z97o ; CUNDIFF, 1973) have clearly demonstrated the advantage of crossbred females in this kind of population. Cows which are crosses between the traditional beef breeds are 15 p. I oo more productive than the same breeds in pure form. Productivity here is measured as weight of calf weaned per cow per year. In the same terms, a further 10 p. 100 can be gained from heterosis in the calf, i. e., by having the sire of the calf from a breed different to the dam. The total effect of systematic crossing is therefore about 25 p. 100 . This evidence is leading to increasing interest in the use of planned crossing programmes for beef cow populations. In populations that breed their own replacement females, this must be in the form of cyclical crossbreeding of some kind. So the role of the specialised beef breeds in their pure form as commercial beef cows is likely to be a declining one. II. -BEEF BREEDS AS COMPONENTS OF THE COMMERCIAL BEEF COW The only product of a suckler cow is her weaned calf. The value of this product is the probability that she produces a live calf in the first instance, multiplied by the weight of calf weaned. Since about 8 5 p. 100 of the feed input to the cow and calf unit goes to the cow, the main cost involved is the cow's annual feed requirement. This can be fairly adequately described as a function of cow body weight. A reasonable measure of the economic utility of a commercial beef cow would therefore be the weight of calf weaned per I oo kg of cow weight. The ways in which the cow contributes to this goal are shown in figure i. There is only one point in this complex where the aims are in conflict. That is between the requirement for small cow body size in combination with high calf growth potential. Since mature body weight and growth rate are highly correlated genetically, it may be difficult to pursue these two aims simultaneously. In order to measure their relative importance, it is necessary to reduce them to the common denominator of money. The maintenance requirement of an extra 100 kg of cow body weight can be calculated from, for example, the work of NE VILL E and M C C ULLOU G H (ig6g) as about 1225 Mcal ME per year. If the cow receives two thirds of her feed as grazed grass and most of the rest as conserved grass, the cost per Mcal. ME in Irish conditions is about o.5 p. This gives an annual maintenance cost of about £ 6. 00 per 100 kg. If weanling calves are valued at 45 p. per kg liveweight, then it takes an extra r 3 kg of calf weaned to cover the increased maintenance cost of the dam. Both feed costs and calf prices will be different in other countries, but these differences are likely to require more rather than less calf output to compensate for cow feed requirements. Most of the American evidence on this shows a lower gain in calf weight per I oo kg increase in cow weight. S INGH et al. ( 1970 ) found 4 . 7 kg ; NEVI L L E (1962) found 7 .o kg ; while Krrox ( 1957 ) found smaller cows giving higher calf weight per unit of cow weight than bigger cows. These studies all concern essentially purebred He y efo y ds. The British MI,C field recording programme gives some information across breeds. Their results are given by KWx!rrrrv and ST OLLARD (ig!3), from whose paper the following graph is reconstructed. Two conclusions may be drawn from these figures. The first is that it pays to have a dairy component in the cow : Angus X Friesian cows give almost 20 kg more calf weaned than Angus type cows, while both types of cow have the same bodyweight. The second conclusion is that cows sired by the large breeds (Red breeds -!-Charolais) compare well with other beef-type cows, but not so favourably with beef X dairy type cows. Thus Charolais cross cows weighed more than 40 kg heavier than Hereford x Friesian cows, but produced 5 kg less in calf weight per 100 kg of cow. Note that all these comparisons use the Hereford type cow as a refeerence base. The overall conclusion, therefore, is that while larger cows produce larger calves, the increase in calf output does not compensate for the increased cow maintenance requirement. However, the non-feed costs in a beef herd tend to be per cow and not per unit of cow weight, and so will favour larger cows. In addition, the increased slaughter value of larger cows will offset some of the increased growth and maintenance costs incurred. The feed requirement needed to produce one extra kg of liveweight and to maintain it for the normal life of a beef cow, say, eight years, amounts to about 100 Mcal ME. In Irish conditions this costs approximately 50 p. The slaughter value of the extra kg of liveweight is about 30 p. The net cost per kg is therefore 20 p., or £ 20 . 00 per 100 kg. If the cow produces five calves, this averages £ 4. 00 per calf, so that even when account is taken of the cull value of the cow, the maintenance cost is likely to exceed the value of the correlated gain in calf weaning weight. On balance, therefore, it appears that smaller cows are more economic as beef dams, and that the larger beef breeds, including the Charolais, should not normally be a component of the commercial beef cow. III. -BEEF BREEDS AS SIRE LINTS This is where the larger beef breeds have the greatest advantage. Whether it is as crosses out of dairy cows or out of commercial beef cows, their progeny are expected to be profitable, single-purpose beef animals. What are the factors that make beef animals profitable ? There are essentially three, and profitability is roughly their product. They are : r. Probability of survival. 3 . Proportion of meat in the carcasse. There are, of course, other factors too, but they are either of small economic importance or vary little between animals or breeds. To put these three factors in perspective, it is necessary again to use the common denominator of money. The question of survival is largely concerned with the birth of the calf. Mortality after the perinatal period tends to be low and random. There are, however, well established breed and bull differences in the rate at which their calves die or cause trouble at birth. Suppose two bulls or two breeds differ by I p. 100 in the perinatal mortality of their calves. Valuing calves at £ 6 0 , this represents a charge of 6 0 p. per head on each surviving calf. In addition, one must take account of the increased management costs on a cow which loses a calf. These are hard to quantify, but it might be reasonable to put them at £ 10 , or io p. per surviving calf. Breeds orbu lls which differ by i p. 100 in calf mortality will differ by about twice that rate in difficult calvings (Federatie K. I. Nederland, i 97 o). Valuing the distress to both cow and farmer at £ 15 means that the two difficult calvings are costing £ 30 , or 30 p. per surviving calf. So one can say, in very round figures, that a I p. 100 difference in calf mortality, with an associated difference of 2 p. IOO in calving difficulties, costs about £ 1 . 00 per calf born. This calculation, of course, does not allow for the fact that this cost is only a theoretical charge on all calves, and must in practice be carried by a single animal. Valuing growth potential is somewhat easier. If two animals differ by i p. 100 in growth rate, the faster growing one will in a given time achieve a i p. 100 higher weight. If cattle are marketed at around 5 00 kg this amounts to 5 kg liveweight. At present Irish prices, this extra liveweight has a gross value of £ 1 .8 0 . Its net value, after subtracting feed cost, is about £ 1 . 00 . These figures have implications first for the choice between breeds, and secondly for the selection goals which should guide improvement within beef breeds. It is very difficult to obtain a precise comparison of breeds for these traits. This is partly because precise experiments with cattle are very expensive, and so not very numerous ; partly because with less precise, but less costly field data it is often difficult to disentangle breed differences from other factors ; and partly because the samples of the breeds involved are different in different countries. With all these qualifications, three hypothetical beef crossing breeds are compared in table i. These figures approximate to what one might expect from Cha y olais (A), Limousin (B) and traditional British breeds (C) under Irish conditions of production, though it should be emphasised that these synthetic figures are contradicted by experience with these particular breeds in some populations. The largest difference of £ ii.2o amounts to about 8 p. IOO of the gross value of a beef animal. These figures can also be evaluated per insemination fairly simply, since in normal circumstances all the crossbred progeny of a beef sire line are slaughtered. Given an 86 p. 100 net reproductive rate in the population, and an 8 p. 100 discount rate, each insemination will result in the equivalent of o.68 net progeny at the time of the insemination. The present value per insemination of breed A over breed C is then £ IL20 X o.68 = £ 7 .6. The systematic use of breed differences of this order is essential in a beef cow population. In a dairy cow population, the position is less clear, though there are also large advantages to be gained. To illustrate this, I wish to compare three strategies for a population of dairy cows. Net reproduction is assumed to be 86 p. 100 , the discount rate is again taken as 8 p. 100 and cows are presumed to last for 4 lac-tations. If the dairy bulls are selected at a rate of I in 5 on progeny test, their average breeding value for dairy production will be about 3 p. 100 of the mean. Let us say this is worth £ 3 . 00 , which it is in Ireland. If their beef breeding value is taken as a base, then assume that it is possible to provide a breed or line of specialised beef bulls which have a beef breeding value of !-! 10 . 00 for overall beef merit. Three strategies can then be compared. I . Breed all cows to dairy bulls. dairy and beef production, and M, the corresponding figure for beef bulls for beef production, then the present financial value of the genetic merit of an average insemination in each case is This is made up of three components as shown in table 3 . Thus, with systematic crossing of a certain proportion of dairy cows to beef bulls the average value of an insemination, in net current terms, can be increased by 102 p. 100 . This means that the amount of beef crossing in European dairy cow populations is likely to increase. This in turn will increase the demand for and benefit from high performing specialised lines of beef crossing bulls.

v3-fos

2019-03-19T13:14:44.974Z

1975-10-01T00:00:00.000Z

237233336

s2

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed. Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery ofbacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed. Bacteria associated with the gut mucosa have been described in a number of mammals (2,5,6) but the phenomenon has received only brief mention in ruminants (7). In a study of the rumen epithelium of sheep with the scanning electron microscope, objects resembling bacteria were observed on the epithelial surfaces. The persistence of these bacteria through extensive and thorough washing of the tissue suggested that they might be firmly attached to the epithelium. The present study was undertaken to investigate the extent and nature of the epithelial bacterial population. MATERIALS AND METHODS Romney wether sheep, aged from 1 to 4 yr and fed on mixed white clover (Trifolium repens L.)-perennial ryegrass (Lolium perenne L.; "Grasslands Ruanui") pasture, were slaughtered by severing the jugular vein. The rumen and reticulum were rapidly exteriorized and pieces of wall about 2 cm square were removed from specific sites. Each tissue sample was washed by shaking vigorously in 500 ml of 0.85% (wt/vol) sodium chloride at room temperature, and when free of adhering rumen contents (approximately 10 a) was immediately fixed in 10% (wt/vol) formalin. To minimize postmortem changes in the tissue, the manipulations were performed as rapidly as possible and all samples were fixed within 3 min of sacrificing the animal. The pieces of fixed tissue were washed further with jets of distilled water from a squeeze-bottle, attention being paid to the areas between the papillae, and were freeze-dried at -30 C. Dried tissue pieces were mounted, suitably orientated, on aluminum stubs with rubber cement. The preparations were coated with 20 nm of carbon, followed by 20 nm of gold-palladium, while being rotated in a vacuum chamber. The coated preparations were examined and photographed with a Cambridge Stereoscan scanning electron microscope, Mk 2a. Each epithelial sample was assessed for degree of bacterial cover by examination of a number of sites on two or more papillae. The degree of cover was scored on a scale from 1 (a few scattered bacteria) to 4 (dense mat of bacteria). The types of bacteria present were also recorded. Magnifications were mainly in the range x 2,000 to 5,000. RESULTS The distribution and degree of bacterial cover on the epithelial surfaces were investigated by examining pieces of tissue from 14 sampling sites in the ruminoreticulum of a single sheep. The sites examined are shown in Fig. 1. With each sample a search was made of surfaces of suitably orientated papillae. An example of the papillae in a typical preparation from the roof of the dorsal rumen is shown in Fig. 2. Bacteria were found on all of the surfaces examined but the numbers varied greatly in different areas of the ruminoreticulum. Most of the bacteria seen were on the dorsal, caudal, and lateral surfaces of the rumen, with the most dense populations on the roof of the dorsal rumen and on the floor of the caudodorsal blind sac (positions 4 and 5, Fig. 1). The rest of the rumen surfaces had only light or moderately heavy cover of bacteria. With the exception of one sample (see Fig. 5b) only scattered bacteria were seen on the reticulum samples. Areas on some of the papillae were covered with populations composed almost entirely of a single morphological type. To substantiate the observations that bacterial populations varied in both density and type on surfaces from various sampling sites, samples were obtained from nine other sheep. In each of these sheep pieces of tissue were taken from four areas in the rumen (positions 3, 4, 5, and 7 as described in Fig. 1). These areas had been found to vary in density and type of bacterial cover in the first sheep examined. A summary of the results for all 10 sheep is shown in Table 1. The differences found in the original animal were confirmed; similar bacterial cover and types were found in the same epithelial sampling sites in all animals. Examples of the degrees of cover observed are illustrated in Fig. 3a and b. In Fig. 3a, only a few bacteria are present on the epithelial surface and the granular projections covering the surface of the epithelial cell can be clearly seen. In Fig. 3b, the surface of the sample from the floor of the caudodorsal blind sac is densely covered with a mixed population. Minute local areas with a "Pure" population were frequently found and Fig. 4a shows such an area on a papilla from the roof of the dorsal rumen. A coccus can be seen colonizing the raised portion of the epithelial surface while the lower surfaces are covered by a population composed almost entirely of a long rod. Figure 4b illustrates the discrete spatial separation of the two forms. Details of structure of the two morphological types are shown in Fig. 4c (14) 3. peared to have a rough, pimpled surface. The rods were 0.3 to 0.5 gum by 3 to 7 ,um, usually curved and thickened in the polar region. Figure 5a represents a degree of cover intermediate between those in Fig. 3a and b, and shows a relatively pure population of a curved rod on a sample from the floor of the caudoventral blind sac. Figure 5b shows a localized high concentration of a short rod on a sample from the mid-reticulum. This was the only dense population of bacteria seen in any reticulum sample. In a number ofsites throughout the ruminoreticulum, pockets of a spiral organism were found. Figure 5c is typical of the heaviest population seen. Figure 5d gives more detail of its morphology. Although this spiral is associated with mixed populations on the epithelial surfaces, it does not correspond with any of the spiral organisms previously reported from rumen contents (1). It has been isolated and cultured and is being characterized. It differs in its small size (0.3 by 4 pum) and in the extremely tight coiling of the spirals, the coils of which are just visible under critical phasecontrast illumination. At high magnifications, thin thread-like structures were frequently seen between bacterial cells (Fig. 6a and b). DISCUSSION The bacteria found on the epithelial surfaces of the ruminoreticulum are occupying a specific ecological niche or are merely contaminants from the bacteria-rich rumen contents. A number of observations indicate that the bacteria may be true inhabitants of the epithelial surface. First, there was variation in bacterial cover in different areas of the ruminoreticulum. Second, when four areas in each of the nine animals were examined in detail, two areas consistently had heavy cover and two were consistently lightly covered. Third, epithelial areas were found with a single morphological type predominating, and these areas were consistent in location in all sheep. Fourth, at least one of the bacteria, the spiral ( Fig. 5c and d), has not been reported previously from rumen contents. However, failure to demonstrate its presence might be explained by its small size. The main types of bacteria found on the epithelium are rod shaped, as is the case in normal rumen contents. It is not possible to identify them on the basis of morphology so comparison with known rumen bacteria cannot be made. Isolation and characterization of some of these bacteria are in progress. Tamate et al. (7), in their study of bovine rumen epithelium, described colonies of a coccus with thread-like structures between the cells. The similar structures observed by us were associated with both coccal and rodshaped bacteria (Fig. 6a and b). The threads appear similar to mucus. However, mucus is not secreted by the epithelium of the ruminoreticulum and it seems unlikely that the material could be derived from salivary mucus. The close association of the threads with individual bacterial cells suggests that they may be an extracellular product of bacterial metabolism. Nothing is known of the functional significance of these bacteria on the rumen epithelium. Indigenous populations of bacteria have been observed, by conventional histological methods and by transmission electron microscopy, on the mucosal epithelia of various regions of the gut of many animals. These animals include man, monkeys, swine, hamsters, rats, mice, and birds (2,5,6). The bacteria involved are believed to interact physiologically with the epithelium (5, 6) but the mechanisms are not fully understood. Some of these indigenous populations are firmly attached to the epithelium and some are merely embedded in mucin; most are in a position to receive nutrients from the adjacent epithelial cells. Interference with alkaline phosphatase has been postulated for bacteria in the duodenal epithelium of mice (8). The epithelium of the ruminoreticulum is an important absorptive surface and passage of some metabolites into the rumen also occurs (3,4). Attached bacteria would thus be in a favorable position to utilize nutrients passing through the wall. Other benefits of attachment to the epithelium can be postulated. An advantage would be conferred on organisms whose growth rate was insufficient to avoid being washed out of the gut. Also, in the rumen, ingestion by ciliate protozoa might be avoided. Further studies of these bacteria, including their isolation and growth in vitro, and determination of their substrate affinities may provide evidence for their role in this niche. ACKNOWLEDGMENTS The microscopy was carried out at the Physics and Engineering Laboratory, Department of Scientific and Industrial Research, Lower Hutt, and we are indebted to the staff of the electron microscopy section for their assistance and guidance. We thank C. S. W. Reid and D. Dellow for slaughtering and sampling the animals.

v3-fos

2018-04-03T00:27:24.563Z

1975-05-01T00:00:00.000Z

12080102

s2

Rumen bacterial degradation of forage cell walls investigated by electron microscopy. The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues. The microorganisms comprising the rumen bacterial population vary in morphology and metabolic activity (7,19). Symbiotic relationships for the degradation and utilization of plant cell wall constituents have been shown by using known cultures of rumen bacteria (10,(13)(14)(15)(16). Investigations of the metabolic activities of these bacteria have helped clarify the complex nutritional system in the rumen. However, the initial steps in tissue digestion involving the mode of association with and degradation of intact plant cell walls by rumen bacteria have not been investigated extensively. Electron microscopic investigations have revealed certain interesting ultrastructural features of rumen bacteria (3,11,23,28). Leatherwood (23), using the scanning electron microscope (SEM), observed "tube-like appendages" on the cellulolytic coccus Ruminococcus albus only when grown on cellulose-containing media. Costerton et al. (11), using the transmission electron microscope (TEM), reported coats external to the outer membrane of three gram-negative rumen bacteria, Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii. Earlier TEM observations from our laboratory of the tropical forage Coastal Bermuda grass degraded by rumen bacteria in vitro revealed attachment, appar-ently by an extracellular matrix, of large cocci to the thick forage cell walls (3). In addition, observations of intact leaf sections by using the SEM have shown differences in the ease and extent of forage tissue digestion by rumen microorganisms (1,2). Investigations using the SEM at the level of microbial attachment should be useful in adding a new perspective to the complex problems of bacterial attachment and degradation of forage tissues by rumen bacteria. The objective of the work reported here was to examine the mode of bacterial attachment to and degradation of intact cell walls in tissues of the temperate forage grass Kentucky (Ky)-31 tall fescue (Festuca arundinacea Schreb.) by TEM and SEM. MATERIALS AND METHODS Substrate. Leaf blades of Ky-31 tall fescue were harvested after 4 weeks of summer regrowth, frozen immediately, and stored at -30 C until used. Sections 2 to 5 mm in length were cut from the midportion of the frozen blades and used as substrate for the bacteria. Microbial inoculum. To obtain a preparation relatively free from particulate debris which would obstruct TEM observation, whole rumen contents were squeezed through four layers of cheesecloth, centrifuged at 250 x g for 1 min, and prepared as previously described (1). Leaf sections were placed in VOL. 29, 1975 RUMEN BACTERIAL DEGRADATION OF FORAGE CELL WALLS flasks with 250 ml of the bacterial buffer suspension and continuously bubbled with CO2 at 39 C for a maximum of 72 h. Control leaf sections were incubated in buffer (9) with constant CO2 bubbling at 39 C for 72 h. For observation of degraded forage tissue by the SEM, rumen microorganisms were prepared by two methods. (i) Whole rumen fluid was strained through four layers of cheesecloth and diluted with an equal volume of McDougall's carbonate-phosphate buffer (24). (ii) Strained, whole rumen fluid was mixed with an equal volume of phosphate buffer (20) and incubated in a separatory funnel for 1 h at 39 C to permit sedimentation of heavy feed particles and large protozoa. Leaf sections were placed in 250 ml of each of these inocula and continuously bubbled with CO2 at 39 C for 4 or 6 h. TEM. Leaf sections incubated with the bacterial inoculum and with control buffer (without microorganisms) were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.0 for 2 to 3 days and postfixed in 1.5% buffered osmuim tetroxide for 4 h. Leaf sections were prepared for the TEM as previously described (3). SEM. Leaf sections incubated in the microbial inoculum for 4 to 6 h were fixed in 4% buffered glutaraldehyde for 30 h and postfixed in 1.5% buffered osmium tetroxide for 4 h. The degraded leaf sections were blotted free of excess moisture and affixed to SEM stubs so that the degraded edge of the cross-section was prominently displayed in a vertical position. The stubs were quick-frozen in dry ice-isopropanol or liquid nitrogen and allowed to reach room temperature as the specimens were dried in a vacuum evaporator before coating with gold-palladium (60:40) alloy wire. The specimens were then observed in a field emission SEM at about 15 kV. RESULTS Control leaf sections observed with the SEM revealed that the buffer did not solubilize the tissues after 72 h of incubation (Fig. 1). As observed with the TEM, even the easily digested phloem cell walls were intact after 72 h of incubation in buffer (Fig. 2). However, an intact leaf section incubated with rumen microorganisms and observed with the SEM revealed differential tissue destruction after 4 h (Fig. 3). Mesophyll (M) and phloem (P) cell walls were removed to a point beyond the depth of focus of the SEM (at x416); remnants of the outer bundle sheath (B) remained although this tissue had lost structural integrity. The epidermis (E) appeared to be partially degraded (arrow), whereas the sclerenchyma (S) and rigid vascular tissue (V) resisted microbial digestion. The bacterium-plant cell wall association investigated at higher magnifications revealed differences dependent on the tissue type. Non- descript areas of degradation were evident in the mesophyll cell walls near the bacteria but not necessarily in the location of bacterial association with the cell wall (Fig. 4, arrows). Similar phenomena were revealed by the TEM in that bacteria were seen near but not necessarily attached to the degraded areas in both mesophyll (Fig. 5, C) and phloem cells (Fig. 6, arrow). However, bacteria appeared at times to be attached by a dense extracellular substance to the mesophyll cell wall (Fig. 5, arrows). The tissues digested after longer incubation times (i.e., the outer bundle sheath and epidermal cells) appeared to require bacterial attachment prior to tissue degradation. Rod-shaped bacteria, lying on the epidermal cell wall, were surrounded at times by an apparent zone of degradation (Fig. 7, arrows). The TEM revealed similar conditions with bacterial degradation preceded by attachment of bacterial cells apparently by an extracellular substance to the epidermal wall (Fig. 8). The zones of degradation were sharply defined in contrast to the diffuse zones in the mesophyll and phloem cells ( Fig. 5 and 6). In addition, bacteria had tightly adhered to the outer bundle sheath cells (Fig. 9, B), and zones of hydrolysis surrounded rodshaped bacteria in the inner bundle sheath cell ( Fig. 9, arrow). Attachment to the outer bundle sheath by a large coccus as shown by the TEM appeared to be mediated by an extracellular substance (Fig. 10, arrow); similar electrondense substances were not apparent with the other three attached bacteria. Observations by SEM revealed a large coccus that appeared to be attached to the epidermal cell wall by rod-like appendages (Fig. 11, arrow). DISCUSSION That rumen microorganisms degrade the mesophyll and phloem cell walls more rapidly than other tissues has been shown for various forages (2). The current observations by the TEM and SEM, as well as previous ones by the TEM (3) on a grass of lower digestibility (i.e., Coastal Bermuda grass) (1), indicated that rumen bacteria at times degrade these tissues without prior attachment. The degradation of mesophyll and phloem cell walls by enzymes free from the surfaces of a specific microbe or group of microbes cannot be ruled out at this time. However, bacteria near degraded areas were diverse in morphology, indicating that no one species alone was involved. In addition, the bacterial attachment prior to degradation of bundle sheath and epidermal cells appeared to Outer bundle sheath cell wall observed by the TEM after 12 h of incubation with rumen microorganisms. Four bacteria appear to be attached to the plant wall. Attachment of the large coccus to the plant wall is mediated by an electron-dense, extracellular substance (arrow), whereas no such structure is apparent with the rods. However, the attachment appears to be so close that the bacterial shape of the attaching side is modified. x occur with bacteria of diverse morphologies. Reports have shown that higher temperatures increase the cell wall constituents of tall fescue with a resultant decrease in in vitro digestibility (4,17). Research should be undertaken to examine the bacterial attachment phenomenon relative to the cell walls of plants that have undergone environmental changes with resultant changes in the rate of digestibility. The secretion of degradative enzymes has been reported for the rumen bacterium R. albus (23,26). Smith et al. (26) reported that extracellular enzymes from R. albus digested up to 65% of a small quantity of ground or blended cellulose. The cell walls of the mesophyll and phloem may be so structurally different from bundle sheath and epidermal cell walls that cell-free enzymes, or fractions of the degradative enzyme complexes (22), can degrade the former tissues. In addition, Leatherwood (23) proposed that in R. albus an "affinity factor" may be necessary to hold the "hydrolytic factor" of cellulase in position to the insoluble cellulose for multiple attacks to occur. Such a phenomenon may indeed be required for hydrolysis of the cell walls more resistant to bacterial degradation where attachment precedes degradation. Previously we reported that large rumen cocci possessed an electron-dense extracellular substance that appeared to adjoin the bacterial and plant cell walls (3). Leatherwood (23), using the SEM, reported tube-like appendages associated with R. albus cells only when grown on cellulose-containing media. We found similar structures in a large coccus that apparently was attached to the epidermal cell wall as shown by the SEM {Fig. 11). The rod-like appearance and amorphous, capsular-like appearance of this extracellular feature by the SEM and TEM, respectively, of the large cocci may be the same structure manifested in various ways by different drying techniques. Springer and Roth (27), using the TEM, had reported that the length and width of fibrils of capsules seen in Klebsiella pneumoniae varied with the dehydrating procedures. Perhaps electron microscopy of critical-point-dried (5) samples would elucidate these extracellular features in rumen bacteria as has been shown recently for other bacterial species (8). Jones et al. (21) and Fletcher and Floodgate (18) have reported the attachment of bacteria-to substrates by extracellular substances. In addition, Shilo (25) showed that close contact be-699 on March 17, 2020 by guest http://aem.asm.org/ tween myxobacteria and the blue-green algae was required for algal lysis, although no capsule-like material was reported. Berg et al. (6) reported that Cellvibrio fulvus and Sporocytophaga myxococcoides cells grown on different types of cellulose media adjoined the cellulose fibers with distinct depressions made in the substrate; S. myxococcoides produced extracellular substances interpreted as bacterial envelopes when grown on cellulose. Costerton et al. (11) reported that three gram-negative rumen bacteria, B. ruminicola, B. succinogenes, and M. elsdenii, possessed extracellular coats outside the cell envelope. Although these authors (11) concluded that the extracellular coats provided protection in a highly competitive environment, our current and previous (3) observations indicated that an extracellular substance may also mediate the attachment of rumen bacteria to particular plant cell walls so that degradation may occur. Although attachment of large cocci to substrate by capsule-like material was noted often, we have not observed extracellular substances in all attached bacteria. It is possible that the coats are thin in some cases (11) and not seen without specific staining (i.e., with ruthenium red). Costerton et al. (12), reviewing the cell envelope of gram-negative bacteria, reported that the degradative enzymes associated with the cell wall provide a "facility unique among unicellular organisms in that complex food molecules are broken down into their component monomers in a zone immediately surrounding the cell." Our observations suggest that attachment to intact plant cell walls, mediated by extracellular substances in at least some rumen bacteria, is required before the hydrolytic fraction of the enzymes can degrade the complex organization of certain forage cell walls. Such a phenomenon would help explain why certain forages, whose microanatomy consists of a high ratio of bundle sheath and epidermal to mesophyll and phloem cells, are less rapidly digested than forages with a lower ratio of these tissues.

v3-fos

2018-04-03T06:22:38.890Z

1975-01-01T00:00:00.000Z

46418049

s2

Comprehensive examination of protein distribution profile in wheat grain. A wheat grain was divided into three portions, endosperm, germ and bran, and the protein profile of each was examined comparatively after sequential extraction with isopropyl alcohol, sodium chloride, lactic acid and KOH solutions. Recovery of the protein in the extracts was 93-96%. The relative protein concentrations of the four soluble fractions of endosperm were very similar to those of bran, but germ showed a distinct distribution of the soluble proteins. Soluble protein fractions of endosperm, germ and bran, particularly their NaC1 soluble fractions, exhibited distinct individuality upon examination by polyacrylamide gel electrophoresis and gel filtration. Indeed, it was suggested that the gel electrophoretic profile could serve as a criterion for the quality evaluation of wheat flour. On the other hand, the KOH soluble proteins of endosperm, germ and bran, which gave indiscrete electrophoretic patterns, existed predominantly in highly aggregated forms which disintegrated upon exposure to 1% SDS or by reduction with 2-mercaptoethanol. Summary A wheat grain was divided into three portions, endosperm, germ and bran, and the protein profile of each was examined compara tively after sequential extraction with isopropyl alcohol, sodium chloride, lactic acid and KOH solutions. Recovery of the protein in the extracts was 93-96%. The relative protein concentrations of the four soluble fractions of endosperm were very similar to those of bran, but germ showed a distinct distribution of the soluble proteins. Soluble protein fractions of endosperm, germ and bran, particularly their NaCl soluble fractions, exhibited distinct individuality upon examination by poly acrylamide gel electrophoresis and gel filtration. Indeed, it was suggested that the gel electrophoretic profile could serve as a criterion for the quality evaluation of wheat flour. On the other hand, the KOH soluble proteins of endosperm, germ and bran, which gave indiscrete electrophoretic patterns, existed predominantly in highly aggregated forms which disintegrated upon exposure to 1% SDS or by reduction with 2-mercapto ethanol. The distribution spectrum of wheat proteins has not been fully established due to the lack of definitive procedures for extraction and fractionation. Wheat proteins are in general fractionated according to their solubilities by sequential extraction of ground samples (1)(2)(3). A problem encountered in this classical method arises from low recovery of proteins. MASS (4) devised an effective method in which a sample thoroughly ground with sand is packed in a column and progressively extracted with appropriate solvents. This method has been recently modified by MATTERN et al. (5). While proteins of endosperm have been extensively investigated in relation to its processing properties (6), the protein distribution profiles of germ and bran are 363 and bran are much higher than that of endosperm. Among the fractions of germ the NaCl soluble fraction is distinct from the others for its low serine and leucine contents. Gel electrophoretic patterns of the soluble proteins of endosperm, germ and bran Figure 1 shows electrophoretic profiles of isopropyl alcohol soluble fractions of (Fig. 3). Lactic acid soluble fraction of endosperm is distinct in that it exhibits several eminent bands at the advancing front. The KOH soluble fractions comprised high proportions of ag gregates which upon gel electrophoresis accumulated at the upper region of the gel columns giving anomalously diffused patterns. It was thought that removal of the large aggregates, which retard migration of other protein components, may improve electrophoretic pattern. Accordingly, the KOH soluble fractions were subfractionated by gel filtration on Sepharose 4B and subjected to gel electrophore sis. The result, as shown in Fig. 4 for endosperm, was not with success yielding indistinct pattern. Gel filtration profiles, however, can provide some information regarding molecular weight distribution of the KOH soluble fractions. As shown in Fig. 5, the elution profiles indicate apparent diversity among the protein fractions of endosperm, germ and bran. The proportion of large aggregates eluted at the void volume of the column is appreciably higher in the KOH soluble fraction of endosperm than in the germ and bran fractions. Disaggregation of the KOH soluble proteins by treatment with SDS and by reduction with 2-mercaptoethanol There may be two possible reasons for the appearance of the indistinct ele ctrophoretic patterns of the KOH soluble fractions, i.e., progressive aggregation through disulfide bond formation and reversible polymerization of physical nature or both. In the experiments that follow, these possibilities were examined. In the presence of SDS, the KOH soluble proteins underwent disintegration should be noted that not all of the proteins comprised in the KOH soluble fraction are susceptible to SDS, since considerable amounts of protein still remained at the top of the gel. However, these aggregates disappeared by reduction which also led to disintegration of the aggregates (Fig. 6). It seems that the three KOH soluble fractions respond diversely to reduction. In conclusion, the KOH soluble protein occur predominantly in aggregated forms which disintegrate into several smaller units upon exposure to SDS or by reduction. The use of the gel electrophoretic profile as a criterion of the quality evaluation of wheat flour The electrophoretic profiles of the soluble proteins of wheat so far presented do not provide a means that allows indisputable differentiation of endosperm proteins from the proteins of germ and bran. The only difference worth noting is in the electrophoretic profiles of NaCl soluble fractions of the three portions (endosperm, germ and bran) (Fig. 2). In order to find the electrophoretically distinct com ponents in the proteins of the three portions, the NaCI soluble fraction of endo sperm was mixed with those of germ and bran separatory, and the two mixtures were subjected to gel electrophoresis. The resultant profiles are presented in Fig. 7, in which E+G and E+B respectively represent endosperm-germ and endosperm bran mixtures. Comparison of the composite patterns with the corresponding Fig. 7. Fig. 8. Fig. 7. Electrophoresis of the mixtures of NaCl soluble fractions of endosperm and germ (E+G), and endosperm and bran (E+B). The experimental conditions are as described for Fig. 1. Fig. 8. Electrophoresis of isopropyl alcohol soluble fractions of endosperm (a), germ (b) and bran (c). Electrophoresis was carried out on 3.5% polyacrylamide gel and the patterns were obtained by staining with PAS reagent. individual patterns (Fig. 2) can reveal contamination of endosperm. However, it requires a close examination to identify the contaminants, germ or bran. In search for a more simple criterion, it was found that isopropyl alcohol soluble fractions of germ and bran, but not endosperm, contained PAS positive components which migrated into 3.5% polyacrylamide gel (Fig. 8). Since only endosperm does not contain this component, single electrophoretic run on a given fl our sample is required to examine its purity. Thus, this procedure can serve as a criterion which permit a rapid evaluation of flour quality. It is of interest to note that NaCl soluble fraction of endosperm, but not those of germ and bran, contained PAS positive components which migrated into 7% polyacrylamide gel.

v3-fos

2018-04-03T06:11:15.500Z

1975-06-01T00:00:00.000Z

45870003

s2

Ultrastructure of cell envelopes of bacteria of the bovine rumen. Most of the bacteria found in rumen fluid samples taken from cows fed hay, or a concentrate diet, had cell walls of the gram-negative type. Most were intact, with only a small proportion of lysed cells, and many of the cells contained electron-translucent cytoplasmic deposits similar to the carbohydrate reserve material described in pure cultures of rumen organisms. All of the bacteria observed in these samples had an external "coat" layer outside the outer membrane when fixed in glutaraldehyde and osmium, stained with uranyl acetate and lead citrate, and examined as sectioned material. These coat layers varied from thin (ca. 8 nm) structures to very extensive fibrous systems, sometimes including concentric arrangements and radial fibers extending up to 1,200 nm from the cell. The thin-coat layers sometimes exhibited a rough periodicity. In all, 10 different types of coat layers were distinguishable on a morphological basis. It is proposed that these external coat layers have protective and adherence functions for the rumen bacteria in the environment. Mixed microbial populations of certain specific environments have been described recently by direct observation with electron microscopy (2)(3)(4)15). Fletcher and Floodgate (15) examined marine bacteria adherent to surfaces, and Casida's group (3,4) described bacteria immediately after their elution from soil. A common finding in these and other studies is that each of the gram-negative bacterial cells that make up most of these populations is enclosed by an extensive and complex capsular structure external to the outer membrane. The marine organisms adhere to their substrate by a fibrous polysaccharide (15), and the capsule surrounding the soil bacteria is often fibrous in nature (2,3). These findings suggest that bacteria living in natural, challenging environments may depend for their survival on the production of external structures on the cell wall that dictate their adhesion pattern and provide a measure of protection for the cells (12). Salmonella typhimurium shows little external polysaccharide in shaken laboratory culture but produces a very extensive (150 nm) lipopolysaccharide microcapsule in infected tissue (26), indicating that cells in laboratory cultures may differ from cells in their natural environment. In contrast, some bovine rumen bacteria, grown in the rumen (6) or in pure cultures that have been repeatedly transferred in the laboratory (11,23), showed the presence of layers of fibrous polysaccharide outside the cell wall or of external patterns of globular units resembling the protein coats of Spirillum (5) and many marine bacteria (29). In one case (23), the fibrous polysaccharide coat of the cells of a rumen bacterium has been shown to mediate their attachment to cellulose fibers in pure culture. In this study, we used direct transmission electron microscopy to determine the extent to which complex cell coats are formed by bacteria within the rumen. MATERIALS AND METHODS Rumen contents were collected from eight fistulated cows fed a daily ration of 5.4 kg of a pelleted all-concentrate diet, or 8.2 kg of alfalfa hay, in two equal feedings. Samples were collected before the morning feeding and 4 and 8 h after this feeding on days 21, 27, and 35 after initiation of each of these diets. Rumen contents were filtered through four layers of cheesecloth and centrifuged at 48,000 x g at 4 C for 20 min. The pellet from this centrifugation contained most of the bacteria in the sample, and this pellet was prefixed for 1 h by the addition of 0.5% glutaraldehyde in 0.067 M cacodylate buffer at pH 6.8. Fixation was carried out by resuspending the material in 5% glutaraldehyde in 0.067 M cacodylate buffer at pH 6.8 for 2 h at room temperature. The material was enrobed in agar by resuspension in 4% agar at about 40 C and expressed by Pasteur pipettes 841 on March 21, 2020 by guest http://aem.asm.org/ Downloaded from CHENG AND COSTERTON as a cylindrical core. The cores were washed five times in the cacodylate buffer, postfixed in 2% osmium in the buffer, washed five times in the buffer, and dehydrated through a graded acetone series before embedding in Vestopal (23). Thin sections were stained with uranyl acetate (2% aqueous) and then lead citrate (25) and were carbon coated before examination, using an A.E.I. 801 electron microscope. Glutaraldehyde and osmium were obtained as concentrated solutions, under argon, and the embedding materials were kept under freon to minimize oxidation and standardize block hardness. RESULTS Most of the bacteria seen in about 100 rumen samples showed the gram-negative pattern of cell wall structure, and very few were seen to have a thick peptidoglycan layer similar to that seen in pure cultures of Megasphaera elsdenii (11) and Ruminococcus albus (23). All of the cells examined had outer membranes of the usual dimensions (about 8.5 nm), and all had some structure external to this double-track layer ( Fig. 1-5). Ten different morphological variants of this external structure were discerned. One of the simplest of the extracellular coats was a single, thin, electron-dense layer, separated from the outer membrane by a regular space ( Fig. 1 and 2a, F). An irregular periodicity ( Fig. 1 and 2a, arrows) similar to that seen in cells of pure cultures of Bacteroides ruminicola (11,12) and short, irregularly spaced connectives between the coat layer and the outer membrane ( Fig. lb, C) were apparent in highmagnification electron micrographs of this structure. The absence of a fibrous coat on these cells is an important observation because it suggests that the fibrous coats seen on other cells are not the result of the nonspecific adhesion of fibers produced elsewhere in the rumen. A relatively simple coat structure was seen in the diffuse coat of particles and fibers that appear to be anchored directly to the outer membrane of some cells (Fig. lb, 2, 3, 4a, G). The particles were intensely electron dense and the fibers moderately electron dense, and the possibility must be considered that the particles are cross-sections of the fibers. These thin fibers extended up to 1.2 gm from the cell surface and, where similar cells were clustered, produced small areas of continuous fibrous material ( Fig. 2a, P). In a few cells, the extracellular coat consisted of a thin deposit of electron-dense material in the outer aspect of the outer membrane ( Fig. 2b, H). More often, cells showed a thicker (50 nm) layer of electron-dense material (Fig. 4a, I). In both cases, this intensely stained material appeared to be composed of fine granules that were aggregated into clusters in the thicker structure. One of the most common forms of the extracellular coat in these rumen organisms was a discrete mat of fibrous material (80 to 200 nm thick) with a distinct outer boundary ( Fig. 1 and 4c, J). This fibrous coat often served to connect the cell to a piece of detritus, to a different cell (Fig. 1), to a similar cell or, rarely, to a series of similar cells (Fig. 4c). Other types of extracellular coats, which were only rarely seen, were a highly convoluted, double-track structure outside the outer membrane with adherent bleblike structures (Fig. 2b, K), a homogeneous electron-dense mass maintained at a constant distance from the outer membrane by radial connective structures (Fig. 4b, L), and a thick electron-dense layer with thick and irregular radiating fibers (Fig. 5, M). Small numbers of cells in these rumen samples were enclosed by a single, thin, electron-dense layer, with apparent periodicity in tangential section. This layer was maintained FIG. 1-5. Electron micrographs of sections of bacteria in embedded samples of rumen fluid from cows with normal digestive processes. The cows whose rumen bacteria are illustrated in Fig. 1, 3, 4b, 4c, and 5 were fed on alfalfa hay, and those whose bacteria are shown in Fig. 2 and 4a were fed on an all-concentrate diet. All of the cells were labeled according to the type of external coat layer they possess, as follows. (F) These cells have a thin electron-dense coat that shows a rough periodicity where the sectioning angle is favorable (arrows). In some areas (C), fine electron-dense connectives can be seen between this layer and the outer membrane. at about 75 nm from the outer membrane by radial fibers that extended to it and beyond it into the menstruum (Fig. 3, N). A few cells were enclosed by two concentric electron-dense layers maintained at considerable distance from the outer membrane, and from each other, by radial fibers (Fig. 4a and 5, 0). Sectioned material is not ideal for the study of adhesion, but the fibrous extracellular coats of bacteria often appeared to mediate an adhesion of these rumen bacteria to food particles. In the bacterial rumen populations, we always found some cells that contained electrontransparent masses (Fig. 2b and 4b) within their cytoplasm. Each of these masses, like the a-1,4 glucan deposits (9) seen in cells of a pure culture of a rumen organism (M. elsdenii), were delimited by a single electron-dense layer. Because cells of all physiological ages were present in these samples, the appearance of their cytoplasm and the degree of condensation of their nucleoids was highly variable, but very few lysed cells were seen. DISCUSSION The relationship between a microbial population and its environment is mediated by the cell envelope of the bacterial cells. The cell envelopes of bacteria growing in the normal bovine rumen are predominantly of the gram-negative type, and all have additional cell coats outside the outer membrane. The bacteria of freshwater environments are also predominantly gram negative (M. Franklin, "Hotpack" lecture of Canadian Society of Microbiologists, Montreal, 1974), as are those of marine environments (17), and many of these bacteria have been shown to possess extracellular coats of fibrous carbohydrate (15,18) or of globular protein (5,29). Many enteric pathogens have been shown to produce externally located carbohydrate materials (16), and lipopolysaccharide, which is a component of the outer membrane, is actively shed into the medium (19,30) in shaken batch culture or accumulated around the bacterial cells in a capsular form in infected tissue (26). Similarly, bacteria eluted directly from the soil are often surrounded by a mat of fibrous material (2-4) that forms an enclosing capsule, and gliding bacteria exude a slime (22) that is important in their motility (13). Thus, it is clear that many bacteria can produce and assemble complex and often extensive coat layers on the outer surface of their already complex gram-negative cell wall (12). These gram-negative cell walls by themselves confer protection from antibodies (24), antibiot-ics (20), and other hazards of microbial life (12) and also maintain a molecular environment so that cell wall-associated enzymes are conditioned (28) and protected (8). Part of this protection is provided by the limited penetrability of the outer membrane, but the Donnan effect exerted by ions bound within the structural molecules that constitute the cell wall is also important in conditioning the molecular environment within the cell wall and in limiting the access of extraneous molecules and ions to the cytoplasmic membrane (12). Coat layers have been observed to confer protection from attack by predatory bacteria (Bdellovibrio) (F. L. A. Buckmire, Bacteriol. Proc., p. 43, 1971) and to inhibit phagocytosis (14). Whether coat layers are composed of carbohydrate or of protein, they must be expected to contain bound ions that would act in the manner of a complex ion exchange resin to further condition the molecular environment of the cell envelope and to limit its penetrability (12). Cell coats are also sometimes important in the adhesion of bacteria to surfaces in their environment (10,18). At least one species of rumen bacteria adheres to cellulose fibers by means of its polysaccharide coat layer (1,23), and the secondary and irreversible attachment of aquatic bacteria to surfaces is a function of their production of a carbohydrate material (10,15,21). That this attachment may be of physiological and ecological significance is indicated by the finding that Myxobacteria must adhere to the surface of blue-green algae for the enzymes associated with their cell wall to digest the cell walls of the algae (27). The predominance of gram-negative bacteria with extracellular coat layers in these environments may also result, in part, from their content of wall-associated enzymes. These enzymes have been shown to be located in the periplasmic space and at the cell surface of gram-negative cells (12), and some rough strains of S. typhimurium release an alkaline phosphatase-lipopolysaccharide complex into their environment (19). Studies of pure cultures of rumen organisms have shown that one "marker" enzyme (alkaline phosphatase) for the wall-associated group of enzymes is tenaciously bound to structural elements in the periplasmic space (7). The retention of degradative enzymes within the gram-negative cell wall and at its surface allows the enzymes access to external "food" molecules, even if these are insoluble polymers, and prevents the loss of these enzymes into the menstruum. The activity of these enzymes provides products that are spatially very close to the permeases that will 848 APPL . M ICROBIOL . on March 21, 2020 by guest http://aem.asm.org/ Downloaded from BACTERIA OF THE BOVINE RUMEN transport them into the cell and that are vital to cellular growth. Thus we find that the predominant bacteria of the bovine rumen have a gram-negative cell wall with an additional external cell coat. This cell coat, which may be composed of protein or of carbohydrate, may function in adhesion of the cells to surfaces, and the whole cell envelope probably functions in the protection of the cell and the retention of cell wall-associated enzymes. This external coat layer takes 10 morphological forms in the material we examined and, although there is a possibility that capsules may change as the cells age (9), further studies indicate that there is an even greater variety of distinct capsular types among rumen bacteria.

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2014-10-01T00:00:00.000Z

1975-10-15T00:00:00.000Z

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Indirect selection in poultry breeding with special reference to single genes SUMMARY The interest of indirect selection in poultry breeding is discussed, with special reference to the utilization of known genes. The most favourable situations are those where the intensity of direct selection is zero (trait impossible or too costly to measure, environment where the pro- gress is desired difficult to realize, or presence of negative genetic correlations suppressing genetic gain) and that where the heritability of the trait to improve is low or zero (selection plateaux). Examples corresponding to these situations are presented : possibilities of indirect selection for feed efficiency of laying hens corrected for body weight and egg production ; selection plateaux for egg laying and possible utilization of a heterozygote advantage at locus Hi ; selection to lower percentage of broken eggs in cages and possible use of dw and 0 genes. INTRODUCTION The idea of utilising known genes in selection is, of course, nothing new. However, in general qualitative visible traits are involved, and breeders are not accustomed to include single genes to modify production criteria in their breeding plans. It may be suggested, in particular cases, that the genotype to specified loci be included in selection indexes. This can sometimes be an appreciable aid to selection. The aim of this paper is to present a few examples in favor of this idea. GENERALITIES ON INDIRECT SELECTION AND GENE UTILIZATION In what conditions is it worth considering specified genes in breeding programs ? Common sense suggests that it will be the more useful as it will concern a trait difficult to improve by direct selection, and as the specified gene (s) will have a larger effect on this trait. This has been studied on a theoretical basis by SMITH ( 19 67). The use of Mendelian factors may be regarded as a particular case of « indirect » selection : selection on a trait B (genotype at a particular locus) to improve a trait A. Such selection will not only be useful but necessary, if direct selection produces a genetic gain per generation AG A = o. t : selection intensity, h', : heritability of trait A, this will be the case if : a) The selection intensity exerted on A is equal to zero or b) if the heritability of A is zero. The first table summarizes several corresponding possibilities. a,) A trait may be expressed in only one sex (e.g. females). Then, individual selection on males has to be made using indirect criteria. Among these might figure specified genes, but as there are other possibilities (selection on performances of sisters, daughters...) the advantage of the former is subject to discussion in each particular case. a 2 ) A more convincing case is that of traits for which direct selection (in both sexes) is too difficult or too costly. For instance, a selection on feed efficiency of laying hens, independent of body weight and egg production, requires controlling feed consumption on an individual, or at least on a family, basis; this control is costly in time and money. Another case would be that of a breeder attempting to reduce the volume of droppings produced by caged birds. a 2 ) In the previous case, the measurement of the trait was difficult or costly to obtain ; also the environment where a genetic improvement is desired may be diificult to realize ; for instance, in selection for resistance to a specific disease because of the cost and risks of exposure, or for adaptation to environmental conditions difficult to realize on a large scale in our countries : Tropical climate, feed including local ingredients, and so on. In these various situations, a selection on males only may be made. This would reduce cost, but research of indirect criteria may be more satisfactory. a,) A case where the realized selection intensity on a trait A may be practically zero is when A has a negative genetic correlation with an economically interesting trait B, so that a selection in favor of both A and B in each generation gives no progress for either. A possible issue is the research of indirect criteria correlated with A but not presenting the unfavorable correlation with B, for instance, single genes having an effect not consistent with overall correlation. _ b) The second case where indirect selection is necessary is when h; = o (« Selection plateaux »). A selection is exerted, but there is no corresponding gain, either because the genetic variability for the trait is exhausted in the population, or because the additive variance alone is suppressed (for instance, unfixed genes with heterozygote advantage may remain). In these two cases, single genes may be of some utility, especially in the former, as the only solution is to introduce a new genetic variability, the controlled introduction of specified genes minimizing the contribution from extraneous genomes. To these situations we can add that (c) of traits with low, but not zero, heritability for which the question of indirect selection may also be settled. In presence of one of these favorable cases, it is not only necessary to have at hand genes with an appreciable effect on the trait to improve, but these genes must be sufficiently easy to detect. An ideal case is that of genes « marked &dquo; by a visible effect, but the frequency of significant associations between such genes and quantitative traits is limited a priori and often these genes are fixed in commercial strains. The case of blood groups presents another difficulty, especially for locus B alleles, because even if they have an appreciable incidence on production traits, their identification is cumbersome and more or less limited to a particular population. Certain genes responsible for biochemical polymorphisms may be easier to deal with, although they are somewhat more costly to detect. Finally, it is preferable to handle genes having a known mode of action (at least up to a certain level), for in this case the existence and generality of their possible pleiotropic effects can be more positively proven. SOME EXAMPLES OF INDIRECT SELECTION AND UTILIZATION OF SINGLE GENES A few examples will be evoked in this paper, with particular reference to our results at Jouy en Josas. Table 2 summarizes a list of these, reviewing the traits already mentioned on the right of table i. Resistance to specific disease corresponds typically to case (as) (difficult environment to realize). It is beyond the scope of this paper to discuss this problem. Some of our results concern single-gene effects on egg-shell breakage in cages. This represents situation (c) ; it might also be viewed as an attempt of indirect selection to improve shell strength at higer ambient temperatures, which corresponds to case (a.). As regards case (a 4 ) (unfavorable genetic correlations), an example is the situation of broiler breeders making efforts to improve feed efficiency and reproductive capacity in maternal lines while continuing to increase broiler growth rate. The unfavorable correlation is between a higher growth rate on one hand, larger adult size and higher feed requirements of the dam on the other. Selection for a modified growth curve may be conceived (e.g. R ICARD , in press) but, as a single-gene effect, the use of the sex-linked dwarf is to be mentioned. Finally, for laying intensity, subject to selection « plateaux » (case b) the problem of the Hi gene (responsible for an agglutination reaction of red-blood cells) will be discussed. Indirect selection, single genes and feed efficiency of laying hens Following By!x!,y ( 1941 ) and other authors, one can express feed consumption in a given time by a multiple regression equation of the form : where F = observed feed intake of an individual hen, W = mean body weight during the period of observation, 8 W = body weight variation, E = total weight of eggs produced, R = « residual » term (deviation of observed feed intake from the expected value given by the regression), a, b, c = appropriate coefficients. The exponent ot is generally of the order .6-.7 (In our own results, taking .5 is a sufficient approximation). Of course, the first factor to be taken account of is body weight (W) ; it is well known that reducing size of laying hens by breeding improves feed efficiency. As concerns single genes, the dwarf gene dw is one of the possible contributions to further progress in this direction. But even with equal body weight there remain differences in feed efficiency which may not be negligeable. In our experimental strains in Jouy en Josas ( 1 ), we observed significant « residual)) » differences in feed intake (« R » variable) between families of half-sisters. The average difference between particular families can ( 1 ) Including a synthetic strain with normal (Drv) and dwarf (drxr) birds, and a strain of Rhode-Island red. represent more than 10 p. 100 of mean feed consumption in the flock (B ORDAS , ME R AT, 1974)-To improve this « residual » feed efficiency, we investigated the possibility of selecting on the R variable estimated by short-term measurement (two weeks) of feed consumption (BoRnAS, 11!RA'r, ig 75 ). The results show a rather high correlation of -!-. 8 4 between individual feed consumption estimated on a short-term and on a longer-term ( 3 months) basis. This remains true after correcting for average body weight and egg production during the « long » period of observation (r = !-.71). However, the individual measurement of feed intake, even over a relatively short period, may be difficult. So, we looked for possible correlations between « residual » consumption and both production traits and body measurements of the same bird. The correlation with water intake suggests limited possibility of improving feed efficiency by reducing the former, if this correlation exists at the genetic level. A very rough evaluation of water consumption may be obtained by visual scoring of the consistency of droppings. This could at least apply to the detection of the « polydipsia » gene (DuNsoN and Buss, 19 68), but this case of excessive drinking may be a special one. Generally speaking our own un published data show no more correlation between water intake and « residual » feed consumption at fixed total feed intake, which would not suggest the former to be associated with modified metabolic efficiency. With wattle length and shank length, the phenotypic regression coefficients of cc residual » feed intake correspond, respectively, to an increase of I IO g of feed consumed per 2 8 days for wattles i cm longer, and of the same order per additional cm for shank length. Any significant relation between these measurements and uncontrolled feed wastage seems to be excluded in our conditions, but as a simple explanation, it may be suggested that heat dissipation increases due to larger unfeathered appendages (S TURKI E, 19 65, evaluates at 15 p. 100 the proportion of heat lost through only comb and wattles). Our results may thus suggest other indirect selection to improve feed efficiency by reducing the size of these organs. Several major genes for wattle and comb size are known. For instance, it is considerably reduced by the P (pea comb) gene. Preliminary data on « residual » feed consumption of P! and pp hens also suggest, that the pea-comb allele causes a slight reduction (between 1 . 5 and 2 p. 100 ) of this trait. Another interesting correlated trait does not concern R (residual feed consumption) but a variable R' defined as the deviation of observed consumption from its expectation given by a regression equation including body weight (W) and egg mass produced (E), but not weight variation OW. This variable is correlated with OW which is very simple to measure. The observed regression of R' on OW shows that a reduction of about 50 gms for AW per 2 8 days should lead to a decrease of about 100 gms of feed consumed during the same period, again if the « genetic n regression is comparable to the phenotypic one (unpblished data). Another single gene effect, at first sight differing from previous cases, concerns several plumage color genes. Comparing full sisters of different genotype at such loci, in our « synthetic » population, we observed repeatedly a slightly but significantly lower « residual » feed consumption for white laying hens than for their colored sisters. This was obtained in two trials for cc hens (recessive white) compared to Cc ones, in two others for Ii birds (dominant white) compared to ii (MA RAT , r 9 68 ; MÉ R AT, B ORDAS , 1971 ). A non-significant result on fewer data concerns ee hens (restricted black) which show a slightly lower R value than their Ee (extended black) sisters (unpublished data). In each case, the average initial body weight and egg production did not appreciably differ for the compared genotypes. This seemingly parallel effect for 3 independent loci suggested the hypothesis of a pleiotropic effect associated with plumage color. Nothing suggests any difference in digestive and metabolic efficiency, nor in behaviour (energy spent in activity). No difference in internal temperature was found. On the other hand, we tried to test the hypothesis of differences in heat dissipation through the plumage. Results of radiation measurement were not easy to interpret (anyway, a direct relation between visible pigmentation and infra-red radiation can hardly be expected). However, two other observations, concerning the I gene may be of interest. i. Preliminary data suggest a higher average total weight of the plumage for white hens (Ii vs ii). 2 . After 5 -6 months of laying, the frequency of featherless zones was found to be significantly higher for ii than for Ii counterparts (MT RAT and B ORD AS, unpublished data). Thus a tentative explanation of our results for feed efficiency would be that white hens have a more insulating feather coverage than colored hens. We plan to check this hypothesis further, and to extend our observations to the 5js locus. It will be of some general interest to see if the quantity and distribution of plumage (not including rather obvious cases such as Na or scaleless genes) has a significant bearing on feed efficiency in our conditions. Of course, an evaluation of plumage weight is not practical at all, but scoring for « naked o areas in the plumage after some months in production may be. Finally, another single gene effect is in relation to short-term regulation of feed intake (B ORDAS , MA RAT , in press). For the last years we recorded individual daily feed intake for 2 week periods. This gives an estimation of feed ingested by each bird on days with or without egg formation and oviposition, with 4 types of days represented by X = days without either egg formation or oviposition, L = days with oviposition only, 0 = with egg formation only, LD = with both egg formation and oviposition (using the symbols proposed by WOOD-GUSH, H ORN E, i 97 o). Normal (Dzv) and dwarf (dw) hens are compared. In percentage, and even in absolute value, the difference between days with and without egg formation is higher for dwarf hens. A proposed hypothesis is that dwarf hens, having smaller skeletal reserves, have to draw from their feed a larger part of the calcium needed for egg shell formation. A closer correlation found for genotype dw between feed intake and shell thickness of the egg being formed also seems to favorize this hypothesis. If so, and if the use of dwarf laying hens is more widespread in the future, a question to be asked is would these hens benefit more than normal ones from separate calcium intake, such as proposed by MorrGirr, S AUV E UR ( 1974 ), for shell strength, and/or bone accidents in cages? Laying intensity : association with a single gene (Hi) In line with the problem of selection plateaux for laying (Cr, AYTON , 19 68), we were interested in a consistent advantage for laying intensity, that we observed, associated with the heterozygous state at a two-allele locus easy to detect, the Hi locus (SC H E INBERG , REC K E L , 19 6 1 ). The dominant allele (Hi) causes an agglutination of red blood cells with certain seed extracts. This reaction is limited to laying hens, for it requires sufficient oestrogen concentration in the plasma. It is absent in hihi birds. The Hi locus seems to be very frequently polymorphic according to samplings made in various populations by S CH E INB E R G, R!EC!r, ( 19 6 1 ), Box!r, (i964), F, RD 6 S ( 19 6 9 ) and by our own laboratory. Since 19 66 the performance of Hihi and hihi birds has been compared every year in our « synthetic » strain at Jouy on pairs of full sisters. Hi Hi and Hi hi were compared on mean performance in families with a different expected proportion of these two genotypes among birds of the (Hi) phenotype (MA R AT, DU R A N D, zg!3). It appears from our data that the number of eggs laidat least during our period of control, including about the first half of the laying yearis higher for Hihi females than for the other genotypes. The superiority of Hi hi birds over hi hi ones is of the order of 9 p. 100 of the mean population, but the most inferior genotype is Hi Hi, with an estimated reduction in average egg number (as compared to the mean of the Hi hi birds) of the order of 25 p. 100 , which is surprisingly high. The observed effect on egg number cannot be explained by a difference in age at first egg. We wondered if « intensity » or « pauses », in Goodale's sense, was more likely to play a role (H UTT , 1949 ). For convenience we considered as « pauses » any interruption of laying for more than 2 consecutive days ; « intensity » is the ratio of egg number to the number of control days after deduction of cc pauses ». We found no significant difference between genotypes at the Hi locus for » « intensity ». On the contrary « pause » days are minimal for the Hi hi genotype (M ARAT and D URAND , in press). Finally, it may be concluded that our population possesses one chromosomal region with heterozygote advantage for egg number, and especially for the absence of « pause ». Whether this is a pleiotropic effect of the Hi gene remains open to question. It cannot be excluded, as we found very comparable results (significant heterozygote superiority for egg number) on 2 generations, in two other populations at the Poultry Research Centre in Nouzilly (M E R AT and D URAND , in press). One population had some common, although remote origin with ours, the other did not. On the other hand, some indication of the physiological effect of the Hi allele and its possible relation with estrogen binding in plasma (SCFnEn!rs!RC, 1971 ) suggests a direction of research for better understanding of possible side-effects. It would be interesting to isolate genes or groups of linked genes with a special effect on « pauses », for we have very little knowledge of their genetic and physiological determination. By comparison, we observed that the dw gene, which reduces egg production in light strains, mainly effects mean. clutch length, and has rather little effect on cc pauses » (table 3 ). Percentage of broken eggs : effect of dw and 0 genes Broken eggs in cages are an important economic problem ; CARTER (1970) discussed various aspects of this problem, including some genetic ones. It seems important to investigate further which other traits of economic value are correlated with per cent of broken eggs. As an illustration, we found (S ILB E R and M ] K RA T, 1974 ) three main correlations for this trait in our flock : -. The correlation with egg shell thickness is easy to understand. With more recent data we found a correlation of comparable magnitude with elastic deformation under a 5 00 g load (S CHOORL , Bo!RS!n, 19 6 2 ), in concordance with previous results in the literature (CARTER, 1970 ). On the other hand, the appreciable correlation with adult weight within our population may be due to weight « per se », as CARTER ( 197 0) showed that different weights applied to cage floors cause differences in breakage by modifying the rigidity of the floor. Finally, the correlation of egg weight with egg breakage was found also by Boc!o!,YUSS! and T SARENKO ( 1973 ) but not by A ND E RSON et al. ( 1970 ). This divergence might possibly result from experimental conditions, including the relative values of the egg mass and the fraction of the floor mass which is involved in the expression of its kinetic energy. Direct selection on shell breakage, or correlative selection on shell deformation and thickness, are quite possible. However, in this respect, it may be of interest fo mention two important singlegene effects. One concerns the sex-linked dwarf gene dw. Dwarf hens in our conditions have less than half the p. 100 of broken eggs which may be attributed to normal size hens of similar origin. Table 3 mentions this (among other traits). R ICARD , CoCHEz (1972) found similar results in a heavier strain. As table 3 suggests, this is not a matter of shell thickness (which shows no significant difference), nor is it due to intrinsic shell strength as estimated by shell deformation (making allowance for the difference in size between eggs from Dw end dw hens). However, there may be two simple reasons, both concerning « mechanical injury » caused to the egg : -a possibly lower mean height of drop at oviposition of dwarfs which have shorter shanks and different attitude ; -a direct effect of body weight, which concords with the general correlation observed within « normal » hens. This represents an additional reason in favour of the use of dwarf laying hens, when considering their advantages and disadvantages (summarized, in table 3 ). It would also hold for broiler dams, if they reproduce in cages in the future ; -1 -a second gene found to have a bearing on egg breakage is the « blue egg » gene O. It was introduced in part of our flock about 10 years ago. Sms!ER and MA RAT ( 1974 ), compared p. 100 of breakage for Oo and oo full sisters in cages. The results are consistent, showing, that blue eggs have half the breakage per cent, compared to normal-coloured eggs. On the other hand, no significant difference was found associated with this gene for growth, body weight at any age, laying performance, egg weight, albumen height and shell thickness. Conversely, deformation is significantly lower for blue eggs. Explanation of the reduced incidence of breakage is symmetrical to that for the dwarf gene. There does not seem to be any effect associated to the 0 gene for « mechanical injury » caused to the egg, but rather a difference in intrinsic shell strength. It is premature to hypothesize on wheter the blue pigment (biliverdin) affects shell texture, is accompanied by differences in the protein matrix of the shell, or whether there is also a difference in the cuticle and/or in the membrane. Preliminary observations would suggest that blue egg shell membrane is thicker. From a practical point of view, the incorporation of the 0 gene in egg laying strains in our market conditions seems problematic, even if at first sight, it has no deleterious effect on performance. But two possibilities may be worth further exploration : -First, introduction of this gene in female broiler strains, if reproduction in cages becomes a current practice in the future. -Second, possible incorporation into egg laying strains intended for hot climates, since some markets may have no a p y iori objection to blue shells, and shell is expected to be more of a problem at high ambiant temperature. Moreover, preliminary data obtained in climatic rooms suggest that hens with the 0 gene are less affected than others in regard to reduction of shell thickness at high temperatures. CONCLUSION These few examples may illustrate the way in which single genes may sometimes bring forth a peculiarity of economic significance. Of course, this is only part of the picture, especially when the desired allele(s) is not present in the commercial populations. Introduction of foreign germ plasm is a serious problem for the commercial breeder and in this sense, the use of individual genes in breeding represents a narrow approach. This is why the examples proposed are preferably in cases where direct selection is not possible or easy. At the research level, single genes however present an additional advantage, for the investigation of their possible associations to quantitative traits requires facilities which are not comparable to those used by large breeders in creating commercial strains and crosses. This is one way in which geneticists can hope to bring, not only ideas, but sometimes a little genetic material for practical breeding. This is especially true in problems of local adaptation which the commercial breeder may not find sufficiently rewarding, although they may be important for the development of poultry production in particular areas.

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2020-12-10T09:05:43.002Z

1975-01-01T00:00:00.000Z

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Incidence of Aflatoxin in California Almonds In a survey of California almonds, aflatoxin was found in 14% of 74 samples of unsorted, in-shell almonds as received by the processor in 1972, but it occurred at very low levels (below 20 parts per billion [ppb]) in 90% of the contaminated samples. The overall proportion of individual nuts contaminated was especially low and is estimated with 95% probability to have been in the range of 1 nut/55,300 nuts to 1 nut/14,700 nuts. Aflatoxin contamination is not restricted to any particular section of the almond-growing region of California. Commercial sorting procedures are effective in removing most aflatoxin-contaminated nutmeats, since none of 26 samples of processed, whole nutmeats contained aflatoxin. In contrast, 13 of 27 samples of diced almonds were contaminated, but nine of these 13 samples contained less than 20 ppb. Only one of 25 samples of sliced nutmeats contained aflatoxin (4 ppb). Thus, aflatoxin incidence in almonds varies greatly with the category of finished product. The apparent high incidence in diced nutmeats is probably due mostly to the more uniform distribution of aflatoxin occurring in this product (because of its small particle size) than that occurring in the other products. Sample size requirements for monitoring aflatoxin in almonds are discussed. In a survey of California almonds, aflatoxin was found in 14% of 74 samples of unsorted, in-shell almonds as received by the processor in 1972, but it occurred at very low levels (below 20 parts per billion [ppb]) in 90% of the contaminated samples. The overall proportion of individual nuts contaminated was especially low and is estimated with 95% probability to have been in the range of 1 nut/55,300 nuts to 1 nut/14,700 nuts. Aflatoxin contamination is not restricted to any particular section of the almond-growing region of California. Commercial sorting procedures are effective in removing most aflatoxin-contaminated nutmeats, since none of 26 samples of processed, whole nutmeats contained aflatoxin. In contrast, 13 of 27 samples of diced almonds were contaminated, but nine of these 13 samples contained less than 20 ppb. Only one of 25 samples of sliced nutmeats contained aflatoxin (4 ppb). Thus, aflatoxin incidence in almonds varies greatly with the category of finished product. The apparent high incidence in diced nutmeats is probably due mostly to the more uniform distribution of aflatoxin occurring in this product (because of its small particle size) than that occurring in the other products. Sample size requirements for monitoring aflatoxin in almonds are discussed. Aflatoxins may occur in food products if certain molds, namely Aspergillus flavus or A. parasiticus, develop on them under appropriate conditions. These molds, like other storage fungi, seem especially likely to be a problem when seeds or nuts are at an intermediate moisture level, i.e., below that required by field fungi and above that inhibitory to fungal growth (4). The minimum moisture level for aflatoxin production at 30 C by A. flavus is equal to the moisture content of a product in equilibrium with 83% relative humidity or higher, depending on the nature of the substrate and the duration of storage (6). For starchy cereal seeds such as maize and wheat, the limiting moisture level for growth of A. flavus is about 18.5% (4), whereas in oily seeds such as peanuts it is 8 (4) or 9% (7). For almonds (Prunus amygdalus), the limiting moisture content for growth ofA. flavus is likely to be similar to that found for peanuts, due to the similarity in composition of these nuts. Infection of tree nuts with aflatoxigenic molds probably occurs most often in the field before and/or during harvest while the kernels are still moist. Tree nuts are generally exposed to field dirt and to possible physical damage by modem I Present address: Santa Clara County Department of Public Health, Occupational Health Chemistry, San Jose, Calif. 95128. methods of mechanical harvesting, which involve knocking the nuts to the ground and later collecting them with a machine that brushes them onto a conveyor belt traveling in front of a collecting bin (8). Insect damage before and after harvest might contribute to the invasion and development of molds. The exact conditions responsible for the contamination of tree nuts remain to be established. Since aflatoxins are highly toxic to most animals and carcinogenic to at least some animal species (12), their presence in various food and feed crops poses a serious threat to the safety of our foods. Foods subject to aflatoxin contamination have been regulated by the U.S. Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act, as amended 1972, Section 402a, which relates to adulterated foods (3). Although no legal tolerance has been established as yet for these toxic compounds in foods, the FDA has set an informal guideline level of 20 ppb of aflatoxin, beyond which they will seize a product (1). The guideline level applies to the total amount of aflatoxins, which by analytical methods used routinely by the FDA includes aflatoxins B1, B2, G1, and G2 (2). In 1971, the FDA alerted the almond industry of California that the results of a recent FDA study indicated that almonds, as well as other 48 tree nuts, are sometimes contaminated with aflatoxins. Therefore, the present study was undertaken with the support of the California almond industry to determine the incidence of aflatoxin in almonds and to determine the effects of commercial handling and processing practices on aflatoxin incidence. This study includes almonds as they arrive from the grower, almonds at various stages of sorting in the processing plant, and almonds as finished products marketed by the processor. Since studies on aflatoxin in peanuts have shown that only a few nuts out of many thousands need to be contaminated to be a serious problem (5,(9)(10)(11), it was assumed that very large samples of almonds would be required for representative sampling and meaningful analysis. The sample size chosen represents a compromise between the estimated appropriate size and a size that is practicable in regards to cost and analysis. MATERIALS AND METHODS Samples. Samples from the 1972 almond crop were collected throughout the harvesting and processing season. The following general categories of almonds were sampled: (i) unsorted in-shell nuts, representing incoming almonds as received by the processor; (ii) "in-process nuts," representing nutmeats at various stages of sorting by the processor; and (iii) processed nutmeats, representing various finished products sold for food use. This last category consisted of diced, sliced, and whole nutmeats, including the whole nutmeat samples from the final stage of the in-process or sorting study. Only Nonpareil variety almonds were used for the study of unsorted in-shell nuts and of whole nutmeats, because this variety represents the major portion of the California almond crop and because this variety seems most likely to have mold damage due to its extra soft, thin shell. With sliced and diced nutmeats it was not possible to limit samples to varieties, since they are usually prepared from mixed varieties. The nut samples were obtained from six almond processors of large and medium size, who together handle most of the California almond crop. The unsorted in-shell nuts and the in-process nutmeats were obtained from all six processors, whereas the sliced and diced nutmeats were collected from only three processors. The geographical origin of each in-shell sample was recorded as it was collected. Altogether 223 samples of in-shell nuts and nutmeats were assayed. Sample handling and preparation. Samples were collected in plastic bags and stored at 32 to 34 F (0 to 1 C) as soon as received at the laboratory to arrest any aflatoxin formation. Storage at low temperature was probably not critical, because all samples received were dry enough (e.g., below 7% moisture) to be fairly safe from mold and aflatoxin development, at least over short periods of storage. The samples were removed from cold storage 1 to several days before being prepared for assay to allow them to reach room temperature and thereby avoid condensation on the nuts. In-shell samples were sorted by hand to remove foreign materials, i.e., loose hulls, loosely attached hulls, rocks, sticks, etc. Removal of the variable amounts of foreign materials made the samples more comparable, was necessary to protect the blades of the equipment used for sample preparation, and avoided interference in the analysis by certain constituents of the hulls. In all but a few cases, there was sufficient sample available to permit 18.0 lb (ca. 8.2 kg) of in-shell nuts or 15.0 lb (ca. 6.8 kg) of nutmeats to be cut and blended in a Hobart vertical cutter-mixer (VCM) (25-qt [about 22.3 liters] VCM). A fine, homogeneous meal was prepared with the VCM by intermittent cutting (e.g., 15 s at a time) at slow speed for 1 min and then at high speed for another 1 or 1.5 min. Allowing the samples to cool between the periodic cuttings avoided problems of overheating, oiling-out, and compacting of the product. In a few cases, it was necessary to use Celite Hyflo Supercel (Johns Mansville) as a cutting aid to attain a finely cut, free-flowing meal. Using a sharp wave-cut blade of the type available for the Hobart VCM contributed to the preparation of a fine nut meal without aid of the diatomaceous earth. Analysis. Aflatoxins B,, B,, G,, and G, were determined by a procedure very similar to Method I for aflatoxins in peanuts given in the Official Methods of Analysis of the AOAC (2). Method I has been found by the FDA and ourselves to be the most reliable method for tree nuts of the several official aflatoxin methods. The analysis was carried out under gold fluorescent lighting to avoid possible loss of the ultraviolet-sensitive aflatoxins. A weighed portion of the finely cut samples of nutmeats (50.0 g) or in-shell nuts (75.0 g, which is equivalent to about 50.0 g of nutmeats) was mixed with 15 g of Celite Hyflo Supercel, 25 ml of water, and 250 ml of chloroform in a 500-ml, glass-stoppered (g.s.) Erlenmeyer flask. If cutting aid was present in the sample, the sample size was increased proportionately. After shaking the sealed flask and its contents on a Burrell wrist-action shaker for 30 min, the extract was filtered rapidly through a Buchner funnel containing filter paper (Schleicher and Schuell no. 595) coated with 10 g of Celite Hyflo Supercel. A 50-ml sample of the filtrate was chromatographed on a silica gel column, and the eluate was analyzed by thin-layer chromatography according to the AOAC procedure. The aflatoxins were estimated quantitatively against aflatoxin standards obtained from the Southern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, La. Analysis of subsamples. The method of analyzing the 50 g of subsamples used to study aflatoxin distribution differed from the usual method slightly in sample preparation and extraction. Thus, 50 g of the sample (whole nutmeats or diced nutmeats) was used as received. The sample together with 15 g of Celite Hyflo Supercel was ground in a Waring blender (1-qt jar [about 0.946 liters]) with 250 ml of chloroform for 1 min. The mixture was transferred to a 500-ml g.s. flask, 25 ml of distilled water was added, and the 49 VOL. 29, 1975 extraction was continued on a wrist-action shaker for 30 min. The extract was filtered and analyzed as described in the method used for the survey. RESULTS AND DISCUSSION Unsorted in-shell nuts. The survey of incoming in-shell nuts was expected to give some idea of the incidence and level of aflatoxin in Nonpareil almonds as received at the processing plant. Although the overall concentration of aflatoxin in these nuts appears to have been fairly low, aflatoxin was by no means a rare contaminant. About 14% of the samples (10 of 74) were contaminated with aflatoxin at 1 ppb or more. The amounts of aflatoxin found in the positive samples are shown in Table 1. It should be noted that the concentrations in this table are expressed on a total weight basis (i.e., kernel plus shell). Whereas only one sample of in-shell nuts exceeded the present guideline of 20 ppb (edible portion), four other samples contained 5 ppb or more of aflatoxin. Since aflatoxin incidence in in-shell almonds might be related to orchard location, the geographical origin of each contaminated sample was examined. The 10 almond samples containing aflatoxin originated in all sections (i.e., northern, central, and southern sections) of the growing area in the Central Valley of California. Although a more extensive study might show some correlation of aflatoxin incidence to climatic conditions or cultural practices within the almond-growing area of California, it is evident that the aflatoxin problem is not restricted to any one district of California. Estimation of proportion of contaminated nuts. It must be emphasized that, although the proportion of samples found contaminated was substantial (14%), the proportion of individual nuts contaminated was probably exceedingly small, assuming aflatoxin incidence in almonds is similar to that found in peanuts (5,10). The proportion of contaminated nuts can be estimated by statistical analysis. The probability of a contaminated sample can be estimated by the Poisson distribution: where: k = number of nuts in the sample and p = proportion of contaminated nuts. A point estimate of the proportion of contaminated nuts (p) can be obtained by equating this function (1) to the proportion of positive samples (x) actually found in n samples. x/n = 1e -kp solving for p: Since (1) defines the probability of one of two possible outcomes, it is appropriate to use the standard formulas for binomial confidence intervals on this function. The resulting upper and lower limits are each solved for p. The average proportion of contaminated nuts (p) and its 95% confidence intervals (upper and lower limits) can be estimated for the unsorted in-shell nuts, if it is assumed that all samples had 240 in-shell nuts/lb (or per 453.6 g). Thus, it is estimated that the average proportion of nuts contaminated with aflatoxin for the unsorted in-shell nuts is 3.78 x 10-s and that the lower and upper 95% confidence limits are 1.8 x 10-s and 6.7 x 10-5, respectively. The reciprocal of the proportion of contaminated nuts, which gives the number of good nuts per contaminated nut, provides a better visual picture of the small porportion of nuts that are contaminated. Thus, it is estimated that on the average only one nut in 26,500 nuts (about one nut in 110 lb [about 49.9 kg] of in-shell almonds) is contaminated; the 95% confidence interval is one nut per 55,300 nuts to one nut per 14,700 nuts. In-process nuts. The survey of nutmeats taken at various stages of commercial sorting and grading shows that normal sorting procedures, which are based on physical appearance, reduce the incidence and levels of aflatoxin in almonds. The results show that manual sorting tends to remove the aflatoxin-contaminated kernels from the sorted (select) kernels, resulting in their concentration in the rejected kernels ( Table 2). None of the 26 samples of sorted kernels showed any sign of aflatoxin, whereas 10 of the 16 samples of hand-rejected kernels (oil stock) were contaminated. Furthermore, nine of the 10 positive samples of manually rejected kernels contained aflatoxin in excess of the guideline level of 20 ppb. The presence of measurable amounts of aflatoxin in only two of the 16 samples (12%) of the unsorted kernels indicates an incidence of aflatoxin that is in fair agreement with that found in the unsorted, in-shell nuts from the growers (14%). In fact, by statistical analysis similar to that used for in-shell nuts, assuming 400 kernels/lb, the average proportion (p) of contaminated kernels in the unsorted kernels is estimated to be roughly the same as that in the incoming in-shell nuts (2.23 x 10-5 versus 3.78 X . Hand sorting appears to be more effective than electronic sorting in removing aflatoxincontaminated kernels. Not only was the number of contaminated samples highest in the handsorted rejects, but the average aflatoxin concentration was also highest in these rejects. The difference in aflatoxin incidence in hand-sorted rejects and electronically sorted rejects could result from the fact that the kernels rejected by electronic sorting contain a high proportion of broken and sheller-damaged kernels, which dilute the aflatoxin-contaminated kernels that are associated with the "seriously damaged" (moldy and insect-damaged) kernels. Too few samples were analyzed to prove statistically that electronic sorting tends to remove aflatoxin-contaminated nuts. Since there is a tolerance for seriously damaged kernels in the United States standards for grades of shelled almonds, some samples of finished product eventually may be contaminated with aflatoxin. The higher the tolerance for seriously damaged kernels, the more likely this will occur. There is little chance of the most seriously damaged kernels being in the finished product, but the present study did not attempt to relate type or degree of defect with aflatoxin. On the basis of the data obtained, it appears that one does not have a very good chance of finding a positive (contaminated) sample in sorted whole nutmeats unless one uses a sample much larger than 15 lb (6.8 kg). For example, if the sorted nutmeats contain 1% of the seriously damaged kernels that are equivalent to those rejected by hand sorting, the probability of finding one or more contaminated samples in the 26 samples of sorted nutmeats used in this study is only 0.225. That is, on the basis of the data (i.e., p = 1.63 x 10-4 for hand-rejected kernels; 1% tolerance for seriously damaged kernels) it can be estimated that 77.5% of the time one can examine 26 samples of 15 lb (6.8 kg) each without finding any contamination. Processed (finished product) samples. The survey of processed nutmeats was made to determine the frequency and amount of aflatoxin contamination in various finished products available to the consumer. The products sampled for this part of the study were from the following basic categories: whole almonds, sliced almonds, and diced almonds. Aflatoxin occurs to a much greater extent in diced almonds than in sliced or whole almonds (Table 3). Thus, whereas none of the 26 samples of whole nutmeats and only one of the 25 samples of sliced nutmeats were contaminated, 13 of the 27 samples (48%) of the diced nutmeats contained aflatoxin. However, only four of these 13 contaminated, diced samples were above the existing FDA guideline of 20 ppb. Although blanching may tend to lower aflatoxin concentration, this factor was not considered in the analysis of the data because of the small number of samples involved. A higher tolerance for seriously damaged kernels used for dicing than for any USDA grade of whole or broken almonds undoubtedly contributes to this finding. To exemplify the situation, a statistical estimate can be made of the proportion of contaminated kernels (whole) in the diced nuts on the basis of the proportion of contaminated samples that were found. Thus, if it is assumed that all diced nut samples were contaminated at the same level and that there were 400 kernels/lb in the raw material from which they were prepared, it can be estimated that the average proportion of contaminated kernels in the diced nuts is 1.09 x 10-' with 95% confidence limits of 5.6 x 10-1 and 1.9 x 10-g. That is, on the average there was one contaminated kernel in every 9,200 kernels used for dicing, and the limits for 95% confidence were a lower limit of 1 nut/17,900 nuts and an upper limit of 1 nut/5,300 nuts. Since no samples of sorted, whole kernels were contaminated, it is not possible to make a similar statistical estimate for whole almonds. However, a statistical estimate of the upper limit for a 95% confidence interval of the average proportion of contaminated kernels can be made for whole almonds, and it is 1.9 x 10-I or no more than 1 nut/52,600 nuts. Since the size of the sample required to include a contaminated kernel is inversely related to the proportion of kernels contaminated, it is evident that much larger samples must be used to survey aflatoxin in whole nutmeats than in diced nutmeats. Distribution study. The most important factor influencing incidence and sample size requirements is distribution (i.e., degree of nonuniformity) of the contaminant in the product. Aflatoxin distribution undoubtedly varies among the different product categories. Thus, distribution is likely to improve as particle size of the product is reduced, so that aflatoxin should be distributed more uniformly in diced nutmeats than in whole nutmeats. Therefore, in several cases the unground materials remaining from the above surveys of aflatoxin were subsampled to examine aflatoxin distribution in whole and diced nutmeats. The size requirements for representative sampling can be estimated on the basis of such distribution studies; however, it was not possible to make a reliable estimate from this study, because it was limited by the amount and nature of the material remaining from the surveys. Nevertheless, this cursory study did demonstrate a vast difference between aflatoxin distribution in whole nutmeats and that in diced nutmeats. For example, the whole nutmeats remaining from two in-process samples found to contain aflatoxin (28 and 47 ppb) were subsampled for distribution analysis. In each case none of the five subsamples (50 g each) contained aflatoxin. The remaining unground, diced nutmeats of three positive samples (5,75, and 119 ppb) were similarity analyzed, with three, five, and seven subsamples, respectively. In contrast to the results with whole nutmeats, all subsamples of the diced nutmeats contained aflatoxin, although great variation in amount did exist (Table 4). Aflatoxin varied from a trace to a level higher than that found in the 15-lb (6.8-kg) analytical sample. Furthermore, the relative amounts of the four aflatoxins (B1, B2, G,, G2) differed among the subsamples. Nevertheless, aflatoxin was distributed more uniformly in diced nutmeats than in whole nutmeats. On the basis of the distribution study, it is evident that the likelihood of finding a contaminated sample in a given lot is much more dependent on aflatoxin distribution than on the proportion of contaminated kernels, which is likely related to the tolerance for damaged nuts. In the survey, the higher proportion of contaminated samples found with diced nutmeats than with whole nutmeats was due primarily to more uniform distribution of aflatoxin in the small nut pieces and only secondarily to the presence of a higher proportion of contaminated kernels (i.e., from the higher tolerance for damaged nuts). Because of the difference between aflatoxin distribution in diced nutmeats and in whole nutmeats, much smaller samples of diced nutmeats than whole nutmeats should be re- quired to monitor aflatoxin. Whereas 15 lb (6.8 kg) may be an adequate size for monitoring aflatoxin in diced nutmeats, it is estimated that a sample of 10 to 100 times this size would be necessary for equal assurance of properly evaluating a lot of whole nutmeats. Further study is necessary before any precise estimate can be made of sampling requirements for monitoring aflatoxin in almonds.

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2020-12-10T09:04:12.729Z

1975-11-01T00:00:00.000Z

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Interaction Between Aflatoxicosis and a Natural Infection of Chickens with Salmonella Broiler chicks with a natural congenital infection of Salmonella worthington required a lower concentration of dietary aflatoxin (0.625 μg/g) to depress growth than uninfected chicks (2.50 μg/g). The possible interaction of aflatoxicosis with infectious diseases has been a question since the discovery of aflatoxin. One of the first reports of aflatoxicosis (14) described the isolation of Salmonella from the internal organs of turkeys that had the disease. Brown and Abrams (4) consistently isolated Salmonella from ducklings and chickens with typical aflatoxicosis. They also discovered a hypoproteinemia, which included the globulin fractions, and proposed that birds were more susceptible to Salmonella during aflatoxicosis. Abrams (1) extended this hypothesis to other bacterial and viral diseases. Experiments designed to prove this hypothesis have yielded contradictory results. Aflatoxicosis and infection with Salmonella gallinarum exerted their effects on body weight and mortality independently and without interaction (16); however, an interaction on body weights and crop weights was found in chickens consuming aflatoxin and infected with Candida albicans (6). Pier et al. (10) reported that aflatoxin did not impair acquired resistance to Newcastle Disease virus, and Adinarayanaiah et al. (2) reported that aflatoxin had no effect on antibody formation against S. pullorum antigen. Richard et al. (12) found no interaction of aflatoxin and Aspergillus fumigatus on mortality, histopathologic lesions, or growth rate in turkey poults despite the two factors together causing the formation of precipitating antibodies against A. fumigatus. In an interesting study Pier and Heddleston (9) found that aflatoxin consumed by turkey poults and young chickens during or after immunization with Pasteurella multocida interfered with the development of resistance to a lethal challenge with the organism. These contradictory results are even more inexplicable in view of the findings that dietary aflatoxin can cause a dose-related inhibition of the reticuloendothelial system (7) and immunosuppression in chickens (17). Thus, natural aflatoxicosis has been associated with the occurrence of Salmonella in birds, whereas laboratory experiments have generally failed to find interactions between aflatoxicosis and infectious agents. One possible explanation for these contradictory results is that the conditions used in the laboratory experiments were too far removed from field conditions. We want to report a laboratory experiment arising from a field case which demonstrates an interaction between aflatoxicosis and salmonellosis. The opportunity to investigate possible interactions arose when an unusual field case was referred to us. It was characterized by a slightly increased mortality rate and greatly depressed growth rate in certain flocks of young broiler chickens. Analysis (11) of feed (broiler starter mash) samples from the affected houses revealed aflatoxin B1 in quantities ranging from trace amounts to 0.600 Ag/g. These observations suggested aflatoxicosis, since these symptoms are associated with the disease (15). Further inquiry revealed that the affected birds came from a single flock of breeder hens. Cloacal swabs from 15 affected birds selected at random were incubated in tetrathionate broth (BBL) for 24 h at 37 C. Then a loopful of the broth was streaked on brilliant green agar (BBL), which was observed for typical Salmonella colonies after incubation. The affected chicks were uniformly infected with S. worthington (identified by the Center for Disease Control, Atlanta, Ga.). The affected birds also had a slight diarrhea and dehydration, which are the primary symptoms usually associated with uncomplicated paratyphoid infections in chickens (3). Before the breeder flock was discarded, we were able to acquire some chicks 870 from it and from an uninfected flock of the same age and genetic stock (Indian River x Indian River) on a nearby farm. The infected and uninfected chicks were brought to the laboratory and divided into groups of 10. They were housed in electrically heated batteries with feed and water available ad libitum. The feed was a commercial broiler. starter ration to which graded amounts of aflatoxin (0, 0.625, 1.25, 2.5, 5.0, and 10.0 ,ug/g of feed) were added. The aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on rice by the method of Shotwell et al. (13). The moldy rice was steamed, dried, and ground to a fine powder, which was analyzed for total aflatoxin content by the spectrophotometric method of Nabney and Nesbitt (8) with the modification ofWiseman et al. (19). The percentages of aflatoxin B1, B2, G1, and G2 (70, 9, 16, and 5%, respectively) were determined spectrophotometrically (8) after separation on thinlayer chromatograms (11). There were four groups of 10 infected birds and four groups of 10 uninfected birds per aflatoxin level, and the experimental design was completely randomized. The birds were fed for 3 weeks and their body weights were determined. The data were submitted to an analysis of variance, and the least significant difference between treatment means was determined (5). The statistical analyses showed that both aflatoxin and a natural infection with S. worthington had a significant (P < 0.05) effect on body weight and that there was a significant interaction between them ( Table 1). The interaction was most clearly revealed in the threshold dose of aflatoxin required to exert an effect on body weight. In the uninfected birds 2.5 ,ug/g or greater was required to depress growth rate. (This same threshold dose in uncompromised broiler chicks has been obtained without exception in over 100 different experiments in our laboratory.) In infected birds even the smallest dose (0.625 ,g/g) caused a significant inhibition of growth. The mortality records revealed that there was no similar interaction on mortality. The observations in this experiment and field case indicate that there can be an interaction between aflatoxicosis and an infection with S. worthington on body weight. In addition, we observed a similar interaction in two other instances in which the commercially obtained chicks were not known to be infected with Salmonella until the laboratory experiments were ended; unfortunately, the epidemiological efforts in the field failed because the feed had been consumed and because the placement of the chicks could not be accurately traced. These observations suggest that the failure of many investigators to find an interaction between aflatoxicosis and infectious diseases may be the result of their failure to create the necessary but as yet unknown conditions in the laboratory. These observations also suggest that interactions exist under field conditions and that the symptoms observed may not necessarily be those expected. This consideration is reinforced by reports that birds with aflatoxicosis (6) and salmonellosis (18) are more susceptible to below normal temperatures, such as frequently occur under field conditions. Together, the interactions make for a difficult epidemiology and point out some of the insidious aspects of mycotoxicoses confronting the animal industries.

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2020-12-10T09:04:20.880Z

1975-09-01T00:00:00.000Z

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Effects of an Abrupt Change in Ration from All Roughage to High Concentrate upon Rumen Microbial Numbers in Sheep When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values. Previous reports have shown that the type and level of ration consumed by an animal affect the numbers and types of bacteria and protozoa in the rumen (1,4,15,17,22,23). When the animal is changed from one ration to another, a period of microbial adaptation occurs, which can be defined as that time interval required for the rumen microbial population to stabilize. However, very little information is available on the absolute microbial changes that occur during this adaptation period (9,18,23). Several investigators have estimated the length of this adaptation period by measuring digestibility data as a relative indicator of rumen microbial activity (10,16,19). In general, results of these studies have indicated that the length of time required for adaptation depends upon how radical a change is made in the ration. A recent study conducted in this laboratory with sheep, using daily dry-matter and cellulose digestibility as criteria, investigated the length of the adaptation period when animals were changed from an all-roughage ration to either a corn silage ration, a 60% cracked corn-40% orchardgrass hay ration, or a chopped alfalfa hay ration and back to the roughage ration (20). The most marked changes in daily digestibility coefficients occurred when the sheep were placed on the 60% cracked corn-40% orchardgrass hay ration. However, in almost all cases the digestibility coefficients for all rations showed little if any further change after 5 days. On the basis of these results, the present study was initiated to investigate the changes in bacterial and protozoan concentrations that occur in the rumen of sheep during this abrupt change from a ration of all orchardgrass hay to one containing 60% cracked corn-40% orchardgrass hay. In addition, concentrations of specific types of rumen bacteria, i.e., cellulose digesters and total volatile fatty acids (VFA), were also measured. Although the sheep were fed at approximately equal dry-matter intakes, the markedly different nature of the rations could affect both rumen volume and rate of passage. Thus, measurement of liquid and drymatter turnover rates and rumen volume was 404 included as a possible help in interpreting any observed microbial changes. MATERIALS AND METHODS Animals. Three cross-bred wethers, weighing approximately 45 kg, were fed a ration of 800 g of chopped orchardgrass hay per day and then abruptly changed to a ration containing 800 g of 60% cracked corn and 40% chopped orchardgrass hay. The animals were fed once daily at 9:00 a.m. and had free access to water. All three sheep were surgically prepared with rumen fistulae, and composite samples of rumen contents for counting the microbial concentrations were collected from various locations within the rumen just before the daily feeding. Nine ewes, four fed orchardgrass hay and five fed 60% cracked corn-40% orchardgrass hay, were used to estimate dry-matter turnover. The rations were fed at least 3 weeks before slaughter. Rumen microbiology: general. The anaerobic culture techniques were similar to those described by Hungate (11). Methods of preparation of media and dilution of rumen ingesta have been described by Dehority (6). Total viable bacterial numbers were determined in roll tubes with 40%.rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA), similar to that described by Bryant and Burkey (3). Total substrate concentration in the RGCSA medium for experiments 1 and 2 was 0.3% carbohydrate, which was divided into 0.125% (wt/vol) each glucose and cellobiose and 0.05% (wt/vol) soluble starch. For experiment 3, a total substrate concentration of 0.1% carbohydrate was used, 0.025% (wt/vol) each glucose and cellobiose plus 0.05% (wt/vol) soluble starch. Colonies were counted with a binocular dissecting microscope after 7 days of incubation at 38 C. Bacterial counts were also determined with differential media, in which either 0.3% xylan, 0.3% pectin, 0.3% soluble starch, or 0.75% ball-milled cellulose replaced the glucose, cellobiose, and starch added to the basal medium. Methods used to solubilize the xylan and pectin were those described by Dehority (6,7). Total and molar percentages of rumen VFA were determined in all samples from sheep 2 and 3 by gas chromatography (8). Protozoan numbers per milliliter and percent generic distribution were determined according to the procedure of Purser and Moir (21). Experimental procedures. Before the start of the experimental periods, all sheep were fed the chopped orchardgrass hay for 6 weeks. Sheep 1 was sampled intermittently over a 29-day period from days designated as t_ to tL21. From tL to to the sheep was fed chopped orchardgrass hay. After a sample was taken on day t0, the animal was changed to the concentrate ration for the remainder of the experiment. With sheep 2, samples were only taken over a 22-day period from t-to t14. Sheep 3 was sampled over the 28-day period from t7 to t21. Other than the cellulose medium, the only differential counts attempted with sheep 3 were with a 1% soluble starch medium. The starch roll tubes were incubated at 38 C for 24 h, and after the colonies present were counted the rubber stoppers were carefully removed and the tubes were filled with Lugol iodine, which had been diluted 1:8 with distilled water. Within 10 to 15 min the agar medium turned a dark brown to purple color with resultant clear zones appearing where starch colonies had grown. Although considerably more colonies appeared with incubation up to 7 days, after 24 h the clear zones overlapped so much that accurate counts could not be made. Rumen volume, fluid, and dry-matter turnover rates. Rumen volume and fluid turnover rate were measured by using polyethylene glycol as a marker. Analytical procedures were similar to those of Hyden (14). Preliminary experiments, following a sampling schedule similar to that proposed by Hyden (14), gave abnormally high estimates of rumen volume. Further studies indicated that for animals fed once daily the magnitude of dilution effects upon concentration of a soluble marker was quite large, giving a very steep slope in the first few hours and a corresponding large rumen volume when the line was extrapolated back to zero time. This dilution effect has also been experimentally demonstrated by Warner and Stacy (24). In the present work, polyethylene glycol was added 1 h before feeding, and sambles for analysis were taken 1 and 24 h later. Dry-matter turnover in the ewes was determined at slaughter according to the procedures described by Hungate (12). Acid-detergent lignin (26) was used as a marker to estimate digestibility. RESULTS Bacterial colony counts. Anaerobic bacterial colony counts per gram of rumen contents for the three sheep during the period when they were abruptly changed from orchardgrass hay to the 60% corn-40% orchardgrass hay ration (day 0) are presented in Fig. 1 and Table 1. Rather marked differences in bacterial concentrations between the three sheep can be seen in Fig. 1. Bacterial concentrations were much lower in sheep 1, and aside from a slight drop on days 1 and 2 after the ration change concentrations gradually increased through day 21. In sheep 2, bacterial concentrations began to increase immediately after the ration change and increased at a much greater rate than for sheep 1. No values were obtained for day 21 in this animal, since it was inadvertently sheared on day 20 and went off feed. Concentrations also began to increase immediately after changing rations for sheep 3; however, a tremendously high peak occurred on day 5, followed by a decrease on days 7 and 14, with an indication of a gradual increase between days 14 and 21. Although the peak observed on day 5 could be the result of a sampling error, concentrations of starch-and cellulose-digesting bacteria (to be presented later) and protozoa all increased in a similar fashion. In the study reported by Potter gestibilities of these same rations were estimated over the same period, most of the change occurred between days 1 and 5. Values appeared to be relatively stable from days 6 through 21. On this basis, means of the bacterial concentrations were calculated for those days before the ration change (to to to), the socalled transition period (t1 to t5) and from t7 on. These data are shown in Table 1, and in all cases concentrations from t7 on were significantly higher (P < 0.01) than those on the orchardgrass ration (t8 to to). In sheep 2 and 3, where days t, to t5 were a period of rapid increase, mean values were not different from either of the other two means. In sheep 1, where most of the increase occurred after day 5, the transition period mean did not differ from the value on orchardgrass hay. Although there were considerable differences between the three sheep, bacterial concentrations increased in all animals after the ration change. Digestibility data (20) had suggested that the transition or change was complete within 5 days; however, this would be in contrast to the present data, where concentrations continued to increase through 21 days and may not even then have reached a plateau. Selective medium colony counts. In addition to total colony counts for sheep 1 and 2, an attempt was made to estimate the concentra-tions of bacteria capable of fermenting starch, xylan, pectin, and cellulose. A selective medium was used in which the substrate under study was the only added energy source. With either xylan, pectin, or starch as substrate, 7-day colony counts were made and results were expressed as a percentage of the colony count obtained with RGCSA medium. For the five samples from sheep 1 while on orchardgrass hay, values for the xylan medium ranged from 70 to 113%; for pectin, 70 to 106%; and for starch, 71 to 136%. Similar results were obtained at the other sampling times and also for sheep 2. The probable explanation for this discrepancy is that the 40% rumen fluid basal medium contains enough energy sources to support growth of considerable numbers of colonies (5). Using the procedure of staining with Lugol iodine, described earlier, the concentration of starch-digesting colonies at 24 h was estimated for sheep 3. The actual number of starch-digesting colonies appearing within 24 h was probably far less than would be obtained at 7 days and represented the faster-growing species. However, the numbers of starch-digesting colonies obtained on days t-5, tU3, t-1, and to were 13, 10, 10, and 18%, respectively, of the colonies observed with RGCSA medium. For days t1, t2, and t3, the percentages rose to 25, 40, and 36%, respectively. Too many colonies were present to count on days ti, and a value of 47% was obtained for the sample taken on day t7. by t14 and t2,, percentages had fallen to 9 and 16M, respectively. These data suggest a temporary marked increase in the starch-digesting species, which grow rapidly enough in artificial media to show colonies in 24 h, during the first week after the change to the high-concentrate ration. The concentration of cellulose-digesting bacteria can be estimated fairly specifically by counting the clear zones in cellulose-agar roll tubes after incubation for 28 days. Cellulolytic colony counts for the three sheep, over this period of ration change, are shown in Fig. 2. The most obvious difference between animals is that the concentration of cellulolytic bacteria in sheep 3 was about 20 times higher than in sheep 1 and 2. For all animals, decreased concentrations were observed immediately after the change in ration. For sheep 1, colony counts decreased up to 5 days and then increased back to nearly the same level as on the orchardgrass hay ration. In contrast, a marked peak was observed on day 5 in sheep 2 and 3, followed by a decrease to a level lower than the colony counts on all roughage. When mean values on all roughage were compared with mean values for day t7 on, the difference was significant only for sheep 3 (P < 0.01). Protozoan concentrations. The concentrations of rumen protozoa determined over the same experimental period are presented in Fig. 3 and Table 2. In general, concentrations were similar in all sheep on the roughage ration, and, as can be noted in Fig. 3, the response to changing from all roughage to concentrate was consistent among the three animals. Concentrations began to increase on day 1 and appeared to reach a plateau about day 5. These data were grouped and the means were calculated in the same manner as for bacterial colony counts ( Table 2). For all sheep, a significant increase in protozoan concentration was found between the all-roughage ration and from day 7 on after the change to concentrate. These data agree with the pattern of increased cellulose and drymatter digestibility reported by Potter and Dehority (20). With respect to percent generic composition of the protozoa, a significant decrease in Entodinium (94.5 to 88.9%) and increase in Diplodinium (1.6 to 5.7%) (P < 0.05) were observed in sheep 1. No changes were found with sheep 2, and the percentage of Entodinium increased significantly (P < 0.05) with sheep 3 (92.4 to 95.1%). These data do not suggest any obvious relationships between ration and percent generic composition. VFA. For sheep 2, a significant increase in total VFA (P < 0.05) was observed when the animal was changed to the concentrate ration; mean millimolar values were 158.5 for days t-8 to to compared with 234.4 for days t1 to t14. A significant decrease (P < 0.05) in the molar percentage of acetate and an increase in propionate also occurred. Although not significant, the molar percentage of n-butyrate showed an increase in the concentrate ration. Changes in total VFA and molar percentages for sheep 3 were almost identical to those for sheep 2; however, in this instance only the decrease in molar percentage of acetate was significant at P < 0.05. All other changes were significant at P < 0.1. No particular trends in either total VFA or proportions with time after the ration change were apparent. These results agree with the VFA changes reported by Latham et al. (15) when animals are changed to a concentrate ration. Rumen volume and fluid turnover rate. Using polyethylene glycol as a soluble marker, rumen volume and fluid turnover rates were estimated for the three sheep on both rations (Table 3). These determinations were made after completing the work on microbial changes, and at least 3 weeks were allowed for adjustment of the animal to the ration. A significant decrease (P < 0.05) was observed in ru-men volume for two of the three sheep; however, rather unexpectedly, fluid turnover rate was not significantly affected by ration. Table 4 presents total bacterial colony and protozoan counts based on rumen volume. Values for the transition period (days t1 to t5) have been omitted, since no estimates of rumen volume were made for that period. These data differ from those in Tables 1 and 2 in two respects: total bacterial numbers in sheep 1 were not significantly affected by the change in ration when rumen volume was considered, and there was a striking similarity between total bacterial and protozoan numbers in sheep 2 and 3. Possible reasons for the failure of total rumen bacterial numbers to increase in sheep 1 in response to an increased energy intake are not immediately obvious. As can be noted in Fig. 1, bacterial concentrations were still increasing on day 21, and possibly if this animal had been sampled at a later date higher concentrations would have been found. On the other hand, protozoan concentrations did increase, but the increase was less than in the other two sheep, and values were fairly stable from days 7 through 21. These data suggest that, for studies comparing rumen microbial numbers, colony counts and protozoan numbers based on unit volume Dry-matter turnover rate. Since both the roughage and concentrate rations were fed at similar dry-matter intakes, it seemed possible that this might explain equal fluid turnover rates. However, because of the readily fermentable nature of the concentrate ration, it would as reported in Tables 1 and 2 can be misleading and more meaningful values can be obtained by using values based on total volume. Also, if marked differences occurred in fluid turnover rates between animals or rations or both, some adjustment of values would be required. 18 Mean and standard error of the mean. d For each parameter, means within a column followed by different superscripts are significantly different at P < 0.05. Tables 1 and 2 multiplied by rumen volume, Table 3. Mean and standard error of the mean are presented. AddFor each parameter, means within a column followed by different superscripts are significantly cantly different at P < 0.01; b, d means are significantly different at P < 0.02. appear that this ration should have a faster rate of passage. Dry-matter turnover was estimated in four ewes fed orchardgrass hay and five ewes fed 60% cracked corn-40% orchardgrass hay (Table 6). Dry-matter digestibility, as estimated from lignin ratios, was significantly higher (P < 0,01) in the concentrate-fed animals; however, turnover of the dry matter was slower. These data agree with the concept proposed by Hungate (12), that turnover rates based on indigestible feed components will be inversely related to digestibility when feed intake is held constant. Hungate further suggests that with such concentrate rations the fluid turnover rate is probably a better index of rumen function. Although of considerable interest, these values for dry-matter turnover in the rumen do not appear to offer information that can be used to better estimate total daily microbial production. DISCUSSION The variability between the three sheep used in these experiments agrees with the observations of Warner and Stacy (24) concerning animal differences; however, most of the responses have been noted in various studies by other workers. For example, the increase in bacterial colony counts in sheep 2 and 3 in response to an increased intake of available energy substantiates the results of Bryant and Burkey (4) and Maki and Foster (17). In contrast, Latham et al. (15) obtained results similar to those observed with sheep 1, in which bacterial concentrations did not increase when available energy intake increased. On the other hand, protozoan concentrations increased in all sheep when the available energy intake was raised, agreeing with previously reported results (1,18,23). Warner (23) has reported the changes in ru-men microbial concentrations of a single sheep when changed from a roughage to a concentrate ration at a fixed level of intake. He observed that about 10 days was required for microbial changes to occur, and concentrations were relatively stable after that time. These findings differ from the present results, in which bacterial concentrations appeared to still be increasing after 14 to 21 days and protozoan concentrations had stabilized after 5 days. When the present observations are considered with respect to the results of Potter and Dehority (20), where digestibility of these two rations had stabilized after 5 days, it would appear that the continuing changes observed in viable bacterial concentrations are not of major significance to overall digestion in the animal. Maki and Foster (17) have reported that viable counts of bacteria in the rumen contents of cows fed a high-roughage ration represented only 3 to 12% of the number determined by direct count, whereas 57 to 73% of the bacteria from cows fed a ration without roughage (grain plus alfalfa meal) could be cultured. A similar increase in the proportion of viable to direct bacterial count was noted in rumen contents from an all-concentrate-fed animal by Bryant and Burkey (4); however, they also observed that the percentage value of viable counts was similar to that of direct counts, about 8.4%, when animals were fed all roughage or a mixture of roughage and concentrate. Although direct counts were not made in the present study, the concentrate ration contained 40% orchardgrass hay, and based on the results of Bryant and Burkey (4) it appears probable that viable counts on the two rations represented a similar percentage of the total bacteria. A possible reason proposed for the discrepancy between direct and viable counts is that the direct count measures dead cells and those metabolizing cells which cannot be grown in the artificial medium (12). The differences between roughage-and concentrate-fed animals must then be attributed to a lower proportion of these two cell types in the concentrate-fed animal, a shift in the population to more organisms that can be grown, or possibly sequestration of the cells in the fibrous material in hay-fed animals that are not readily disrupted by mixing. One additional point to be considered is that in the studies of Bryant and Burkey (4) and Maki and Foster (17), viable colonies were counted after 3 and 4 days, respectively. More recently, Bryant and Robinson (5) have reported that their 3-day counts averaged only 67% of 7-day counts. Rumen contents for their study were obtained from a cow fed a 79%o alfalfa-21% grain ration. A similar increase in colonies has been observed in our laboratory between 5 and 7 days. If those organisms which appear after 3 to 5 days of incubation are specifically associated with roughage digestion, the differences observed between direct and viable counts on roughage-and concentrate-type rations may be less than previously reported. Adjustment of rumen microbial concentrations for rumen volume in sheep 2 and 3 revealed a striking similarity in the microbial protoplasm supported by a fixed energy intake. In sharp contrast, however, were the low concentrations in sheep 1. After adjustment for rumen volume, total bacterial numbers were the same on the two rations. Obviously other factors must be affecting bacterial concentrations in this animal. The decrease in rumen volume for two of the three sheep, when changed to equal dry-matter intake of the concentrate ration, probably reflects differences in salivary flow and digestibility. Any attempt to estimate total rumen microbial production per day must take into account the rate of passage of bacteria and protozoa from the rumen. Several investigators have studied this problem; however, because ofdifferences in methods and rations, rather marked differences in results were obtained (13,25). The primary questions that must be answered are: what is the relationship between fluid flow and microbial passage, and how is this affected by type and amount of ration and frequency of feeding. Since fluid turnover rate did not change between the two rations, we have assumed microbial passage rates to be similar. Further studies may show this assumption to be unwarranted. Although rumen dry-matter turnover of the roughage and concentrate rations was found to differ, no obvious effect on microbial numbers was evident. A recent report by Akin et al. (2) has confirmed the attachment of rumen bacteria to plant tissue, suggesting that rate of passage for certain species may be influenced by quantity of rumen dry matter. In the present study, rumen dry matter decreased by 21% in animals maintained on the concentrate ration. Concentrations of cellulolytic bacteria were observed to decrease in sheep 1 by 22% and in sheep 2 by 28%, indicating a fairly close relationship between dry matter and cellulolytic bacteria. In contrast, cellulolytic bacterial concentrations in sheep 3 were almost 20 times higher on the roughage ration and decreased by about 77% with the ration change. LITERATURE CITED

v3-fos

2020-12-10T09:04:20.714Z

1975-03-01T00:00:00.000Z

237231519

s2

Influence of Carbohydrates on Growth and Sporulation of Clostridium perfringens Type A Growth and sporulation of Clostridium perfringens type A in Duncan and Strong (DS) sporulation medium was investigated. A biphasic growth response was found to be dependent on starch concentration. Maximal levels of heat-resistant spores were formed at a starch concentration of 0.40%. Addition of glucose, maltose, or maltotriose to a sporulating culture resulted in an immediate turbidity increase, indicating that biphasic growth in DS medium may be due to such starch degradation products. Amylose and, to a lesser extent, amylopectin resulted in biphasic growth when each replaced starch in the sporulation medium. A level of heat-resistant spores approximately equal to the control was produced with amylopectin but not amylose as the added carbohydrate. Addition of glucose or maltose to a DS medium without starch at stage II or III of sporulation did not alter the level of heat-resistant spores as compared with the level obtained in DS medium with starch. Omission of starch or glucose or maltose resulted in an approximately 100-fold decrease in the number of heat-resistant spores, although the percentage of sporulation (90%) was unaffected. The role of starch and amylopectin in the formation of heat-resistant spores probably involves the amylolytic production of utilizable short-chain glucose polymers that provides an energy source for the completion of sporulation. In the course of a recent investigation of the influence of pH and temperature on sporulation and enterotoxin production by Clostridium perfringens type A, a biphasic growth pattern was observed. The secondary turbidity increase occurred 11 or 12 h after inoculation of Duncan and Strong (DS) sporulation medium either when the pH was controlled at 7.0 or not externally controlled by addition of NaOH (4). In spite of the renewed growth, no increase in the number of heat-resistant spores after 6 h was noted. Microscopic examination showed that vegetative cells were responsible for the turbidity increase. In conjunction with the release of free spores, which occurred at 7 to 8 h, the net affect was a drastic reduction in the specific activity of intracellular enterotoxin protein. In this report we attempt to elucidate the factors involved in this biphasic growth and their relationship to the sporulation process. MATERIALS AND METHODS General. C. perfringens NCTC 8798 (Hobbs serotype 9) was used throughout. Cultural conditions, measurement of the levels of sporulation, heat-resistant spores, and turbidity were as previously described (4) except that 1 liter of DS sporulation medium was used and the incubation temperature was 40 C. No attempt was made to externally control the pH. Total cell counts were determined by using a Petroff-Hauser counting chamber. Vegetative cell numbers were obtained by multiplying the total cell counts by the percentage of the nonsporulating population. Chemicals. Amylose was purchased from Sigma Chemical Co., maltotriose and amylopectin from Calbiochem, and amylase from Worthington Biochemical Corp. One unit of amylase activity is the amount that liberates from starch 1 mmol of reducing groups, calculated as maltose, per minute at 25 C. Starch-iodine reaction. The levels of starch were determined as follows. A stock iodine solution was composed of 5 g of KI and 0.5 g of I,. Five milliliters of culture was centrifuged at 27,000 x g for 10 min. To 0.5 ml of the supernatant fluid was added 5 ml of the diluted (1:10) stock iodine solution. The optical density (620 nm) of the resulting blue solution was immediately read by using a Perkin-Elmer doublebeam spectrophotometer. RESULTS Growth of NCTC 8798 in DS sporulation medium. When an 11to 13-h fluid thioglycolate culture of NCTC 8798 was inoculated into DS sporulation medium, vegetative cell multiplication occurred for 2 h (Fig. 1). Vegetative cell numbers are actually estimates since the earliest stage of sporulation that could be de-tected by light microscopy was stage II. By 4 h the number of vegetative cells decreased concomitant with the onset of sporulation. The vegetative cell level remained approximately constant for the next 4 h, while most (about 90%) of the population sporulated. After approximately 8 h vegetative cell multiplication resumed, resulting in the characteristic biphasic pattern. An increase in Klett units was not seen until 2 h later because sporangial lysis also occurred at 8 h, as indicated by the initial appearance of spores free of their sporangium. The level of heat-resistant spores increased between 3 and 4 h and usually remained stable for the next several hours. Little decrease in the starch-iodine reaction occurred until the onset of sporulation. The achroic point (point where starch and iodine no longer give a color reaction) was reached in 14 to 15 h. Influence of starch on biphasic growth. The influence of starch concentration on growth of C. perfringens in DS sporulation medium is shown in Fig. 2. The concentration of starch routinely used in the sporulation medium is 0.4%. This formulation will subsequently be referred to as DS control. The plateau at 8 h and subsequent biphasic growth in control DS were TIME (hr) also observed at a starch concentration of 0.3%, though the secondary turbidity increase was somewhat less pronounced. Nevertheless, the dependence of biphasic growth on starch concentration was evident. No biphasic pattern was observed at starch concentrations of 0.1 or 0.2% (not shown). Given such a concentration dependence, it seemed that the observed biphasic growth might be due to products of starch hydrolysis. During the first 2 h of incubation, there was little decrease in optical density of the starch-iodine reaction (Fig. 1). Energy for vegetative cell growth during this period probably came from proteose peptone and yeast extract. After commencement of sporulation (2 to 2.5 h), there was continued decrease in the iodine staining ability until about 15 h, just before the end of diauxic growth (Fig. 1). This suggested that the latter was due to a sudden increase in utilizable carbohydrates resulting from amylolysis. If short-chain carbohydrates produced as a result of starch hydrolysis were responsible for biphasic growth at 10 to 11 h, then addition of maltose, for example, should also result in an immediate turbidity increase. This, in fact, did occur when maltose, glucose, or maltotriose was added at 7 h (Fig. 3a). Similar results were obtained when these carbohydrates were added at 4 h (Fig. 3b). However, subsequent biphasic growth also resulted, probably due to initial utilization of the added carbohydrates and subsequent starch hydrolysis. Further evidence for the involvement of amylase in the biphasic growth pattern is shown in Fig. 4. Addition of amylase to a 7-h culture resulted in an immediate decrease in the starch-iodine color reaction and concomitantly a dramatic turbidity in- germination and subsequent outgrowth) to the renewed vegetative growth characteristic of the secondary turbidity increase. Cleaned NCTC 8798 spores were incubated in the presence of 0.2% maltose. Germination, measured by a decrease in the optical density of the spore suspension, did not occur even if sterile culture filtrate from a 10-h sporulating culture was included. Thus it appears that germination and subsequent outgrowth of the spore population is not responsible for biphasic growth. Influence of amylose and amylopectin on growth and sporulation. Since starch was responsible for biphasic growth in DS medium, we sought to determine which starch fraction, amylose or amylopectin, was the responsible component (Fig. 5). When each fraction replaced starch in the sporulation medium, two distinct results were obtained. Addition of both 0.8 and 1.6% amylose resulted in secondary turbidity increases whose onset was, respectively, 10 and 3 h later than that occurring in control DS. A concentration of 0.4% amylose resulted in a slight but noticeable turbidity increase between 19 and 41 h. Incorporation of 0.30% amylopectin in DS also yielded a slight secondary turbidity increase at 15 h. However, the extent of the latter (60 Klett units) was considerably less than that occurring in the control DS (about 150 Klett units). Addition of greater than 0.30% amylopectin into DS medium was precluded by solubility difficulties. Only a slight turbidity increase occurred between 24 and 36 h, when 0.075% amylopectin replaced starch in DS medium. Although 1.6% amylose used in the above experiments is four times the starch concentration in DS medium, the growth response of C. perfringens NCTC 8798 in the presence of this carbohydrate more nearly mimicked the biphasic response of this organism in control DS than did amylopectin. To determine the function of starch during sporulation in DS, the effect of varying concentrations of soluble starch on sporulation was determined ( (80 to 90%) was quite high regardless of the level of starch. Up to 10' heat-resistant spores per ml was produced even if starch was omitted. However, most spores produced under these conditions were not fully refractile. The results in Table 1 do show that the formation of heatresistant spores was dependent on starch concentration up to 0.4% added starch. Increasing the starch concentration to 0.6% failed to increase the heat-resistant spore level. We attempted to determine whether the effect of starch on sporulation was due to the amylose or amylopectin fraction ( starch. However, the 4-h spore levels were the same. The formation of heat-resistant spores in the presence of amylopectin is, as in the case of starch, concentration dependent. Inclusion of 0.3% amylopectin in DS medium resulted in heat-resistant spore population levels almost equal to that of the controls. Percentage of sporulation was again quite high regardless of the added carbohydrates. Considering the 4-h level (80 to 90%) of sporulating cells in DS without starch, and considering that these were only at stage II or III (Table 3) of sporulation, the influence of starch, amylose, or amylopectin on the early sporulation process in this medium was minimal. An added carbohydrate in this medium became important after sporulating cells had reached stage II or m. This was not an absolute requirement, however, since about 10' spores per ml of culture (between 0.1 to 1% of the control level) was formed without starch, amylose, or amylopectin. At 6 h about 98% of sporulating cells in DS control had proceeded beyond stage m as compared with 72% of sporulating cells in DS without starch (Table 3). Since there was a greater than 2-log difference in the level of heat-resistant spores even at 12 h ( _-, Not determined. the amylolytic production of utilizable carbohydrates that could be used for energy during completion of the sporulation cycle. To test this possibility, 0.01% glucose or 0.01% maltose was added 2.5 h after inoculation of DS medium without starch and replenished at 0.5-h intervals thru 5.5 h (Table 4). A number of heatresistant spores essentially equivalent to the control was produced under these conditions. Since there was no decrease in percentage of sporulation between 3 and 6 h, the added carbohydrates were apparently preferentially utilized by the sporulating population. DISCUSSION When C. perfringens NCTC 8798 is grown in DS sporulation medium, a biphasic growth response is observed. Results reported here indicate that this is due to the production of utilizable carbohydrates resulting from amylolytic reactions. Amylase is known to be produced by bacilli and clostridia at the end of exponential growth (9). The almost negligible decrease in the starch-iodine reaction during the first 2 h when vegetative cell multiplication occurred also implicates the involvement of amylase at the end of exponential growth. The appearance of new energy sources resulting from amylolysis allows the nonsporulating population to renew vegetative cell division. The dependence of heat-resistant spore levels on starch concentration suggests that starch also plays a role in the sporulation process. Data reported here suggest that biphasic growth and sporulation are separate functions of the two starch components amylose and amylopectin. The rates of hydrolysis of these two components probably determine their role in the tw processes. Conceivably, early fission products' of amylose are oligosaccharides that are only slowly hydrolyzed to metabolizable G1, G2, or G, units (glucose, maltose, or maltotriose). Such a mechanism of action of amylase on amylose has been proposed for some time (1). Since the 4-h level of heat-resistant spores in the presence of amylose was equal to the control, early hydrolysis products of amylose may also include a limited amount of G,, G2, or G. units. With regard to amylopectin, such shortchain glucose polymers could be early and major amylolytic products that could provide an energy source for the completion of sporulation. Differences in early hydrolysis products are likely related to differences in molecular structure between amylose and amylopectin. One (amylose) is a straight-chain polymer of glucose units joined a-1,4. The other (amylopectin) is a polymer of glucose units linked a-1,4, but with branch points linked a-1,6. In the case of both carbohydrates, resistant oligomers could be further hydrolyzed to G,, G2, or G. units at a time corresponding to the onset of biphasic growth. Such a two-phase reaction occurs in the case of malt alpha-amylase hydrolysis of amylose and corn starch (5) and by the action of Bacillus subtilis alpha-amylase on amylose and amylopectin (8). In the latter case early fission products of amylose and amylopectin included G2 or G, units. The increased level of heatresistant spores using amylopectin as the carbohydrate source and the similar effects of maltose when added early during the sporulation process indicates that hydrolysis of the amylopectin moiety of starch releases G,, G2, or G, units at about stage III of sporulation of C. perfringens in DS medium. Furthermore, since the replacement of starch with 0.4% amylose resulted in only minor biphasic growth, amylopectin probably also functions in this phenomenon. Attempts to demonstrate the levels of glucose or reducing sugars were only partially successful due to the complex nature of the sporulation medium, which gave irreproducible results, high blank values, or blocked color reactions of such assays. A complicating factor in these experiments is that the starch we used had been commercially solubilized by hydrolysis with dilute acid (soluble starch). This could account for the longer lag before biphasic growth when pure amylose was the substrate. Nevertheless, we conclude that energy for the completion of sporulation is derived mainly from amylopectin but that both amylopectin and amylose contribute to a secondary turbidity increase, which probably results from the slow amylolysis of oligomers produced during early starch hydrolysis. Although little work has been done on detailed action patterns of bacterial amylases, it is known that the modes of attack of plant alphaamylases depend on the source of the enzyme and substrate. Clearly a more detailed account of the action of C. perfringens amylase, including its designation as alpha or beta, await its purification. Both carbohydrates from yeast extract and the utilization of proteose peptone may provide energy for the brief vegetative growth phase in DS medium. Exhaustion of either energy source would result in the end of vegetative growth and signal the onset of sporulation and production of amylase. As mentioned above, the latter would result in G, to G, units for sporulation beyond stage III. Energy for stages I to III could come from amino acid utilization via the Stickland reaction (6) or utilization or organic acid intermediates produced during vegetative growth. The latter mechanism has been reported to occur during sporulation of clostridia (2, 7) and bacilli (3). However, in a recent report (4) we failed to detect an increase in pH, which is characteristic of secondary metabolism of such compounds. The requirements for an exogenous energy source during sporulation by this organism are in sharp contrast to the aerobic bacilli, in which endotropic sporulation can occur. We conclude that the function of starch and amylopectin and, to a lesser extent, amylose is to provide utilizable carbohydrates so that the majority of the sporulating fraction can become heat resistant.

v3-fos

2018-04-03T01:01:27.795Z

1975-02-01T00:00:00.000Z

27681655

s2

Studies on Batch Production of Bacterial Concentrates from Mixed Species Lactic Starters Optimum growth conditions for mixed species starter FDs 0172 at constant pH in skim milk, whey, and tryptone medium were investigated. Growth rate and maximum population were optimal at 30 C. pH values between 5.5 and 7.0 did not influence the growth rate and maximum population obtainable. Lactic acid-producing activity declined rapidly after reaching the end of the exponential growth phase. The bacterial balance was found to be influenced by the growth parameters: media, pH, temperature, and neutralizer. Skim milk or whey medium at 25 C, pH 6.5, and neutralized with 20% (vol/vol) NH4OH kept the bacterial balance almost constant throughout the cultivation. Grown in tryptone medium at constant pH, the changes in bacterial balance and other metabolic activities were striking compared to the other two media tested. The effect of lactate as an inhibitor was found to be complex, changing with the growth conditions. Concentrates made from mixed species starters FDs 0172, FD 0570, CH 0170, CHs 0170, and T 27 were comparable to controls when cultivated at the optimum conditions found and thereafter centrifuged. Aroma production, proteolytic activity, and CO2 production did not change significantly compared to controls when cultivated at optimum conditions in skim milk or whey medium. Optimum growth conditions for mixed species starter FDs 0172 at constant pH in skim milk, whey, and tryptone medium were investigated. Growth rate and maximum population were optimal at 30 C. pH values between 5.5 and 7.0 did not influence the growth rate and maximum population obtainable. Lactic acid-producing activity declined rapidly after reaching the end of the exponential growth phase. The bacterial balance was found to be influenced by the growth parameters: media, pH, temperature, and neutralizer. Skim milk or whey medium at 25 C, pH 6.5, and neutralized with 20% (vol/vol) NHOH kept the bacterial balance almost constant throughout the cultivation. Grown in tryptone medium at constant pH, the changes in bacterial balance and other metabolic activities were striking compared to the other two media tested. The effect of lactate as an inhibitor was found to be complex, changing with the growth conditions. Concentrates made from mixed species starters FDs 0172, FD 0570, CH 0170, CHs 0170, and T 27 were comparable to controls when cultivated at the optimum conditions found and thereafter centrifuged. Aroma production, proteolytic activity, and CO2 production did not change significantly compared to controls when cultivated at optimum conditions in skim milk or whey medium. Interest in frozen lactic starter concentrates has grown in the dairy industry during the last 5 years. A lot of research work has been done on the preparation of these concentrates from starter bacteria used in the dairies. Most of these studies have been carried out on single strain cultures, primarily used in the Anglo-Saxan countries for cheddar cheese production (6,13,17,18). The increase in yield of biomass when neutralization was used was confirmed first by Kosikowski (12) and later by Bergere and Hermier (2). This, together with a centrifugation procedure, has subsequently been the method of choice for the production of concentrated starters. Starters used in the northern European countries for their cheese varieties are chiefly of the mixed species type. The starter used in Sweden for most of our fermented dairy products is of this type and consists of mixtures of mesophilic lactic acid bacteria. Streptococcus cremoris and Streptococcus lactis are the main lactic acid producers. Streptococcus diacetilactis and Leuconostoc citrovorum are responsible for aroma and gas production in the products. There are at least two possible ways to make concentrates of mixed species starters. One way would be to make concentrates of the isolated strains that compose the mixed strain starter by methods outlined in the literature (3) and mix these concentrates afterwards to a mixed type starter concentrate. Another way would be to make a concentrate directly from the mixed species starter by mass cultivation (constant pH) and centrifugal concentration (20). The first method, although most attractive at first glance, would have some problems and disadvantages compared to the second method outlined. Sorting out single strains and species, isolated either from commercial mixed species starters or from other sources, which will grow in symbiosis with each other without strain dominance and changes in important metabolic activities would be very troublesome and time consuming. The greater fermentor capacity needed for this type of production is another disadvantage of that method. Although cultivation procedures were thought to be more difficult to optimize when making concentrates from mixed species starters, it was nevertheless chosen in this study because, if successful, most of the problems and disadvantages with the other method would be overcome. This paper presents a study of the optimal conditions for making a mixed species starter concentrate directly from a mixed strain starter, with preserved strain balance and metabolic activities, such as lactic acid production, aroma formation, proteolytic activity, and gas production. MATERIALS AND METHODS Cultures. Five mixed species starters were used in this study. FDs 0172, FD 0570, CH 0170, CHs 0170, and T 27 were all commercial mixed species starters obtained from Flora Danica, Odense, Denmark, Christian Hansen Laboratories, Copenhagen, Denmark, and Viesby Laboratories, Denmark, respectively. They were all composed of strains of S. cremorisiS. lactis, S. diacetilactis, and L. citrovorum. To keep the composition constant, the starters were inoculated in skim milk at 2% (vol/vol) on reaching our laboratory and immediately frozen and stored in liquid nitrogen in stainless-steel tubes to serve as a stock culture throughout this investigation. The effect of this preservation procedure on lactic starter FDs 0172 can be seen in Table 1. The other starters used were equally stable towards freezing. When used for making concentrate, a tube with inoculated frozen starter was thawed at 25 C for 10 min and incubated for 16 h at 22.5 C. The starters were subcultured twice before use. Mass cultivation. The starters [2% (vol/vol) inoculum] were cultivated in laboratory benchtop fermentors of 1-and 10-liter capacity (Biotec, Sweden, and Marubishi Ltd., Japan) equipped with pH-stat assemblies (Radiometer, Denmark) to control the pH at a desired value by titration with 20% (vol/vol) NH4OH or 5 M NaOH throughout the cultivation. Temperature and stirring speed were controlled at desired levels. No aeration was needed for cultivation of these bacteria. Samples to be analyzed were taken from the fermentor through a sampling port with a sterile pipette. Media. Three types of media were used for the cultivation at constant pH. (i) Reconstituted skim milk, 9% reconstituted nonfat milk solids (Semper, Sweden), was sterilized at 110 C for 10 min. (ii) A medium based on whey with filtrate of papain-treated skim milk, yeast extract (Difco), and MnSO4 was prepared according to the following procedure: 70 g of whey powder (spray dried) and 50 g of skim milk powder in 1,000 ml of water were treated with 0.5 g of papain (12,000 U/g) at 30 C for 20 min, 50 C for 20 min, 75 C for 15 min, and 95 C for 15 min. The enzyme-treated suspension was cooled to 75 C, centrifuged (30 min, 5,000 x g), and filtered through a membrane filter (Seitz K 7). After addition of 5 g of yeast extract and 0.14 g of MnSO4, the medium was sterilized at 121 C for 15 min. This medium was, when properly prepared, transparent with a slight opalescence. (iii) A third medium prepared according to Bergere (1) with 40 g of tryptone (Difco), 14 g of yeast extract (Difco), 90 g of lactose, and 0.14 g of MnSO4 in 1,000 ml of water was also used. The lactose was sterilized in one-half the water volume at 115 C for 10 min, and the other components were sterilized at 121 C for 15 min. Centrifugation. At harvesting time the cell suspension from the mass cultivation was cooled to +5 C and centrifuged at +5 C in a Sorvall SS-3 superspeed centrifuge at 34,000 x g with equipment for continuous centrifugation. The supernatant was discarded and the concentrate was used for analyses. Analytical methods. (i) Bacterial estimates. Both microscopic and plate count methods were used to estimate the total number of bacteria. In the milk substrate, the number of cells, stained with methylene blue, was counted directly in the microscope with a special equipment according to Skar (19). In the transparent media, direct microscopic count was made in a counting chamber (Petroff-Hausser) with a phase contrast microscope. All microscopic counts were true total counts, i.e., the single cells in the streptococcal chains were counted. Total viable counts were made with a pour-plate technique on calcium-citrate agar according to Nickels and Leesment (14). The results were expressed as colony-forming units per milliliter. All counts were made in triplicate. Cell mass in the transparent media was as a control estimated by turbidostatic measurements in a Beckman model C calorimeter. (ii) Bacterial balance. The bacterial balance in the mixed species starter cultures was estimated on calcium-citrate agar. The different strains were identified according to the scheme of Nickels and Leesment (14). (iii) Lactic acid production. The acid production (mainly lactic acid) was estimated throughout the constant pH cultivation by reading the amount of alkali added at intervals during the fermentation. The readings were converted into grams of lactic acid produced. (iv) Lactic acid-producing activity. The ability of mixed species starters and mixed species starter concentrates to produce lactic acid was measured as pH drop versus time with a six-channel registrating pH apparatus (Radiometer, Copenhagen). The measurement was made in reconstituted skim milk at 30 C with magnetic stirring. The amount of inoculum was 2% (vol/vol). Lactic acid-producing activity was calculated from the time to reach pH 5.5 on the pH curve and the initial number of bacteria in the vessel according to a procedure of Bergere (1). This method was shown to be applicable to mixed strain starters, in which the lactic acid-producing strains dominated. (v) Proteinase activity. Proteolysis of starters and starter concentrates was determined by the method of Hull (11), modified by Citti et al. (5). Cultures or concentrates were inoculated in reconstituted skim milk to give approximately 106 cells/ml, 2.5 ml of toluene per 100 ml of skim milk was added, and the amount of tyrosine and tryptophane liberated at 32 C, determined as tyrosine, was measured at intervals up to 24 h. The result was expressed as milligrams of tyrosine per 100 g of inoculated milk. (vi) Aroma production. The amount of diacetyl formed by the activity of S. diacetilactis and L. citrovorum in the mixed species starters and concentrates was determined according to the method of Owades and Jakovac (15), modified for milk cultures by Pack et al. (16). The sample to be tested was inoculated in skim milk [1% (vol/vol)] and incubated for 16 h at 22.5 C. Twenty grams of the 16 h-culture was used for the diacetyl determination. The results were expressed as micrograms of diacetyl per microliter in the milk culture after 16 h. (vii) CO, production. The CO.-producing ability of concentrated mixed species starters was determined by the Warburg technique. The bacterial concentrate was diluted in sterile, quarter-strength Ringer solution to a concentration of 101 to 5 x 108 cells/ml. Five-tenths milliliter of this bacterial suspension was placed in the sidearm of a Warburg cup, which held 2.5 ml of reconstituted skim milk. After connection to a manometer and tempering to 30.0 C, the suspension in the sidearm was mixed with the skim milk. Manometer readings were made every 30 min up to 4 h. The CO2 production was calculated as microliters of CO, liberated per cell at inoculation time (21). RESULTS AND DISCUSSION Conditions for optimal growth and lactic acid-producing activity. Optimal growth conditions for single strain mesophilic lactic acid bacteria have been thoroughly examined previously (2,3,6,17,18), and the effect of controlled pH on the growth rate and maximum cell yield is also well documented (2,17). There was good reason to believe that growth conditions for a mixed species starter composed of mesophilic strains or species of lactic acid bacteria should be in good agreement with the results obtained with single strains. The parameters examined in this study to determine the optimal growth conditions for cultivation of mixed species starters were chosen in the following intervals: 20 to 30 C, pH 5.5 to 7.0, and three media, namely skim milk, tryptone, and whey media. Apart from these variables, the effect of different neutralizers for constant pH was examined. Preliminary trials to see if there were marked differences in maximum cell yields with and without pH control were in good agreement with previous work (1,2). From Table 2 it can be seen that cultivation of the mixed species starters at constant pH results in maximum yields 4 to 20 times greater than those which are obtained with static milk cultures. Preliminary trials also showed that the responses to different growth conditions for the mixed species starters used in this study were quite the same. The mixed species starter FDs 0172 was therefore used in the fundamental optimization studies, and the results obtained for this starter were then tested on the other mixed species starters. The comparison in the optimization studies was made with the fresh commercial starter (serving as inoculum) with respect to bacterial balance and important cell activities. In Fig. 1-3 growth curves and lactic acid-producing activity for harvested cells at 20, 25, and 30 C are illustrated. The results show that the growth rates for mixed species starter FDs 0172 increased with increasing temperatures in the interval examined, and that the maximum cell yield increased with the temperature especially significantly when the starter was cultivated in skim milk. There was no significant difference in growth rate and maximum cell yield with different constant pH in the three media tested. This is illustrated for skim milk in Fig. 4. Comparison of the different media used shows that the growth rate and maximum cell yield are greatest in the whey and tryptone media; the cell yield is about three times greater in these media than in skim milk. The variation in growth rate of the entire cell mass of the mixed species starter at temperatures in the interval 20 to 30 C, with an optimum at 30 C, is in good agreement with results obtained for pure cultures (3,6,18). The insignificant effect of pH on the growth rate and maximum cell count is in accordance with the results of Peebles et al. (17) and Pont and Holloway (18), but in contrast to the results of Bergere and Hermier (2) and Cogan et al. (6). The small effect of pH in this study was probably only a coincidence because the different strains and species composing the mixed species starter were affected in different ways by the constant pH values tested ( Fig. 5 and 6). From Fig. 1 and 2, it can further be seen that the relative lactic acid-producing activity of the mixed species starter in skim milk and whey media (compared to frozen stock culture) declined rapidly after the cells had reached the end of the exponential growth period. These findings show that the harvesting time for concentrate making is very critical for mixed species starters, in accordance with the results of Bergere (3) and Lloyd and Pont (13), but in contrast to the results of Bergbre and Hermier (2) and Cogan et al. (6). The greater sensitivity of strains Qf S. cremoris, which dominated this starter, to inhibitors (lactate, D-leucine) during prolonged growth has been shown by Bergere (3). This was confirmed by differential counts (14) made in this study during the growth period (0 to 20 h) under different conditions. The effect of the cultivation -pH on lactic acid-producing activity shows (Fig. 4) activity at pH 7.0 was constant over a longer period of time. This could perhaps be explained by the differential counts (14), which revealed that S. cremoris/S. lactis decreased less rapidly at pH 7.0 than at the other pH values. In tryptone medium (Fig. 3) the lactic acid-producing activity exceeded 100% and declined less markedly after reaching the end of the exponential growth phase. Great changes in the mixed species starter occurred during the growth at constant pH with respect to bacterial balance and other cell activities (Table 3; Fig. 6). From this it can be concluded that the bacterial balance had changed very much during the cultivation procedure, and this would be the explanation for the greatly increased counts FDs 0172, CH 0170, and T 27 were grown in skim milk at pH 6.5 at 25 C. and relative activities shown in Fig. 3. This danger of bacterial changes in the cultivation of mixed species starters in media very different from milk has been postulated by Stadhouders et al. (20). Changes in the bacterial balance during cultivation. When optimizing the growth conditions for a mixed species starter, both the growth rate and maximum cell numbers are important. The balance between individual strains and species composing the starter is extremely important for metabolic activities of the final cell mass. That balance changes in mixed species starters occur was postulated by Stadhouders et al. (20) and has been shown to be the case by using the phage-typing technique by Gilliland (7) on concentrated starters. No systematic survey of the changes in bacterial balance during constant pH cultivation of mixed species starters in different media has hitherto been undertaken. The method used in this study, differential counting on calcium-citrate agar (14), could merely give a rough picture of what happens to the strains of S. cremorisiS. lactis, S. diacetilactis, and L. citrovorum during cultivation at constant pH. This had to be complemented with measurements of important metabolic activities (Table 2). Growth curves separated on S. kactisiS. cremoris, S. diacetilactis, and L. citrovorum revealed areas of exponential growth for the three species from which specific growth rates were calculated. These are referred to as "apparent" specific growth rates in this paper because of the unknown number of strains of each species composing the starter. In Fig. 5 and 6 changes in bacterial balance due to medium, pH, and temperature are illustrated. Growth in skim milk at different pH values and temperatures imposed no significant changes in bacterial balance except for growth at pH 7. When grown in whey medium the balance in the mixed species starter did change during constant pH cultivation at most of the temperatures and pH values tested. This was even more evident for growth in tryptone medium. The optimal growth conditions for FDs 0172 in whey medium with respect to bacterial balance were found to be pH 6.5 and 25 C. In tryptone medium no acceptable optimum was found. The nearest optimum at which a minimum balance change occurred was at pH 6.0 and 25 C. This supports the conclusions of Stadhouders et al. (20) and Gilliland (7). Not only the growth rates for strains composing a mixed species starter, but also the time to reach maximum population is of importance for the balance in the final concentrate. These times were found to be almost equal in this study, i.e., the differences in apparent growth rates illustrated in Fig. 5 and 6 gave a good picture of the strain composition of the final starter suspensions to be further concentrated. Effect of neutralizer on growth and activity. Two neutralizers, NaOH and NHOH, which are reported to affect lactic acid bacteria in different ways when used to control pH for concentrate making (4,10,13,17), were tested in this study to see if such an effect could be observed on mixed species starters. From Table 3 it can be seen that the growth rates and maximum cell populations did not differ significantly when either of the two neutralizers was used. NaOH, however, had a negative effect on bacterial balance and lactic acid-producing activity. The latter is contradictory to the results of Lloyd and Pont (13), who found an increase in activity when using NaOH compared to NH4OH. The decrease in lactic acid-producing bacteria compared to aroma bacteria when NaOH was used instead of NHOH could be due to the effect proposed by Zarlengo and Abrams (24) and Peebles et al. (17), that NH40H (free NH,) could more easily penetrate the cell wall and thereby control the internal pH more accurately. When using NaOH the sensitive S. cremoris cells die more rapidly due to too low internal pH at times. The size of the cells compared by microscopic examination by the Skar method (19) did not seem to differ significantly when either of the two neutralizers was used. Factors affecting maximal population. In this study, as well as in those reported in the literature (2,3,10,18), it is not the deficiency of lactose that stops the growth at a certain population but instead the increased concentration of certain inhibitors. Lactate has been reported to inhibit growth from concentrations of 2% and higher and definitely stops growth at about 6% (18) in a pH-controlled cultivation. Gilliland and Speck (9) found an amino acid, D-leucine, which inhibited growth of lactic streptococci. This amino acid was apparently a metabolite of the streptococci produced under certain conditions. In Fig. 7 the maximum lactate concentration when growth ceased at different conditions of cultivation is illustrated. From this it can be seen that the lactate concentration at maximum population depended on all parameters investigated: temperature, pH, and media. The results of Bergere and Hermier (2) and Bergere (3) that a lactate concentration of 6% is inhibitory when S. lactis C 10 is cultivated in tryptone medium at 25 C, pH 6.5, are, however, in good accordance with the results presented here under the same conditions. The conclusion to be drawn from the results is that the inhibition of lactic starter growth at certain populations under controlled pH is more complex in nature than previously found. A possibility is that other inhibitors involved (for example, D-leucine) are produced at different rates depending on the conditions of growth. This, however, must be further investigated before definite conclusions can be drawn. Lactic starter concentrates. In view of the results obtained, mixed species starter bacterial concentrates were made from the mixed species starters FDs 0172, FD 0570, CHs 0170, CH 0170, and T 27. Cells cultivated at constant pH were harvested at the late exponential phase of growth and were concentrated by centrifugation. The cell concentration in the concentrate varied between 5 x 1010 and 1011 cells/ml. The results of these trials are summarized in Table 2. Optimal conditions for controlled pH cultivation of FDs 0172 with respect to growth rate, maximum population, bacterial balance, and lactic acid-producing activity seemed to be valid for cultivation of the other starters examined. Analysis of aroma production, diacetyl (15,16), proteolysis (5,11), and CO2 production (21,22) were added to this investigation to determine whether optimal conditions, according to the results above, changed other important metabolic activities necessary for a mixed species starter. From Table 2 it can be seen that cultivation in skim milk and whey medium gave starter concentrates with almost unchanged properties compared to the control grown at falling pH in skim milk. In tryptone medium there were marked changes in the parameters examined, compared to the control. The percentage of aroma bacteria, particularly S. diacetilactis, increased by a factor of 2 to 3, all in agreement with the results in Fig. 6. The amount of diacetyl produced by the tryptone medium-cultivated concentrate in skim milk was very low after 16 h, probably due to the higher numbers of S. diacetilactis present in this concentrate or due to the lack of citrate in this medium, which was shown by Gilliland et al. (8) to impair subsequent diacetyl production in milk medium. Gas production increased with the increasing amount of aroma bacteria up to 10 times the value of the control. Proteolytic action of the cell mass was lowered by cultivation in tryptone medium in accordance with the results of Williamson and Speck (23). The danger of bacterial balance changes and consequent changes in metabolic activities when mixed species starter concentrates are made as postulated by Stadhouders et al. (20) and shown for cheddar cheese cultures by Gilliland (7) has been shown to be valid for mixed species starter concentrate production, prepared directly from mixed species starters composed of acid-producing bacteria and aroma formers. But, as shown in this study, mixed species starter concentrates with unchanged properties compared to inoculum could be produced by choosing the proper conditions for cultivation, such as medium, constant pH, temperature, neutralizer, and harvest time.

v3-fos

2020-12-10T09:04:22.953Z

1975-08-01T00:00:00.000Z

237230550

s2

Isolation and Some Properties of Glucoamylase from Cephalosporium charticola Lindau High glucoamylase (α-D-/1 → 4/glucan glucohydrolase, EC 3.2.1.3.) activity was obtained in the cell-free culture fluid of Cephalosporium charticola. Glucoamylase seems to be the only amylolytic enzyme produced by C. charticola. The enzyme, purified on diethylaminoethyl-cellulose, was homogeneous by disc gel electrophoresis. The optimum pH on starch was 5.4, and optimum temperature was 60 C. Starch was degraded more rapidly than several other substrates; maltose was hydrolyzed about one-fifth as rapidly as starch. The molecular weight was 69,000, as determined by Sephadex G-100 filtration. The enzyme is a glycoprotein and contains about 6.6% sugars (mannose and glucosamine). Recently, a culture of Cephalosporium charticola Lindau was found in our laboratory to produce high amylolytic activity. The results of preliminary experiments suggested that an extracellular glucoamylase was present in the culture. In this paper, the purification method and some properties of the glucoamylase will be presented. MATERIALS AND METHODS Production of fungal glucoamylase. A strain of C. charticola Lindau, obtained from the Botany Department of the University of Warsaw, was used as a source of glucoamylase. The fungus was cultivated on malt wort agar slants at 28 C for 96 h. Slants were rinsed with a minimal amount of water, and the resulting spore suspension was used to inoculate 100-ml portions of the following medium in Erlenmeyer flasks: NH4HPO4, 9.0 g; MgSO4 7H20, 0.5 g; KHPO4, 1.0 g; KCl, 0.5 g; FeSO4 7HO, 0.001 g; starch, 20 g; vitamin B,, 0.004 g; and tap water to 1,000 ml. Enzyme purification. After 5 days of incubation at 28 C with shaking (140 rpm), the mycelium was removed by centrifugation. Two volumes of cold acetone (-10 C) was added to the culture fluid, and the precipitate was removed by centrifugation. The precipitate from 827 ml of culture fluid was dissolved in 150 ml of 0.02 M phosphate buffer, pH 7.0. This solution was applied to a diethylaminoethyl-cellulose column (2 by 45 cm) equilibrated with the above buffer, and the column was eluted with a linear gradient of NaCl, 0.0 to 0.5 M, with a flow rate of 0.7 ml/min; 5-ml fractions were collected. Enzyme assay. For assay of the glucoamylase activity, 0.5 ml of enzyme solution and 0.5 ml of 1% starch in 0.02 M phosphate buffer, pH 7.0, were incubated for 30 min at 30 C. Reducing sugars were estimated according to Nelson's method (11). The amount of enzyme that produced reducing sugars equivalent to 1 lmol of glucose per min was adopted as one unit. Estimation of protein. Protein was estimated according to the method of Lowry et al. (8). Paper chromatography of enzymatic products. To characterize the products of hydrolysis of starch, enzyme reactions were stopped after 10, 30, and 60 min of incubation by inserting the tubes in a boiling water bath. The resulting hydrolysates (100 Al) were chromatographed on Whatman no. 1 filter paper at room temperature by descending method, the chromatograms being developed with a solvent consisting of butanol-acetic acid-water (4:1:5, vol/vol/vol), and the papers were sprayed with anisidine phthalate. Hydrolysis of polyand oligosaccharides. Glucoamylase (2 ml, 0.28 mg of protein/ml) was incubated with 2 ml of 0.2% solutions (in 0.02 M phosphate buffer, pH 5.4) of starch, amylopectin, and amylose for 60 min at 30 C. An identical procedure was performed with maltotetraose, maltotriose, and maltose using a 2-h incubation. Molecular weight determination. The molecular weight of the enzyme was estimated by filtration through Sephadex G-100 (1). Pepsin, a-chymotrypsin, egg albumin, and bovine serum albumin served as the reference proteins. Elution was performed with ethyl-cellulose purification step was equal to 23 U/mg of protein, the yield was 71%, and purificolumn chro-cation was 30-fold. ilase. Symbols: Only one peak of amylolytic activity could be -@) protein; eluted from the diethylaminoethyl-cellulose column with about 0.2 M NaCl in phosphate buffer (Fig. 1). No further amylolytic activity was eluted when a linear gradient of 0.5 to 1.0 M NaCl was applied. The obtained enzymatic fraction appeared to be homogenous when checked by polyacrylamide gel electrophoresis (Fig. 2). The only band visible was the protein moving toward the anode at pH 8.4 and displaying glucoamylase activity when eluted from the gel. Similarly, single forms of glucoamylases have been isolated from culture filtrates of Coniophora cerebella (5) and E. capsularis (3), whereas two or more forms have been isolated from A. niger (7), A. phoenicis (6), and A. oryzae (10). Paper chromatography of enzymatic products. The products of enzymatic hydrolysis of soluble starch after various times of incubation were followed by means of paper chromatography. Glucose was the only liberated product in all samples tested after 10, 30, and 60 min of hydrolysis. Similar results were obtained, regardless of whether the enzyme was a purified preparation or the crude culture fluid (Fig. 3). f C. charticola Glucoamylase apparently is the only amylolytic carried out in enzyme secreted by C. charticola under the Fube for 30 min. conditions used. lycine buffer, Optimum pH and optimum temperature. The optimum pH was 5.4 (Fig. 4), and the d. optimum temperature was 60 C, when the incuhydrates in the bation time was 30 min (Fig. 5). the phenol-sul-Relative rates of poly-and oligosaccharide is (2). To estab-hydrolysis. When the velocities of hydrolysis of moiety of the different carbohydrates were compared (Table 164 KRZECHOWSKA 2), starch was digested most rapidly and then, successively, amylopectin, amylose, maltotetraose, maltotriose, and maltose. Maltose was hydrolyzed one-fifth as rapidly as starch and two and three times less rapidly than amylose and maltotriose, respectively. Glucoamylase from C. charticola hydrolyzed amylose, maltotetraose, and maltose at relative rates similar to glucoamylase from E. capsularis (3). Two forms of glucoamylase of A. niger (7) were observed to act on maltose and maltotriose at about the same rate. Molecular weight. The molecular weight of glucoamylase, estimated on Sephadex G-100, was 69,000 (Fig. 6); this is similar to the molecular weight of the glucoamylase from A. oryzae (10), but is less than the value of 97,000 daltons reported for the glucoamylase of A. niger (14). Some glucoamylases have smaller molecular weights; e.g., A. phoenicis (6) and Endomyces species (4) glucoamylases are reported to have molecular weights of 63,600 and 55,000, respectively. Carbohydrate component. Our enzyme contains 6.6% sugars. They were identified by means of paper chromatography as mannose and glucosamine. In comparison, the glucoam-

v3-fos

2019-03-31T13:41:47.048Z

1975-11-13T00:00:00.000Z

87646881

s2

The typification of Lycium inerme As the p iotologue ol Plectronia ventosa L., M ant. 1: 52 (1767), was based on discordant Oliniaceous and Rubiaceous elements, and as P . ventosa has been typified in an Oliniaceous sense, it is no longer possible to use the name Canthium ventosum (L.) K untze for a Rubiaceous plant. The correct name for the Rubiaceous plant, previously but incorrectly called C. ventosum , is Canthium inerme (L.f.) Kuntze, with Lycium inerme L.f., Suppl. 150 ( 1781) as the basionym. The holotype o f Lycium inerme is housed in the T hunberg Herbarium in Uppsala. The protologue of Plectronia ventosa L., Mant. 1:52 (1767), was based on two discordant elements: (1) Burm., Rariorum Africanarum Plantarum X: 257, t. 94 (1739), which is what has until now been called Canthium ventosum (L.) Kuntze, and (2) a specimen (No. 277.2) in the Linnaean Herbarium in London. As the latter is an Olinia, it is clear that the protologue of P. ventosa was based on a Rubiaceous element and an Oliniaceous element. In the International Code of Botanical N o m en ­ clature: 339 (1972), the genus Olinia Thunb. is conserved over Plectronia L. in an Oliniaceous sense, the specimen in the Linnaean Herbarium referred to above being regarded as the lectotype of Plectronia and the genus Plectronia as a synonym o f Olinia. The specimen in the Linnaean Herbarium is not annota ted by Linnaeus but it is annotated "Plectronia ventosa” by Linnaeus filius. As it is not possible to date the specimen and therefore establish whether or not Linnaeus definitely saw it, it seems that one must assume that Linnaeus could have seen the specimen. Since it is difficult to be absolutely certain which of the two elements typifies P. ventosa it seems advisable to follow the choice of the specimen in the Linnaean Herbarium already made in the Code, and there is every reason to believe that this choice is correct. (It is as well to bear in mind that the specimen in the Linnaean Herbarium is a flowering specimen, while both flowers and fruits are depicted in B urm an’s t. 94). Analysis of the protologue o f P. ventosa reveals that there is certain information in it which Linnaeus could not have obtained from B urm an’s t. 94. For example, Linnaeus mentioned that the stems were tetragonal: this is not apparen t from Burman's t. 94 but the stem of the specimen in the Linnaean Herbarium is clearly tetragonal. The generic description of Plectronia in Mant. 1 : 6 p ro ­ vides even better evidence. In the generic description the perianth is described thus: “ Perianthium monophyllum, turbinatum, obsolete quinquedenta tuni , clausum sinubus S, squamis 5 villosis: persistens.” This reference to 5 scales seems very significant as Linnaeus could not have obtained this information from Burman's t. 94, and, in any case, the plant depicted (a Canthium) does not have 5 scales in the mouth o f the perianth. Olinia, on the other hand, does have 5 scales and these are present in flowers o f the specimen in the Linnaean Herbarium. Analysis o f the generic description of Plectronia suggests that it was probably based very largely on a specimen with the exception of * Botanical Research Institute, D epartm en t o f Agricultural Technical Services, Private Bag X101, Pretoria. details of the fruit and seed and for these Linnaeus clearly makes reference to Burman: "Per. Bacca oblonga, bilocularis. Burm. Sem. Solitaria, oblonga, compressa. Burm.” De Candolle, Prodr. 4: 475 (1830), typified Plec­ tronia in a Rubiaceous sense but his decision need not be followed, particularly as it appears that the decision was in conflict with Linnaeus’s concept. Plectronia could only now be used in a Rubiaceous sense if it was proven that the Burman illustration was actually the type of P. ventosa, and, in view of the above facts, this seems unlikely. Cufodontis, in Osterreich Bot. Zeitschr. 107: 106 (1960), published the new combination Olinia ventosa (L.) Cufod., specimen No. 277.2 in the Linnaean Herbarium being regarded as the lectotype of the basionym Plectronia ventosa L. The name Olinia ventosa must be adopted for the plant that has until now been called O. cymosa (L.f.) Thunb. As Plectronia ventosa is typified in an Oliniaceous sense, it is obvious that the name Canthium ventosum (L.) Kuntze can no longer be applied to a Rubiaceous plant. The next available name for the Rubiaceous plant hitherto but wrongly called C. ventosum is Canthium inerme (L.f.) Kuntze, with Lycium inerme L.f., Suppl. 150 (1781) as the basionym. The protologue of Lycium inerme is as follows: “ inerme. Lycium inerme, glabrum, foliis oblongis glabris, floribus aggregatis pedunculatis, stipulis barbatis. Habitat in Cap. bonae spei. Thunberg." There is no Thunberg specimen named Lycium inerme in the Linnaean Herbarium in London or in the Linnaean collections in Stockholm, and the name Lycium inerme does not appear in the microfiche index to the Thunberg Herbarium in Uppsala. No specimen named Canthium inerme or Plectronia inerme appears either. However, in H.O. Juel, Plantae Thunbergianae 430 (1918), there is a cross reference under Lycium inerme to Serissa capensis Thunb., Gen. Nov. PI. 9: 131 (1798). There are two specimens named Serissa capensis in the Thunberg Herbarium in Uppsala, namely. Nos. 5314 and 5315, and, through the courtesy of the Director, Institute of Systematic Botany o f the University, Uppsala, these two specimens were received on loan. I had hoped to find the name Lycium inerme L.f. written somewhere on at least one of the Thunberg specimens named Serissa capensis but unfortunately this is not the case. However, it is quite clear that there 492 T H E T Y PIF IC A T IO N O F LYClUM INERME was a name in the bottom right-hand corner of Thunberg 5314 (reproduced here as Fig. 1) at some stage but the name was subsequently erased and Serissa capensis written in its stead. Unfortunately the name was so carefully erased that it is impossible to form any idea of what was written there previously. So, there is the following circumstantial evidence that one or both of these Thunberg sheets now named Serissa capensis may have been the one(s) on which Linnaeus filius based his description of Lycium inerme: 1. Juel's cross reference under Lycium inerme to Serissa capensis in his Plantae Thunbsrgianae 430 (1918) and his citation on page 419 of both L. inerme and S. capensis as synonyms of Plectronia ventosa L. 2. The arrangement of the specimens in Thunberg's Herbarium, i.e. Thunberg 5314 and 5315 follow consecutively after the specimens of Lycium. 3. The fact that L. inerme has always been accepted to be Rubiaceous. For example, in Index Kewensis and C. H. Wright in Fl. Cap. 4 (2): 109 (1904). F i g . 1.— Thunberg 5314, one of the specimens named Serissa capensis in the Thunberg Herbarium, Uppsala. Analysis of the protologue of Lycium inerme reveals that it was almost certainly based entirely on Thunberg 5314. Some of the details in the proto­ logue, for example, “ stipilus barbatis” , could not have been obtained from Thunberg 5315 as the stipules in 5315 are not as described, while the ‘‘floribus aggregatis pedunculatis” is unlikely to have been taken from 5315 as the specimen has only a few rather inconspicuous young inflorescences in amongst the leaves. In addition, the petioles in 5315 are clearly pubescent above and there are “ pockets" of hairs on the lower surface at the points where the main veins K tw n e g a t iv e In the International Code of Botanical Nomen clature: 339 (1972), the genus Olinia Thunb. is conserved over Plectronia L. in an Oliniaceous sense, the specimen in the Linnaean Herbarium referred to above being regarded as the lectotype of Plectronia and the genus Plectronia as a synonym of Olinia. The specimen in the Linnaean Herbarium is not annotated by Linnaeus but it is annotated "Plectronia ventosa" by Linnaeus filius. As it is not possible to date the specimen and therefore establish whether or not Linnaeus definitely saw it, it seems that one must assume that Linnaeus could have seen the specimen. Since it is difficult to be absolutely certain which of the two elements typifies P. ventosa it seems advisable to follow the choice of the specimen in the Linnaean Herbarium already made in the Code, and there is every reason to believe that this choice is correct. (It is as well to bear in mind that the specimen in the Linnaean Herbarium is a flowering specimen, while both flowers and fruits are depicted in Burman's t. 94). Analysis of the protologue of P. ventosa reveals that there is certain information in it which Linnaeus could not have obtained from Burman's t. 94. For example, Linnaeus mentioned that the stems were tetragonal: this is not apparent from Burman's t. 94 but the stem of the specimen in the Linnaean Herbarium is clearly tetragonal. The generic description of Plectronia in Mant. 1 : 6 pro vides even better evidence. In the generic description the perianth is described thus: " Perianthium monophyllum, turbinatum, obsolete quinquedentatuni, clausum sinubus S, squamis 5 villosis: persistens." This reference to 5 scales seems very significant as Linnaeus could not have obtained this information from Burman's t. 94, and, in any case, the plant depicted (a Canthium) does not have 5 scales in the mouth of the perianth. Olinia, on the other hand, does have 5 scales and these are present in flowers of the specimen in the Linnaean Herbarium. Analysis of the generic description of Plectronia suggests that it was probably based very largely on a specimen with the exception of (1830), typified Plec tronia in a Rubiaceous sense but his decision need not be followed, particularly as it appears that the decision was in conflict with Linnaeus's concept. Plectronia could only now be used in a Rubiaceous sense if it was proven that the Burman illustration was actually the type of P. ventosa, and, in view of the above facts, this seems unlikely. As Plectronia ventosa is typified in an Oliniaceous sense, it is obvious that the name Canthium ventosum (L.) Kuntze can no longer be applied to a Rubiaceous plant. The next available name for the Rubiaceous plant hitherto but wrongly called C. ventosum is Canthium inerme (L.f.) Kuntze, with Lycium inerme L.f., Suppl. 150 (1781) as the basionym. The protologue of Lycium inerme is as follows: " inerme. Lycium inerme, glabrum, foliis oblongis glabris, floribus aggregatis pedunculatis, stipulis barbatis. Habitat in Cap. bonae spei. Thunberg." There is no Thunberg specimen named Lycium inerme in the Linnaean Herbarium in London or in the Linnaean collections in Stockholm, and the name Lycium inerme does not appear in the microfiche index to the Thunberg Herbarium in Uppsala. No specimen named Canthium inerme or Plectronia inerme appears either. However, in H.O. Juel, Plantae Thunbergianae 430 (1918), there is a cross reference under Lycium inerme to Serissa capensis Thunb., Gen. Nov. PI. 9: 131 (1798). There are two specimens named Serissa capensis in the Thunberg Herbarium in Uppsala, namely. Nos. 5314 and 5315, and, through the courtesy of the Director, Institute of Systematic Botany of the University, Uppsala, these two specimens were received on loan. I had hoped to find the name Lycium inerme L.f. written somewhere on at least one of the Thunberg specimens named Serissa capensis but unfortunately this is not the case. However, it is quite clear that there was a name in the bottom right-hand corner of Thunberg 5314 (reproduced here as Fig. 1) at some stage but the name was subsequently erased and Serissa capensis written in its stead. Unfortunately the name was so carefully erased that it is impossible to form any idea of what was written there previously. So, there is the following circumstantial evidence that one or both of these Thunberg sheets now named Serissa capensis may have been the one(s) on which Linnaeus filius based his description of Lycium inerme: 1. Juel's cross reference under Lycium inerme to Serissa capensis in his Plantae Thunbsrgianae 430 (1918) and his citation on page 419 of both L. inerme and S. capensis as synonyms of Plectronia ventosa L. 2. The arrangement of the specimens in Thunberg's Herbarium, i.e. Thunberg 5314 and 5315 follow consecutively after the specimens of Lycium. 3. The fact that L. inerme has always been accepted to be Rubiaceous. For example, in Index Kewensis and C. H. Wright in Fl. Cap. 4 (2): 109 (1904). Analysis of the protologue of Lycium inerme reveals that it was almost certainly based entirely on Thunberg 5314. Some of the details in the proto logue, for example, "stipilus barbatis" , could not have been obtained from Thunberg 5315 as the stipules in 5315 are not as described, while the ''floribus aggregatis pedunculatis" is unlikely to have been taken from 5315 as the specimen has only a few rather inconspicuous young inflorescences in amongst the leaves. In addition, the petioles in 5315 are clearly pubescent above and there are " pockets" of hairs on the lower surface at the points where the main veins depart from the midrib. Thunberg 5314, on the other hand, agrees well with the protologue in every respect, although it is perhaps odd that there is no mention in the protologue of fruits. As Thunberg 5314 clearly agrees with the protologue of Lveium incline I am persuaded to regard this specimen as the holotype. It remains to add that Thunberg 5314 matches material of what has until now but wrongly been called Canthium ventosum. The correct name for this taxon is therefore Canthium inerme (L.f.) Kuntze, Rev. Gen. 3: 545 (1898), with Thunberg 5314 (UPS) as the holotype of the basionym, Lycium inerme. The specific epithet " inerme" is rather inappropriate as the plant often has spinescent branchlets. It is clear that Thunberg 5314 and 5315 belong to two different taxa and that 5315 is in fact a specimen of Canthium mundianum Cham. & Schlechtd. The protologue of Serissa capensis Thunb., Gen. Nov. PI. 9: 131 (1798), seems to have been based very largely, if not entirely, on Thunberg 5314 as there is certain information in it that could not have been gleaned from 5315. For example, once again the description of the leaves and stipules matches those of Thunberg 5314 rather than those of 5315. In the generic description of Serissa there is reference to fruits which quite clearly must have been taken from Thunberg 5314 as 5315 lacks fruits. However, S. capensis is an illegitimate name because Thunberg cited the earlier Lycium barbatum Thunb., Prodr. FI. Cap. 37 (1794) in synonymy, and because it was based in part at least, if not entirely, on the type specimen Lycium inerme L.f. This is stressed because if it was ever argued that S. capensis was based entirely on Thunberg 5315 the illegitimacy of the name S. capensis would prevent the adoption of the specific epithet "capensis" for the taxon to which Thunberg 5315 belongs.

v3-fos

2018-04-03T00:15:57.834Z

1975-03-01T00:00:00.000Z

10283566

s2

Oxidative degradation of squalene by Arthrobacter species. An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K2HPO4 to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, beta,beta'-dimethylacrylic, geranic, and (plus)-(R)-citronellic acids. Among these acids, alpha,beta-saturated carboxylic acids were found to be predominant in quantity. Squalene is a naturally abundant acyclic triterpene, formation of which is thought to occur by the tail-to-tail condensation of two farnesylpyrophosphate (C,,) molecules via presqualenepyrophosphate (8,10), and is an important precursor of steroids and triterpenes. The mechanism of its cyclization to lanosterol via 2,3-oxidosqualene in mammalian livers has been reported by several workers (5,13). Zander et al. (14) have reported another nonoxidative cyclization of squalene in Tetrahymena pyriformis. By this organism, squalene is cyclized to form tetrahymanol by a hydration reaction. Recently, 12,13-dehydrosqualene formation by Staphylococcus aureus (11,12) and also by Halobacterium cutirubrum (9) has been reported, in which the molecule of squalene is dehydrogenated at its center of symmetry. In spite of these findings, no information on microbial degradation of squalene is available. We now wish to report the isolation and identification of the products of degradation of squalene by Arthrobacter sp. This organism is an isolate from soil capable of utilizing squalene and belongs to the genus Arthrobacter. MATERIALS AND METHODS Microorganisms and culture conditions. A strain of Arthrobacter sp. isolated from soil was used throughout this study. A detailed description of the bacterium will be presented elsewhere. The soil samples were inoculated into test tubes, each containing 3 ml of the selection medium consisting of 1.5% squalene, 0.2% yeast extract, and 0.4% NH4NO3, and incubated at 27 C for 3 days on a reciprocating shaker. After extraction of the culture broth with dichloromethane and evaporation of the solvent, the products were analyzed by thin-layer chromatography, using n-hexane-ether (95:5) as the solvent system. Single colonies were isolated from the culture broths that accumulated products of squalene degradation, as evidenced by thin-layer chromatography. A 0.5-ml portion of fresh seed culture was inoculated into 500-ml Sakaguchi flasks (a globular flask with flattened shoulder) containing 50 ml of medium. Medium A contained 2% corn-steep liquor and a specified amount of squalene and was adjusted to pH 6.9 with NaOH; medium B consisted of 2% glucose, 0.5% KNOI, 0.02% MgSO4 7H2O, 0.1% yeast extract, 0.1% KH2P04, and 0.5% squalene and was adjusted to pH 6.5. Flasks were incubated at 30 C on a reciprocating shaker. Assay procedures. Growth was monitored by absorbance at 550 nm after the removal of oily materials from the culture broth by extraction with 3 volumes of a solvent mixture (ethanol-butanol-chloroform, 10:10: 1), centrifugation of the cells at 12,000 x g for 10 min, and resuspension in water. The quantities of squalene and its degradation products were estimated by gas chromatography. Samples (50 ml) of culture broth were extracted three times with 30-ml portions of dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The oily residue was weighed and subjected to gas chromatography. The content of geranylacetone was estimated from the peak height by comparison with that of a known amount of an authentic sample. SQUALENE DEGRADATION BY ARTHROBACTER SP. Analytical methods. Gas chromatography was performed on a Hitachi gas chromatography 063 instrument equipped with a thermal conductivity detector. Separations were performed on a stainlesssteel column (100 by 0.3 cm) containing SE-30. Helium served as carrier gas at a flow rate of 21 ml/min. Temperatures of the column injection port and detector will be specified below. Infrared spectra were recorded on a Hitachi model 215 grating infrared spectrometer. Oily samples were run on neat liquid films between NaCl plates. The nuclear magnetic resonance (NMR) spectra in CDCl, were recorded with a JEOL model PS 100 spectrophotometer, with tetramethylsilane as the internal standard. Mass spectra were obtained with a Hitachi RMU-6E mass spectrometer. Chemicals. Squalene was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo) and used without further purification. Geranylacetone used as the authentic sample was a gift from Takasago Perfumery Co. Ltd. Isovaleric acid was prepared from #,#'-dimethylacrylic acid by catalytic hydrogenation. Other chemicals were obtained from commercial sources. RESULTS Biotransformation of squalene by Arthrobacter sp.: identification of geranylacetone. Geranylacetone was isolated from the culture broth as follows. After 38 h of culture in medium A supplemented with 242 mg of squalene per flask, the culture broths were combined (500 ml) and extracted three times with 300-ml portions of dichloromethane. The extracts were combined and dried over anhydrous sodium sulfate. After evaporation of the solvent, 10 ml of n-hexane was added to the residue, and the resulting precipitate was removed by filtration. The oily residue (1.740 g) obtained after evaporation of the solvent contained 55% geranylacetone and 35% unchanged squalene when analyzed gas chromatographically at the following temperatures: column, 310 C; injection port, 340 C; detector, 340 C. The yield, calculated on the basis of two molecules of geranylacetone from one molecule of squalene, was 55.87%. Pure geranylacetone (0.917 g) was obtained by distillation of the oily residue under reduced pressure. The sample thus obtained had a bp of 90 to 95 C at 7 mm of Hg. Elemental analysis showed: C, 80.21%; H, 12.03%; the calculated values for C13H220 were: C, 80.35%; H, 11.41%. The infrared spectrum of the sample was almost identical to that of authentic geranylacetone (cis-trans mixture) and had values of vn.axtl'm at 1,720, 1,440, 1,380, 1,360, and 1,155/cm. The 1,720/cm band is attributed to the >C=O group of the molecule. This observation was confirmed by mass spectral analysis (70 eV) where the parent ion was observed at m/e 194. Other peaks were observed at m/e 151, 136, 125, 107, 69, 43, and 41; m/e 151 was assigned as The NMR spectra of the compound in deuterated chloroform showed singlet signals at bppm of 1.59, 6H (cis-C,-CH3 and cis-C 0-CHO); 1.69, 3H (trans-C lo-CH3); and 2.11, 3H (CO-CH3) and a triplet band at 5.09 (olefinic protons at C5 and C9). Evidence for the trans geometric isomerism of the C,5-C. double bond was that the NMR signal of two methyl groups at C, and C,0 appeared at the same 6 value, 1.59 (2). Preparation of geranylacetone semicarbazone. To confirm the molecular formula, the semicarbazone derivative was prepared. After recrystallization from an ether-cyclohexane mixture, the semicarbazone had an mp of 94 to 95 C. Elemental analysis of the compound showed the following percent composition: C, 66.48; H, 10 The infrared spectrum of the sample was identical to that of the semicarbazone derived from authentic geranylacetone (cis-trans mixture) and had absorption bands at Vmaxfnujol values of 3,540, 3,300, 1,690, and 1,580/cm. The main mass spectral peaks observed had m/e values of 251 (parental ion), 182, and 69. On the basis of these data obtained from the isolated compound and its semicarbazone, the carbonyl compound produced from squalene by Arthrobacter sp. was identified as trans-geranylacetone. Time course of the production of geranylacetone. Figure 1 shows a typical time course of growth and geranylacetone formation in medium B. After a long lag period (12 to 20 h), cell growth and geranylacetone accumulation started. Geranylacetone formation was found to be parallel with the cell mass increase in the logarithmic growth phase and continued after the logarithmic growth phase at a higher rate than growth. The pH value of the culture broth remained constant during the culture period. The yield of geranylacetone obtained to squalene added was 16% (wt/wt), which was much smaller than that obtained in medium A (39%). Isolation and identification of carboxylic acids produced by Arthrobacter sp. Preliminary experiments had shown that several spots other than squalene and geranylacetone were observed when cells were cultured in medium A supplemented with 1% K2HPOi. For the isolation and identification of these products, the following experiments were carried out. Twelve flasks, each containing 75 ml of medium A supplemented with 1% K2HPO, and 0.65 g of squalene, were inoculated and incubated for 65 h. After acidification of the combined broths to pH 2 with 3 N HCl, the oil fraction was prepared according to the procedure described previously. Yield was 5.21 g. The oil was dissolved in dichloromethane and extracted with 10% sodium carbonate solution. The alkaline solution was acidified with 3 N HCl and extracted with dichloromethane. After drying over anhydrous sodium sulfate, the solvent was evaporated to give carboxylic acids in a yield of 0.54 g. The yield of neutral compounds that were not extracted with sodium carbonate solution was 4.3 g. The gas chromatogram of this neutral moiety showed that it contained 44% geranylacetone and 50% unchanged squalene. The carboxylic acids thus obtained were separated into five peaks by gas chromatography (temperatures: injection port, 250 C; column, 170 C; detector, 260 C). These peaks were designated A, B, C, D, and E in the order of retention time (Fig. 2). Fractions corresponding to peaks A plus B, C plus D, and peak E were collected at the detector outlet of the gas chromatograph instrument and used for identification experiments. Identification of isovaleric and f34Tdimethylacrylic acids. Infrared spectra of the mixture of acids A and B in film showed P,=, absorption at 1,640/cm. When the mixture was hydrogenated on palladium-charcoal (5%) in methanol under atmospheric pressure, a pure carboxylic acid, which was identical with peak A carboxylic acid by gas chromatography, was obtained. The infrared spectra of the carboxylic acid obtained by hydrogenation of the mixture coincided perfectly with that of isovaleric acid. This suggests that the carboxylic acid of peak B has a skeleton of isovaleric acid. Mass spectra of the A-B mixture showed two parental molecular ions at m/e of 102 and 100, which correspond to isovaleric acid (A) and carboxylic acid (B), respectively. The NMR spectra in CDC13 of the A-B mixture showed signals of (CH3)2-CHmethyl protons at 6 0.96 (doublet), CH3-Cmethyl protons at 6 1.90 and 2.15 (singlet), and an olefinic proton at a 5.62 ppm. From these data carboxylic acid B was identified as A,:'dimethylacrylic acid. The ratio of the quantities of isovaleric and f,f3'-dimethylacrylic acids estimated from the NMR spectra was 3:1. Identification of citronellic and geranic acids. The mass spectra of the C-D mixture To confirm the molecular structures of acids C and D, isolation of these two acids in the form of their methyl esters was performed. A mixture of carboxylic acids (A, B, C, D, etc.) (320 mg) was methylated with diazomethane in ether. From the reaction mixture, 214 mg of methyl esters was obtained by the standard method. The methyl esters of acids C and D were separated by gas chromatography (temperatures: injection port, 220 C; column, 150 C; detector, 215 C) (Fig. 3). Peaks C' and D' correspond to carboxylic acids C and D, respectively. Peak E' was a mixture of carboxylic acid esters which were not identified. Fractions corresponding to peaks C' and D' were collected separately and used for identification experiments. Identification of methyl (+ )-R-citronellate. The infrared absorption spectra of the methyl ester C' in film showed an ester carbonyl group absorption band at 1,740/cm and no strong absorption in the region of 1,600 to 1,700/cm, which indicated that the ester C' was an a,3- This ester showed a plain optical rotatory dispersion curve in the region of 250 to 600 nm. The optical rotation values obtained were [a]30025 = + 204°, [a ]3525 = +91°, and [a ]40025 = +68°(C = 0.0044 in methanol). This suggested that the ester is methyl (+ )-R-citronellate (6,7). Identiflcation of methyl geranate. The infrared absorption spectra of the methyl ester D' in film showed an ester carbonyl group at 1,720/cm and a strong absorption band of C=C conjugated with a carbonyl group at 1,645/cm. The mass spectra of the ester D' showed a peak of parental molecular ion at m/e 182 and other fragments at 151 (M+ -CH30), 123, 113, 83, 69, 55, and 41. These spectral data suggested that the ester D' is methyl geranate. DISCUSSION Arthrobacter sp. which was isolated from soil can oxidatively decompose squalene (C30) (I) into geranylacetone (C,,) (II). The yield of geranylacetone to squalene consumed was 56% on the basis that one molecule of squalene gives two molecules of geranylacetone. This fact suggests that squalene molecules in part were cleaved at the two sites shown by wavy lines in the structural formula in Fig. 4. In mammalian liver, squalene is first oxidized at its terminal double bond to give squalene-2,3-oxide (5, 8), which is then cyclized. In the case of Arthrobacter sp. the central part of squalene is attacked, and fission occurs symmetrically at C 1o=C11 and C 14==C 5, although the cleavage mechanism has not been elucidated. Another central attack of the squalene molecule to form 12,13-dehydrosqualene by S. aureus and by H. cutirubrum has been suggested (12,14). When medium A was supplemented with a high concentration of dipotassium phosphate, squalene was oxidized to carboxylic acids in addition to geranylacetone. The carboxylic acids comprised up to 10% of the total recovered Though several efforts to oxidize geranylacetone using the intact cells were not successful, it is probable that these carboxylic acids are derived from squalene by a "central attack" mechanism which is common to geranylacetone formation. The fact that all acids accumulated have beta-substituted methyl groups might suggest that this configuration is resistant to biological oxidation by this organism. The a,f3-saturated form was found to be predominant for both C5 and Clo acids. The oxidative cleavage of squalene by Arthrobacter sp. is a useful method of obtaining pure trans-geranylacetone. The supposed intermediate of the oxidative degradation of squalene by this organism, which retains a C30 skeleton, probably has functional groups such as hydroxyl groups in its central position. As has been suggested by others (1,3,4), flexible linear molecules, such as squalene, which have no specific groups could hardly be the object of selective chemical reactions, especially in central parts of the molecule. Thus, the application of this microorganism to introduce functional groups into central parts of the squalene molecule is interesting from the viewpoint of the synthesis of squalene derivatives.

v3-fos

2014-10-01T00:00:00.000Z

1975-10-15T00:00:00.000Z

11592412

s2

Effect of genes affecting tan colour on productivity in icelandic sheep An account is given of the effect, within the Icelandic Sheep breed, of pelt class of sire and pelt class of daughter on lambing records of 1270 daughters at i, 2 and 3 years of age and on carcass score of 1023 daughters at the ages 2 and 3 years. Daughters of 141 sires were included in the study. The pelt classes were : A, pure white lustrous, long and even fleece, B, pure white with lower quality fleece, C, white with tan colour on outskirts of fleece, and D, white with tan fibres in the fleece. Sires in pelt classes A and B had heavier weaning weight of daughters than sires in other pelt classes. Daughters in pelt classes A and B were lighter at weaning than those in other pelt classes due to lowered selection intensity for weaning weight. The pelt class of sire did not significantly affect any of the production traits. No effect of pelt class of sire or pelt class of daughter on ewe fertility could be demonstrated. Daughters in pelt classes A and B had lower score for carcass production than daughters in pelt classes C and D. INTRODUCTION The research work to be described here has been aimed at improving the Icelandic sheep breed with respect to pelt colour and pelt quality of lambs at slaughter and wool colour and quality of adult sheep. The sheep referred to in the present study have been either white or tan coloured. A description of the origin of the Icelandic sheep breed and its characteristics has been given by A D A LS TEINSSO N ( 19 66,ig7o). The fleece of the white or tan Icelandic sheep is doublecoated, with very fine, short and soft undercoat and long, rather coarse outercoat. In addition to the undercoat and outercoat fibres, white and red (tan) kemp fibres also occur (ADAr, 5T!INSSON, 1975). .. It has been shown by L AUVERGNE (ig 7 g) that the tan colour appears in sheep which carry the gene A W h , and the tan colour is converted to pure white colour in sheep which are homozygous for the gene for piebaldness S b . The investigations to be reported on here have been aimed at two main aspects. The first of these was the occurrence and inheritance of tan fibres in the pelt of lambs at slaughter, and the other aspect has been the connection between the occurrence of tan colour and other production caracteristics of economic importance such as ewe fecundity and lamb weights. Information on the above aspects has been obtained during the process of selection against tan colour. In a previous paper !ADAI,STEINSSON, 1975 ) a review has been given of several investigations regarding the occurrence and inheritance of tan fibres in the fleece, and of some preliminary investigations regarding the connection between tan colour and production characteristics. The main conclusion drawn from those earlier investigations was that the heritability of the amount of tan colour was high or around 0 .5 5 (ADA L STE INSS O N , I(!7I b), and no definite connection had been found between the occurrence of tan colour and production characteristics. ' In the present paper the relationship between pelt quality of autumn lambs and ewe productivity, in terms of number of lambs and weight of lambs, is investigated further. MATERIAL AND METHODS The data on which the present investigation is based were collected on four State farms in Iceland during the period i965-i9!4. The sheep flocks and husbandry practices have been described earlier (A DALSTEINSSO N, 1971 b). All white and tan female lambs kept for breeding during the period 19 6 5 to 1971 and which had been on record up to and including the age of 3 1/2 years were included in the analysis. The female lambs had all been scored for pelt quality at weaning time into one of the four classes shown in table i. Records were also available on the age of the selected female lambs in days on ist October and on their weaning weight, which was taken around that date. Also available was the age of the dam, the identification number of the sire and the sire's pelt class, and finally whether the lamb had been born as a single (r), twin ( 2 ) or a triplet ( 3 ), and whether it had been reared as a single (i), twin ( 2 ) or occasionally as a triplet ( 3 ). The data on each female lamb up to and including weaning were combined with the production records of the same animals in later life. The production records included number of lambs born at ewe ages 1 , 2 and 3 years and scores for carcass weight of weaned lambs at ewe ages 2 1/2 and 3 1/2 years. All ewes which weaned one or more lambs were given a score for carcass weight production according to the system described earlier (AD ALSTEINSSON , 197 11 a). The average score for all ewes on the farm in a given year is 5 . 0 , and the theoretical standard deviation r. 43 which should ascertain that 99 ,g6 per cent of the scores lie between the values o and 10 . The ewes which reared no lamb were not given any production score. A rearing score of i was given to ewes which reared a lamb, and obtained a lamb weight score, while ewes which reared no lamb and ewes with abnormal records were given a rearing score of o. The analysis was carried out by the least squares technique assuming all effects to be fixed. The effects which were taken into account and for which least squares constants were fitted simultaneously will be described in connection with the results from the various analyses. The least squares analysis was carried out on an IBM 370/135 computer in Reykjavik using the LSMLMM programme by H ARVEY (r9!2). RESULTS AND DISCUSSION Altogether z a8o white or tan ewes selected for breeding during the years i 9 65 to 1 971 were included in the analysis. The distribution of the sires and daughters by pelt classes is given in table 2 . Table 3 shows selected comparisons among pelt classes for sires and daughters and their t-values, with respect to weaning weight of daughters. The records have been corrected simultaneously for farm, year of birth, age of dam, type of birth and rearing, and age of selected daughter in days on ist October. The comparisons in table 3 show a significant difference in weaning weight of ewes between white (A + B) and tan (C + D) sire groups in favour of the white sires. The opposite effect is found for pelt class of daughters, where daughters in pelt classes A and B are significantly lighter at weaning than daughters in the tan classes C and D. The reason for the difference between sire pelt classes could be a result of confounding of sire pelt class with time. Genetic progress in live weight might then be expected to bring about this difference. The difference between the daughter pelt classes was to be expected, since selection against tan colour was carried out during the period, resulting in appreciably less selection intensity for live weight among the white than among the tan female lambs. The differences in weaning weight of daughters shown in table 3 would be expected to have some effect on production ability of the ewes in later life. A seperate analysis was therefore carried our in order to estimate the effect of weaning weight on number of lambs born per ewe on record and per ewe lambing at the ages 1 , 2 and 3 years. The effect of weaning weight on rearing score (RS) of 2 year old ewes and carcass weight score (CS) of 2 and 3 year old ewes was also assessed. The regression coefficients and F-values for the regression coefficients are shown in table q.. The effects of farm, year of birth, age of dam, pelt class of sire, pelt class of daughter and type of birth and rearing were estimated simultaneously with the regression coefhcients shown in table 4 . ' As can be seen from table 4 , the weaning weight of the daughters has a significant effect on number of lambs born at i and 2 years of age, and also on carcass weight score at 2 and 3 years of age. On basis of the results obtained in tables 3 and 4 it was decided to use weaning weight as a regression variable in the analysis of the effect of pelt class of sires and daughters on production traits, except for rearing score of 2 year old ewes. The other effects for which constants were fitted when analysing the production traits were farm, year of birth, pelt class of sire, pelt class of daughter and type of birth and rearing of daughter. Table shows the difference in number of lambs born and rearing score for selected comparisons of pelt classes of sires and daughters. The comparisons in table 5 show that the only significant effect of sire pelt class on the traits in table 5 is lowered number of lambs of 3 year old daughter of sires in pelt class A. As this effect is only found to be significant at one age level one is inclined to ascribe it to chance. It can at any rate not be ascribed to absence of tan colour, because the daughters of sires in pelt class B of the same age are just above average. In table 6 are shown differences in number of lambs and carcass scores for selected comparisons of pelt classes for ewes with carcass scores at 2 and 3 years of age. The comparisons with regard to number of lambs in table 6 show much the same picture as those in table 5 , except that the deficiency of number of lambs among daughters of sires in pelt class A is no longer significant. The effect of pelt class of sire on carcass score is in all cases non-significant. The results therefore show that there have been no marked differences between the sire groups under study with respect to their transmitting ability for fecundity and carcass score of daughters. The tan daughter group (C -i-D), on the other hand, shows a significantly higher carcass score than the pure white daughter group (A + B) for both 2 and 3 year old daughters. The discrepancy between pure white sires and pure white ewes in this respect indicates that the lowered carcass score of the pure white ewes is of incidental rather than causal nature. The increased selection intensity for pure white colour has lowered the selection intensity for live weight at weaning within ewe lambs in pelt classes A and B, as seen from tables 3 and 4 . It seems likely that the lowered carcass score of daughters in pelt classes A and B is a reflection of lowered selection intensity for carcass score of dams of these ewe lambs. En race ovine Islandaise on a cherché à mesurer l'influence de la catégorie dans laquelle on peut ranger la fourrure des béliers et de leurs filles sur les performances d'agnelage des 1 2!o dites filles âgées de x, 2 et 3 ans et de 1 023 filles âgées de 2 ou 3 ans. Il s'agissait des descendantes de 1 4 1 béliers. L'échelle de notation comporte les catégories A : blanc pur avec une toison longue et homogène ; B : Blanc pur avec une toison de qualité moindre ; C : blanc avec des fibres rouges sur les bords de la toison ; D : blanc avec des fibres rouges à l'intérieur de la toison. Les filles notées A et B ont des notes de carcasse plus faibles que les filles classées C et D.

v3-fos

2018-04-03T04:09:34.975Z

1975-06-01T00:00:00.000Z

37866015

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Improved Medium for Lactic Streptococci and Their Bacteriophages Incorporation of 1.9% β-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO4·7H2O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems. medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO4.7H20, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems. Lactic streptococci are nutritionally fastidious and require complex media for optimum growth (9,10,11,14,16). In synthetic media, all strains require at least six amino acids and at least three vitamins (2, 27). Their homofermentative acid-producing nature requires that media be well-buffered for reasonable growth response; in this regard Hunter (12) observed that more growth and larger colonies (0.7 to 1.0 mm in diameter after 48 h) resulted in a medium containing lactose, yeast extract, peptone, and beef extract to which 0.05 M sodium phosphate had been added. Bacteriophages for lactic streptococci usually are assayed by the agar overlay technique described by Adams (1), using one of the several complex media cited above. During a study of the plating efficiency of lactic streptococcal phages, Lowrie and Pearce (19) observed that not all bacterial strains, especially those of Streptococcus cremoris, grew well when inoculated into the most widely used of the complex media then available. They devised a new 'Present address: Department of Microbiology, Oregon State University. Corvallis, Ore. 97331. medium, designated M16, which overcame this problem; it was unique in containing a plant protein extract (Phytone) but lacked phosphate, relying on peptone and acetate for buffering capacity. The omission of phosphate was intentional to allow calcium supplementation for phage assays. However, Thomas et al. (30) incorporated phosphate into this medium for their study of streptococcal proteinases, calling the more-buffered medium T5. In the present investigation, a correlation was obtained between restriction in sizes of bacterial colonies and phage plaques and a rapid decline in pH with the M16 medium. Attempts were made, therefore, to improve the buffer strength of the medium without resorting to the use of phosphate, well known for precipitation problems in bacteriological media due to its ability to sequester alkaline earth metals (5). The recent reports by Douglas et al. (8) and Douglas (7) suggested that glycerophosphate (GP) would be suitable for this purpose, especially since its use allowed large plaques on S. lactis to develop (8). The present report describes the resulting new medium, designated 807 M17, and its use in demonstrating improved growth of lactic streptococci and their bacteriophages. MATERIALS AND METHODS Medium. M17 broth medium is made by adding the following ingredients to 1,000 ml of glass-distilled water in a 2-liter flask: polypeptone (BBL, Cockeysville, Md.), 5.0 g; Phytone peptone (BBL), 5.0 g; yeast extract (BBL), 2.5 g; beef extract (BBL), 5.0 g; lactose (May and Baker Ltd., Dagenham, England), 5.0 g; ascorbic acid (Sigma Chemical Co., St. Louis, Mo.), 0.5 g; fl-disodium GP (grade II, Sigma Chemical Co.), 19.0 g; and 1.0 M MgSO4.7H2O (May and Baker, Ltd.), 1.0 ml. This concentration was optimum for growth and prevented the pH of S. cremoris cultures from falling below 5.9 after growth for 15 h at 30 C. Broth is dispensed (10 ml) into tubes and autoclaved at 121 C for 15 min; the pH of the broth (22 to 25 C) is 7.15 4 0.05. Bottom agar used for assay of bacterial colonies or phage plaques is prepared by adding 10.0 g of Davis agar (Davis Gelatine Ltd., Christchurch, N.Z.) to 940 ml of glass-distilled water and heating the mixture to boiling to dissolve the agar. The remaining ingredients, except lactose, are added to the dissolved agar and the mixture is autoclaved at 121 C for 15 min. After cooling to 45 C in a temperature-controlled water bath, a sterile solution of lactose (5.0 g in 50.0 ml of glass-distilled water and sterilized at 121 C for 15 min) to which has been added 10.0 ml of sterile 1.0 M CaCl .6H2O is gently added to the melted agar basal medium. The calcium addition is necessary only when the bottom agar plates are to be used for growing phage, but its addition has no adverse effect on use of the medium for plating bacteria; usually, slight cloudiness develops when the calcium is added. After mixing carefully to avoid bubbles, 15to 18-ml quantities are added to sterile petri plates. The bottom agar plates are held overnight (15 to 18 h) at 22 to 25 C to dry and then checked for any contaminating colonies; they then are stored at 2 to 5 C until used. Top overlay agar is prepared by adding 4.5 g of Davis agar to 1,000 ml of glass-distilled water and heating to boiling until the agar is dissolved. The remaining broth ingredients, including lactose but excluding calcium chloride, are then added and the medium is dispensed (50-ml quantities) into prescription bottles and autoclaved (121 C, 15 min). Top agar is used for carrying diluted phages and bacteria to bottom agar plates for determining titers of virus preparations and colony counts in bacterial cultures. M16, T5, and lactic broth were prepared as described previously (9,19,30). GP-SM. Severe protein denaturation and browning occurred when the GP was autoclaved with skim milk (SM). Therefore, a stock solution containing 9.5 g of GP per 10.0 ml of glass-distilled water was sterilized (121 C, 15 min) separately, and 0.2 ml was added per 10 ml of sterile SM, providing a final concentration of 1.9% GP. Molskness of Oregon State University. Bacteriophage strains. Two bacteriophages were used, both isolated from cheddar cheese whey. Phage 799 is virulent for S. cremoris AM2, and strain 690 is virulent for S. cremoris SKI,; each phage, however, will form plaques on hosts other than those on which they were originally isolated. Growth measurement. Bacterial growth was assayed by recording absorbance readings (600 nm) at 30-to 60-min intervals of the various strains inoculated (1.0%) into 10.0 ml of the various media in flasks fitted with a side arm accommodated by a Bausch & Lomb Spectronic 20 colorimeter. Colony-forming units per milliliter of culture were determined after blending (60 s) of 1:100 dilutions in 10% M17 broth (21) followed by serial dilution, as appropriate; aliquots (0.1 ml) were poured on the surface of M17 bottom agar plates after being mixed with 2.5 ml of top agar as described below for the phage assay procedure, except calcium chloride was omitted. Culture activity in milk. The influence of daily subculturing in M17, M16, and LB for 10 days on acid-producing activity in SM was measured. Strains were maintained in the three broth media by inoculation at. 1% and incubation at 30 C for 24 h. Each day, the 24-h broth cultures were each inoculated in duplicate (1%) into 10 ml of SM containing 9.5% solids (100 g of powder plus 910 ml of distilled water; sterilized at 121 C for 15 min) and incubated, one tube at 30 C and the other at 22 C. Tubes at 30 C were tested for pH after 6 h, and tubes at 22 C were tested for ability to coagulate milk when held for 15 h. Studies also were carried out to determine the influence of culturing strains in SM containing GP on their subsequent acid-producing activity when inoculated into sterile SM. Strains AM1, AM2, ML,, and ML, were incubated at 22 C for 15 h in GP-SM and SM. Each strain was subcultured from these two types of milk into SM and incubated at 30 C in a temperaturecontrolled water bath; pH measurements were taken at hourly intervals. Bacteriophage assays and stocks. To ensure homogeneity, bacteriophage stocks were renewed by single-plaque isolation (3,4,22). Aliquots (0.1 ml) of an overnight (15-h) MW7 broth culture of the appropriate bacterial host were placed in sterile test tubes (10 by 75 mm) fitted with aluminum caps. One drop (0.05 ml) of sterile calcium chloride (1.0 M) was then added to each tube followed by 0.1 ml of phage previously serially diluted in 10% M17 broth so that about 20 plaques per plate resulted. After 3 to 10 min at 22 to 25 C (room temperature) to allow for phage adsorption, melted and cooled (45 C) M17 top agar (2.5 ml per tube) was then added, and the tube contents were immediately poured on the surface of hardened M17 bottom agar in sterile plastic petri plates (10 by 90 mm). Plates were incubated at 30 C and observed periodically for isolated plaques from 3 h onward. When they appeared, usually between 3 to 5 h, two or three well-isolated plaques were MEDIUM FOR STREPTOCOCCI AND THEIR PHAGES picked by touching the top layer with sterile 152mm applicator sticks; the plaque was then transferred to 0.5 ml of chilled (2 to 5 C) M17 broth contained in a test tube (10 by 75 mm) and held in the refrigerator overnight. (The titer of these young plaques is 106 to 106/ml.) Incubation of phage plaque plates was continued until the next morning when they were examined to ensure that no other plaques developed which were partially coincidental with the plaques originally selected and that the plaques were typical in morphology and size for the particular phage-host system. One or more of the M17 broth phage-containing tubes were then used to prepare the phage stock. This was done by adding the entire contents of the tube to 10.0 ml of a 3.5-h M17 broth culture (absorbance = 0.10 to 1.15 at 600 nm) of the appropriate bacterial host growing at 30 C; 0.1 ml of CaCl .6H2O (1.0 M) also was added. With continued incubation, lysis occurred from 2 h onward, usually by 4 h. If lysis did not occur, the stock was discarded and prepared from another plaque isolate. Overnight (15to 18-h) incubation of phage-infected cultures would sometimes yield turbid cultures due to emergence of phage-resistant mutants. After lysis, the phage-laden culture was centrifuged at 4,500 rpm for 10 min in a bench-top clinical centrifuge. The supernatant was then filter sterilized by passing through a sterile syringe-mounted membrane filter (0.45 Mm; Millipore Corp.) into a sterile screw-capped tube. Titer of the stock was determined by counting plaques that developed in M17 top agar when the serially diluted sterile lysates were plated as described above. Stocks were stored at 2 to 5 C. Titers ranged from 108 to 1010 plaque-forming units per ml and would occasionally increase two-to threefold during the first week of storage. The phages were relatively stable when stocks were prepared in this manner, declining in titer only 5 to 10% over 6 months of storage. RESULTS Bacterial growth. Figure 1 shows the buffering capacity of M17 broth in comparison to three other media. Although T5 medium was almost as well buffered as M17, it was unsuitable for bacteriophage assays because of calcium precipitation and, therefore, was excluded from further study. The well-buffered nature of M17 under growth conditions also was apparent. For example, five lactic streptococcal strains tested gave pH values ranging from 5.78 to 6.10 after 24 h of growth at 30 C in M17 broth; these strains grown in M16 and LB, however, gave pH values from 4.70 to 4.87 and 4.42 to 4.70, respectively. When cells of S. cremoris AM, were grown either in M16 or M17 broth (Fig. 2) and inoculated into M16, M17, or LB media, the best growth response occurred in M17 medium. By 9 h, the absorbance readings of M16-grown cells revealed 49 and 52% more growth in M17 as compared to M16 and LB, respectively; for M17-grown cells, these in- creases were 30 and 45%, respectively. Also, a 1to 2-h lag occurred when M16-grown cells were used, whereas except in LB the lag was eliminated with M17-grown cells. These data are typical for all 12 lac+ strains included in the study. In support of the absorbance data, M17 consistently allowed higher total cell counts in each case. For example, with S. cremoris AM1, total counts after 15 h at 30 C in M16, M17, and LB were 1.0 x 108, 1.6 x 108, and 5.6 x 107, respectively, and for S. cremoris AM2 they were 3.7 x 107, 2.0 x 107, 2.0 x 108, and 5.0 x 106, respectively. The medium also proved useful in selecting carbohydrate mutants, especially those unable to ferment lactose. For example, colonies of lac+ strains measured 3 to 4 mm in diameter at 5 days, whereas lacmutants, growing only on the small amount of glucose provided by the yeast extract (5) in the medium, reached colony sizes of less than 1.0 mm. The large colony size was typical of all 12 lac+ strains tested except S. cremoris P2 (an X-ray derivative of S. cremoris HP), which developed more slowly because of a requirement for carbon dioxide for rapid growth on agar plates (29). It should also be mentioned that colonies of all lac+ strains tested other than P2 were clearly visible for counting within 15 h after plating and incubation at 30 C. The milk-acidifying ability of lactic strepto-VOL. 29,1975 cocci was best preserved by maintenance of strains in M17 medium as compared to M16 or LB. Data for two strains typical for all strains Table 1. Furthermore, the M17-derived S. cremoris cultures coagulated milk almost every time 24-h-old broth cultures were inoculated into SM and incubated at 22 C for 15 h, whereas this occurred only rarely for strains derived from M16 or lactic broth cultures after the second transfer. S. lactis strains, however, did coagulate milk even though the 6-h test showed them to be impaired in acidproducing activity. The pH values attained for strains AM,, AM2, ML,, ML8, and C2 in SM by 15 h at 22 C after maintenence for 10 days in the three broth media appear in Table 2. Maintenance of strains in SM containing GP also preserved their rapid-acid-producing abilities in milk, especially early in the growth period. Data for S. cremoris ML,, typical for three other strains tested, appear in Fig. 3. It may be seen that for at least 5 h the SM culture was 1 h slower in achieving the same degree of acidity as compared to the culture grown in the GP-SM, although approximately the same final acidity was achieved by each culture by 15 h. Bacteriophage development. The M17 medium was superior to the three other media for observing bacteriophages. This is illustrated in Fig. 4, where an M17 broth-derived stock of phage 799 replicating on S. cremoris 368 was assayed on M17 and M16 agars. The more clearly defined plaques on the M17 agar are evident. The same was true for whey-derived phage preparations when plated on M17 and TALE 1. Acid-producing activity of S. cremoris AM, and S. lactis ML. as revealed by pH attained after 6 h at 30 M16 agars, as well as for all other virus preparations assayed, although the titers on both media were similar. During this investigation into the efficiency of plating of lactic streptococcal phages on various hosts in M17 agar, it became clear that the medium, because it supported better host growth, allowed the demonstration of phenomena commonly associated with other bacterial virus systems but not previously reported for lactic streptococcal phages. Three examples appear in Fig. 5, where extremely large clear plaques, turbid plaques, and plaques exhibiting diffusion of phage lysin to surrounding uninfected cells are evident. DISCUSSION It is clear from the data presented that the growth of lactic streptococci in M17 medium is improved over that attained in two other com-monly used media, M16 and LB (Fig. 1). The buffering action of GP, as evidenced by the higher final pH in mature M17 broth cultures, apparently allows more total growth and reduced cell death and injury caused by the lower pH reached in other media. Maintenance of lactic streptococci in M17 with daily subculture 811 VOL. 29,1975 and incubation even for 24 h at 30 C before inoculation had little deleterious effect on their subsequent acid-producing ability in milk at either 30 or 22 C (Tables 1 and 2). Maintenance in M16 or LB, however, yielded cells with impaired acid-producing ability, no doubt due to cell death and injury caused by the lower pH attained in these poorly buffered media. These findings suggested that maintenance of the organisms in milk even with daily subculture might cause impaired acid production when cells were reintroduced into milk. This apparently was the case since cells of four widely used starter strains showed improved acid-producing properties when initiating growth in milk if the cells originated from milk containing 1.9% GP (Fig. 3). Since early rapid acid production is highly desirable in such products as cheddar and short-set cottage cheese, future practical value may be found in buffering bulk starter milk with GP. This may prove to be an economical step, since GP is inexpensive and widely used in foods and as a carrier in certain medicines. Slow acid production by lactic streptococci in milk may be due to loss of the ability to use lactose, presumably a rare event (20,23,26), or more frequently to loss of proteolysis, which limits the ability of the organism to obtain nitrogen from milk protein at sufficient rate to allow rapid cell growth (6). Recent data suggest that the genetic determinants for both of these cellular activities (lac and prt) are carried on plasmids (23,25,26), although direct proof is lacking. Reasons for the apparent difference in stability of lac and prt also have not been shown. Since proteinase is localized in the cell wall (30), it is likely that prolonged exposure to acid, which lactic streptococci experience in both milk and nonmilk media other than M17, alters cell wall integrity and encourages loss of proteinase activity. Studies on the influence of different pH levels on the frequency of appearance of prttypes would be revealing; incorporation of GP in media may minimize the loss, especially when, as shown herein, acid-producing activity of cells is improved by minimizing their exposure to low-pH conditions. Since prtappears to be a stable state inherited by descendant cells (6), the effect of the acid environment at the genetic level deserves consideration. Preliminary data obtained in our laboratory indicate that GP addition to milk protects cells from freezing damage. Frozen concentrates of lactic starter cultures are now widely used in the United States, especially for direct inoculation of milk for buttermilk manufacture and to inoculate bulk starter milk intended for use in manufacture of cheddar and cottage cheese. Direct inoculation of vat milk with frozen concentrates, however, has not yet materialized, since at least 107 cells per ml is required to initiate acid production in the milk at a rate to ensure cheese manufacture in the accustomed time (18). The large volume of concentrate presently required to achieve such a cell density makes their use for this purpose impractical. Lyophilized cell concentrates may be applied in this manner in the future (28), and use of GP as a growth medium-neutralizing agent will no doubt prove useful. The usefulness of M17 medium in selecting carbohydrate-requiring mutants also deserves mention. Since wild-type colonies grow to a large size, differences between mutants and parent cells are maximized. The medium, therefore, is finding extensive use in our laboratories to isolate and study such mutants and no doubt will be of value to others for the same purpose. It also is likely that media for other acid-producing bacteria, especially lactobacilli, will be improved by incorporation of GP. In this regard, we have found that S. thermophilus and Lactobacillus bulgaricus strains grow well in the medium, especially if the pH is adjusted to about 6.8 prior to inoculation; comparative growth studies in other media have not yet been made. Few meaningful studies on plaque morphology and lysogeny in lactic streptococci have been reported (13,15,17), presumably because the media usually used allow little differentiation of plaque types because of poor buffering capacity. Nyiendo et al. (24) found that buffering medium was necessary to achieve high titers of lactic phages, and we have found that whey phage stocks at 109 to 1011 plaque-forming units/ ml can be prepared from SM containing GP. It is noteworthy that the pH of M17 medium does not fall below 5.7 even upon incubation of lactic streptococcal cultures for 24 h at 30 C. Thus, not only are differences in plaque size, as determined by the phage host interaction, distinguishable, but other phage phenomena such as lysogeny as visualized by turbid plaques (Fig. 5) became demonstrable. In a subsequent publication, we will report on use of M17 medium to demonstrate widespread lysogeny in the lactic streptococci.

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2020-12-10T09:04:20.430Z

1975-02-01T00:00:00.000Z

237230181

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Polarographic Assay of Hydrogen Peroxide Accumulation in Microbial Cultures A method is described for determining low concentrations of hydrogen peroxide by using a polarographic oxygen electrode to measure the oxygen released into solution on addition of catalase. A sample can be assayed directly without prior manipulation in 3 min. The method is capable of assaying hydrogen peroxide concentrations as low as 7 μM. The method has proved extremely useful for the assay of hydrogen peroxide secreted into milk by lactic acid bacteria. A method is described for determining low concentrations of hydrogen peroxide by using a polarographic oxygen electrode to measure the oxygen released into solution on addition of catalase. A sample can be assayed directly without prior manipulation in 3 min. The method is capable of assaying hydrogen peroxide concentrations as low as 7 ItM. The method has proved extremely useful for the assay of hydrogen peroxide secreted into milk by lactic acid bacteria. Hydrogen peroxide is produced in microbial cultures due to the activity of certain oxidoreductase enzymes. The extracellular concentration is often quite low, especially in cultures which possess strong catalase (EC 1.11.1.6) or peroxidase (EC 1.11.1.7) activities. Special sample preparation is needed for the spectrophotometric assay methods capable of measuring such low concentrations, especially if the growth medium is opaque (1,4,5). Modern oxygen electrodes are capable of measuring small variations in the dissolved oxygen concentration of aqueous liquids. We have taken advantage of this fact to develop a simple assay for hydrogen peroxide in culture media. The addition of excess catalase to a small sample of the untreated culture containing hydrogen peroxide caused the liberation of oxygen, which could be measured with an oxygen electrode, and related to the concentration of hydrogen peroxide using a standard curve. MATERIALS AND METHODS Apparatus. Culture samples were assayed for H,O, in a specially constructed cylindrical glass cell ( Fig. 1) of approximately 4-ml capacity, sealed at the lower end by insertion of the electrode portion of a Beckman oxygen analyzer (model 77701). The cell was waterjacketed to maintain a constant sample temperature of 25 C. The cell contents were continuously stirred during the assay by means of a Gallenkamp overhead stirrer (model SS540) equipped with a nichrome wire loop as a paddle. The stirrer was set at a speed which provided a flow rate of the sample of at least 55 cm/s, over the surface of the electrode. The adequacy of the flow rate was routinely checked by observing that a steady oxygen meter reading was obtained when no change in dissolved oxygen content was occurring in 'Present address: Department of Animal Sciences, University of Kentucky, Lexington, Ky. the cell. The oxygen analyzer meter was routinely calibrated to give a reading of 21% saturation in air. Polarographic assay method. Medium containing H,O, (2.9 ml) was added to the glass cell and stirred to give the desired flow rate. On establishment of a steady reading on the dissolved oxygen meter (or coupled recorder), 0.1 ml of catalase solution containing 50 ,g of purified beef liver catalase (Sigma London Chemical Co.) in 0.05 M phosphate buffer, pH 7.0, was pipetted quickly (using a 0.1-ml constriction pipette) into the bottom of the cell. The continuous stirring ensured rapid mixing of the catalase with the other assay constituents. The immediate rise in dissolved 0, level was noted on the analyzer or recorder. Test media. The method was tested in a minimal medium, a complex medium, and reconstituted skim milk. The minimal medium contained 2 g of glucose, 1 g of NHCl, 0.2 g of MgSO4.7HO, 0.01 g of FeSO,.7H,O, and 0.01 g of CaCl, per liter of 0.05 M Na-K phosphate buffer, pH 7.0. The complex medium consisted of 10 g of yeast extract per liter of minimal medium, and the reconstituted skim milk contained 100 g of nonfat milk solids per liter. The media were sterilized in an autoclave at 15 lb/in for 5 (reconstituted skim milk) or 15 (minimal and complex media) min. Stability of HO,. The stability of H,02 was measured in sterile reconstituted skim milk and minimal glucose (0.2%) and complex media. HO, was added to a concentration of 0.134 mM to 100-ml Erlenmeyer flasks containing 50-ml amounts of each medium. The flasks were held at 20 C without agitation for 5 h, and the HO, concentration was estimated periodically. The rate of 02 loss from supersaturated solutions. The rate of 03 loss from supersaturated solutions to the air above the assay cell was measured. HO, was added to the assay cell containing reconstituted sterile skim milk or minimal glucose (0.2%) or complex media. The concentrations of HO, used (0.26 mM) were such that on addition of catalase the dissolved 0. level in the cell increased to approximately 38% saturation. The dissolved 0. concentration gradually decreased towards the normal calibra- Growth of test culture. Streptococcus lactis C10 was used to demonstrate HO, accumulation during growth. Cultures were grown in 2-liter quantities of sterile reconstituted skim milk in a New Brunswick bench top fermenter at 25 C and agitated at 250 rpm. The pH was maintained constant at pH 6.6 by the automatic addition, on demand, of 1 M NaOH from a burette. The lactic acid developed by the culture was calculated from the amount of NaOH needed to maintain the pH at 6.6. Samples were withdrawn periodically to determine HO, concentrations. RESULTS Assay time. Figure 2 is a typical recorder tracing showing the increase in dissolved 02 content on addition of purified catalase to a sample of reconstituted skim milk containing H202. The level of 02 in freshly sterilized media (or growing cultures) was often less than that of air (21% saturation). However, a steady reading in the oxygen analyzer meter was invariably established in 1 to 2 min, after which 50 Mg of catalase was added. The maximum increase in dissolved 02 content was reached in approximately 1 min, making a total assay time of 2-to 3-min duration. Standard curves. Standard curves were constructed from data relating the increase in dissolved 02 content on addition of catalase to known concentrations of H202, as determined by sodium thiosulfate titration. The H202 solutions were in minimal or complex media or in reconstituted skim milk. Similar graphs were obtained for the minimal and complex media used (Fig. 3). The relationship between increases in dissolved 02 content and H202 concentration was linear as far as 2.0 mM. H202 concentrations as low as 7 AM could be accurately estimated (standard deviation of 5.4% at 6.8 AM). The skim milk standard curve obtained was similar to the other two, except that the increase in dissolved 02 content was slightly greater at any given H202 concentration. The relationship between increases in dissolved 02 content and H202 concentration in skim milk was found to be linear as far as 2.0 mM. The fact that the dissolved 02 increase, on addition of catalase to identical concentrations of H202, was greater in sterile reconstituted skim milk than in sterile minimal glucose or complex media was investigated further. The higher readings were not due to a greater stability of H202 in skim milk than in the other media. H202 was considerably less stable in skim milk than in either minimal glucose or complex media (Fig. 4). A possible explanation for the relatively high readings in skim milk is its greater capacity to retain 02 than either minimal glucose or complex media. Supersaturated to 3 min. Samples of cultures can be assayed directly without prior treatments such as removal of cells or production of clear filtrates from opaque media, such as milk, steps which are essential in spectrophotometric assays (1,4,5). Spectrophotometric assays have the additional disadvantage that complex media ingredients may absorb significantly at the wavelengths employed in such assays, thus causing interference. The sensitivity of the polarographic assay method described compares favorably with that of reported spectrophotometric assays. Concentrations as low as 7 iM could be reliably measured, which means that the assay is five to ten times as sensitive as the methods of Gilliland (5) and Ferrier et al. (4). The establishment of a steady reading for dissolved 02 content of assay samples within 1 to 2 min (Fig. 2) may seem surprising. Some samples had 02 levels lower than that of the air above the assay cell (21% saturation). 02 would be expected to diffuse into the stirred sample from the air above the cell. The steady dissolved 02 reading attained before catalase addition, within 1 to 2 min after starting an assay, indicates that 02 diffusion into the assay cell does not contribute significantly to the dis-solved 02 level of assay samples over the short assay time. The present work indicates that calibration curves constructed from data obtained using a particular medium are only applicable to media of similar composition. Dissolved oxygen increases, obtained from the breakdown of given concentrations of H202 in reconstituted skim milk, were greater than those in chemically defined or broth media. This is not due to a greater stability of hydrogen peroxide in skim milk. As other workers have previously shown (2,5), H202 was quite unstable in sterile skim milk (Fig. 4) and much more unstable than in minimal glucose or complex media. The most likely explanation for the higher dissolved 02 concentrations in skim milk, compared with the other two media, is the former's greater capacity to retain 02 in supersaturated solution (Fig. 5). From Fig. 3 it is evident that the greatest discrepancies between skim milk 02 levels and minimal and complex media 02 levels occurred at the higher concentrations of dissolved 02, i.e., where the degree of supersaturation was greatest. It is likely that more 02 is lost from solution during assay from minimal and complex media than from skim milk, and that the discrepancy increases as the concentration of H202 in the assay cell increases. The method described has been successfully applied to the assay of H202 in cultures of lactic acid bacteria such as S. lactis C10 (Fig. 6). The concentrations produced were well within the range of the assay. The pattern of H202 accumulation resembled those previously obtained for aerated cultures of S. lactis (6) and a Lactobacillus plantarum strain (8). The concentration of H202 accumulated in the aerated skim milk culture after 7 h was 0.1 mM, which is similar to that reported by Hogg and Jago (7) for the same strain. The data of Fig. 6 also show that the rate of acid production by S. lactis C10 in aerated skim milk culture was being inhibited at a very low developed acidity. Other workers (3) have had a similar experience with this strain, growing under aerobic conditions, and attributed the inhibition to H202 accumulation. The concentration of H202 at the time that acid development was being inhibited in the C10 culture of the present study (Fig. 6) ranged from 0.03 to 0.1 mM. Work at this (M. Keane, M.Sc. dissertation, University College, Cork, 1973) and other laboratories (3,9) has shown that addition of H202 at concentrations ranging from 0.029 to 2.3 mM inhibited acid production by several strains of lactic streptococci, including S. lactis C10 growing in milk. The polarographic method allows, therefore, the easy detection of H202 concentrations reported to inhibit lactic acid bacteria. In addition, this assay method could find application in food industries which routinely use H202 as a sterilizing agent. Low, but still unwelcome, levels of residual H202 could be easily detected in any liquid or solid food after emulsification.

v3-fos

2018-04-03T05:23:55.905Z

1975-10-01T00:00:00.000Z

22568043

s2

Gas Chromatographic Presumptive Test for Coliform Bacteria in Water A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively. A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively. Detection and enumeration of coliform bacteria of fecal and nonfecal origin are among the most commonly used microbiological indicators of quality in food and water. Whereas there are numerous analytical methods for detecting coliform bacteria quantitatively, the primarily used techniques require up to 2 days before results are available. Such delays impose obvious health hazzards, and, in the case of food, economic burdens since microbiological results often determine safety for human consumption or storage time. There is, then, need for techniques which make possible rapid assessment of the microbiological quality of water and foods. In an earlier report from this laboratory the detection of Escherichia coli by gas chromatography was described (3). The method was based on the Eijkman concept and detection was accomplished by analyzing for the presence of metabolically produced ethanol in cultures incubated at 44.5 C. According to the Eijkman concept coliform bacteria can be differentiated on the basis of growth at elevated temperatures. Coliform bacteria of fecal origin grow and ferment lactose at 44 to 46 C and are usually + + --or -+ --IMViC types, whereas coliform bacteria from other sources are rarely able to grow in the 44 to 46 C temperature range (4). In this report we describe the application of the Eijkman principle to a gas chromatographic method for the presumptive detection and estimation of coliform bacteria. By incubating cultures inoculated with water samples at 37 or 44.5 C total coliform and fecal coliform concentrations can be estimated. MATERIALS AND METHODS The pure cultures used in this study were a laboratory strain of Escherichia coli B and coliform bacteria isolated from the effluent of the local sewage treatment plant. In experiments involving water samples, the samples were inoculated directly into culture medium, and the indigenous coliforms were allowed to grow out. The growth medium was M-9 salt mixture (6) supplemented with 1.0% lactose, 0.1% Casamino Acids, and 0.1% yeast extract at pH 7.2. The medium was made double strength and sterilized by membrane filtration. For experiments 1 ml of medium was inoculated with an equal volume of water sample, and the cultures were incubated in water baths at 37 or 44.5 C. Best results were obtained when the cultures were stirred with small Tefloncoated magnets and an immersible magnetic stirrer. Coliform concentrations in water samples were determined by the multiple-tube most-probable number method (MPN) in lauryl sulfate tryptose broth (LST) or by the membrane filter procedure (MFC) with M-endo broth both as described in Stand- RESULTS An important consideration in high-sensitivity gas chromatography is a sample free ofinter- U O la fering compounds contributed by the culture medium. In preliminary experiments it was found that autoclaving the culture medium resulted in thermal degradation of lactose and yeast extract which resulted in numerous volatile compounds which chromatographed with metabolically produced ethanol. Filter sterilization minimized interfering compounds contributed by the medium although as shown in the 0-to 420-min panel in Fig. 2 some background peaks were present. The compounds associated with these peaks were found to be associated with lactose; fortunately, the peaks did not interfere with ethanol analysis (Fig. 2). As indicated, one of the chromatographable medium compounds was identified as acetic acid. Also shown in Fig. 2 are the chromatographic changes in the culture medium caused by coliform metabolic activity. It can be seen that concomitant with ethanol formation the acetic acid peak also increased in size. However, acetic acid formation was not used as an indicator of coliform bacteria since it was difficult to reliably detect small increases in peak size. Occasionally a water sample contained microorganisms which grew in the test cultures but did not produce ethanol or other detectable chromatographic changes in the medium. Subculture showed these organisms were non-lactose fermentors. Apparently utilization of other medium constituents did not produce metabolic end products which were detectable under the analytical conditions used in this study. Similarly, when lactose was omitted from the culture medium no chromatographic changes in the medium as a result of microbial activity were detected in 24-h cultures incubated at 37 or 44.5 C. Whereas our initial research indicated that laboratory cultures of E. coli produced ethanol from lactose fermentation, it was of interest to determine if this was also true for coliform bacteria isolated from contaminated water. Water samples taken over a 10-day period were inoculated directly into duplicate LST broth tubes, and the tubes were incubated for 24 h at 37 and 44.5 C. Coliform bacteria were isolated from positive tubes on EMB agar, and the isolates were IMViC typed. The coliform-isolates were inoculated into the M-9 lactose medium described above and incubated at 37 and 44.5 C. Results summarized in Table 1 show that at 37 C all the organisms grew and produced ethanol. At 44.5 C all the + + --group and 16 out of 18 of the -+ --group grew and produced ethanol. Only 8 out of 68 coliform isolates with --+ + characteristics grew and produced ethanol at 44.5 C. The data in Table 1 show that relatively few --+ + coliform bacteria grew at 44.5 C in the M-9 lactose medium. However, when temperature-tolerant organisms with nonfecal IMViC classifications are present in water samples a positive ethanol test results. Consequently, in gas chromatographic experiments ethanol-positive 44.5 C samples were streaked on EMB agar and colonies with the typical sheen tested on citrate agar or IMViC typed. Only when a positive culture was shown to contain citrate-negative coliforms or coliforms with + + --or -+ --characteristics was the test considered positive. In practice we did not find any 44.5 C positive samples which did not contain fecal coliforms, although mixed flora composed of both fecal and nonfecal types were not uncommon. The kinetics of formation of ethanol were the same for pure cultures of E. coli and for cultures inoculated with water samples (Fig. 3). However, as would be expected the time period between inoculation of the cultures and the formation of detectable amounts of ethanol was dependent on the initial coliform concentration of the inoculum. Under the analytical conditions used in the study the minimum detectable ethanol concentration in cultures was 1 to 2 ng. Theoretically the minimum detectable ethanol concentration was 0.1 ng; however, we were not able to attain this level of sensitivity due to base line instability. To determine the relationship between the initial coliform count and the incubation time required for ethanol formation, 1-ml aliquots of diluted water samples were inoculated into equal volumes of 2 x culture media and incubated at 37 or 44.5 C. Total coliform concentrations in water samples were determined by MPN tests in LST broth at 37 C after 48 h and fecal coliform concentrations were determined by the MFC method at 44.5 C after 24 h. Typical 37 C results ( b Tubes were inoculated with 1 ml of each dilution. c Ethanol was determined by gas chromatography. Cultures contained 1 ml of water sample and 1 ml of 2 x M-9 lactose medium. ments (Fig. 4) show that initial coliform counts were exponential functions of the incubation times required for ethanol formation. Coliform detection required less time at 37 than at 44.5 C for reasons which are discussed below. With respect to sensitivity, at 37 C it was possible to consistently detect differences in the incubation time required for ethanol formation when the initial number of organisms differed by a factor of two or more at initial counts of 10 per ml or higher. When the initial count was less than 10 coliforms per ml of sample, variability tended to reduce reliability. Detection of small numbers of fecal coliforms at 44.5 C was more variable than total coliform detection at 37 C. In part this was due to longer lag times at 44.5 C. In addition we found that when the initial fecal coliform count was less than 10 per ml the cultures frequently failed to grow at all. Apparently, fecal coliforms in water samples have difficulty in adapting to 44.5 C; however, we found that once adapted to this temperature the organisms grew at the same rates as at 37 C. It is worth noting that in the experiments summarized in Fig. 4 the presence of coliform bacteria was confirmed in 79 out of80 water samples positive for ethanol. As might be expected, coliforms in water samples cultured at 37 C were usually mixed IMViC types, whereas at 44.5 C fecal IMViC types predominated. At 37 C the tests could be terminated after 12 h ofincubation since cultures negative for ethanol after 12 h were negative for coliform bacteria at 24 and 48 h. At 44.5 C the cutoff time for ethanol formation was 14 h. DISCUSSION The gas chromatographic procedure described here could serve as a useful presumptive test in cases where a specific coliform limit is established. For example if the limit was 10 coliforms or less per ml then a test culture Relationship between the initial coliform concentration in water samples and the incubation time required for ethanol formation. Cultures contained 1 ml of water sample and 1 ml of2 x medium. Total coliforms were determined by the MPN test in LST broth at 37 C, and fecal coliforms were determined by the MFC test in M-Endo broth at 44.5 C. Samples for gas chromatography were taken at 30min intervals. containing 1 ml of the sample should not contain detectable ethanol after 9 h of incubation at 37 C or 11 h at 44.5 C. Cultures which were positive for ethanol could be confirmed by conventional methods. If greater sensitivity or shorter detection times are required samples can be concentrated by centrifugation or membrane filtration. We have obtained the same detection times with concentrated samples as with unconcentrated samples both at the same initial coliform density. Shorter detection times would also be possible by operating the gas chromatograph at maximum analytical sensitivity. If this had been possible coliform detection times would have been shortened by 1 h or more. With respect to rapidity the gas chromatographic method detection times are comparable with those reported for radiorespirometry (2,5) and calorimetry (5). At present, incubation at 44.5 C is not completely selective for fecal coliform bacteria since mixed coliform populations were occasionally isolated from water samples cultured at 44.5 C in M-9 lactose medium. This was also seen when samples were cultured in LST broth for MPN determinations at 44.5 C. It is not known ifthe nonfecal coliform organisms were fermenting lactose and producing ethanol at 44.5 C or growing synergistically with fecal types. An advantage of the method described here is that coliform analyses can be done without sample preparation. Direct analysis of culture medium is possible since the column packing has ideal characteristics for separating polar compounds such as water and alcohols. Columns are relatively unaffected by accumulation of nonvolatile culture medium constituents or bacterial cell components since peak shapes and retention times were the same after 2,800 analyses on the same column. We are aware of the possibilities of automating the gas chromatographic procedure and this problem is currently under investigation. We are also investigating application of the method to determine coliform contents and total bacterial counts in foods. The results of these studies will be reported later.

v3-fos

2018-04-03T01:32:10.151Z

1975-03-01T00:00:00.000Z

29795567

s2

Detection and Growth of Enteropathogenic Escherichia coli in Soft Ripened Cheese The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 10-5 at room temperature and over 10-3 at 4 C within 10 days. With higher initial counts, levels up to 10-9 were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination. In the United States, enteropathogenic types of Escherichia coli (EPEC) are usually associated with cases of infantile diarrhea. Studies from India and Vietnam by Gorbach et al. (6) and Japan by Sakazaki et al. (9) showed that EPEC cause disease not only in children but in adults as well. Dupont et al. (2) performed studies with several types of EPEC to determine the effects of these organisms on animals and human volunteers. Their work demonstrated that EPEC can cause disease in man by at least two mechanisms: by production of a choleralike enterotoxin and by an invasion of intestinal epithelial lining. Before November 1971, there were reports of water-borne outbreaks of EPEC in the United States, but no confirmed outbreaks were associated with foods (1). However, from 30 October to 10 December 1971, over 200 persons in more than 90 separate outbreaks suffered acute food poisoning symptoms after eating imported Camembert or Brie cheese. Our laboratory investigated several complaints of food poisoning in which Camembert cheese imported from France was implicated. None of the common food poisoning organisms were detected; however, high levels of E. coli were encountered and, with the methods used, were frequently the only organism found. This led to the serotyping of these organisms and their subsequent identification as EPEC. However, no in vivo tests were performed to confirm pathogenicity. Therefore, the term EPEC as used in this paper refers only to those strains belonging to serogroups having known association with diarrheal disease as outlined by Ewing (4). The most frequently detected type was a strain of 0124:B17 E. coli that did not ferment lactose within 48 h. This strain was found usually by direct plating of the cheese on agar media and was also recovered from individuals who had contracted the disease (7). These isolates all failed to ferment lactose broth within 48 h; however, after that time fermentation did occur with some strains. Since not all isolates were tested after 48 h, the term "lactose negative" will be used throughout the remainder of this text. Methods for analysis of E. coli normally used fermentation of lactose as a selective factor. Therefore, slow or nonfermenting strains could remain undetected particularly when present in low numbers. With this in mind, existing methods for analysis of E. coli were evaluated and a tentative procedure for the detection of EPEC was developed that would not exclude lactose-negative strains. In addition, studies were initiated to determine whether the high levels of E. coli in the cheese were due to improper processing or simply to the ability of these organisms to grow in the product under normal conditions of handling. Media. Lauryl tryptose broth, brilliant green lactose bile broth, EC broth, GN broth (Hajna) and MacConkey agar were obtained from Difco and prepared according to the manufacturer's instructions. A modification of MacConkey agar was also used. In this, 5 g of lactose and 5 g of arabinose were substituted for the 10 g of lactose per liter normally present. A modification of GN broth was also used in which 1 g of lactose and 1 g of arabinose per liter were substituted for the glucose and D-mannitol. Antisera. Isolates were screened as presumptive EPEC by typing with E. coli OB poly A and poly B and with E. coli OK poly C antisera, all from Difco. Monovalent E. coli 0, OB, and OK antisera (Difco) were used in the identification of the specific serogroups present. Difco polyvalent fluorescein-labeled anti-E. coli globulin (poly A, B, and C) was used for the screening of cheese samples by the fluorescent antibody (FA) method. A 1:4 dilution of these conjugates was determined to be an appropriate working dilution by the method of Goldman (5). Identification. E. coli isolates were identified as possible enteropathogenic types on the basis of biochemical characteristics and serological titrations as outlined by Ewing (4). Enumeration of E. coli. Samples of imported soft ripened cheese collected at the time of entry at the Port of New York were analyzed for the numbers of E. coli present immediately upon receipt in the laboratory. Serial dilutions out to 10-7 were prepared in potassium phosphate buffer at pH 7.2. The same enumeration procedure was used in studies on the growth of E. coli in stored cheese samples. Each dilution was inoculated into three tubes of lauryl tryptose broth containing inverted durham tubes. Cultures producing gas in 24 and 48 h at 37 C were then subcultured into EC broth and incubated in a constant-temperature water bath at 45.5 C. The presence of gas was recorded after 24 and 48 h. The most probable number (MPN) of E. coli was then calculated per gram of original cheese. FA procedure. Three smears were prepared from GN broth cultures by using a 2-mm loop onto multiwell Teflon-coated slides (5). Smears were air dried; the slides immersed in xylene for 3 min, drained and placed in Kirkpatrick fixative solution (ethyl alcoholchloroform-formalin [60:30:10]) for 3 min, and air dried. Treatment of the slides with xylene removed lipids that interfered with fixation. The slides were then rinsed in 95% ethyl alcohol and air dried. The three smears were then treated separately, each with one drop of poly A, B, or C E. coli conjugate. The slides were placed into moist chambers and incubated for 30 min. Excess conjugate was drained and the slides were rinsed in phosphate-buffered saline (pH 7.5). The slides were washed twice in a phosphate-buffered saline bath for 5 min and rinsed in distilled water. After air drying, a drop of buffered glycerol saline at pH 7.5 was placed on the slide and covered with a no. 1 cover slip. Slides were examined with an American Optical Series 20 Microstar microscope equipped with a 50-W mercury vapor lamp. A cardioid dark-field condenser, BG 12 exciter filter, and an OG 1 barrier filter completed the system. Fluorescent cells were rated on a scale of 1 + to 4+. Slides with cells rated 3+ or 4+ (bright cell outline with clear lumen) were considered positive. RESULTS In preliminary studies it was found that strains of EPEC grew well in GN broth. It was thought that selectivity for EPEC might be improved by substituting lactose and arabinose for the glucose and D-mannitol normally present in this medium as carbon sources. According to Edwards and Ewing (3), Edwardsiella, Providencia, Serratia, and Proteus for the most part will not ferment either lactose or arabinose. However, 91% of E. coli ferment lactose and 99% use arabinose. Thus, chances of encountering strains of E. coli incapable of growth on one or the other of these sugars should be minimal. GN broth, the modified GN broth, and several other media normally used for selection of E. coli were compared for ability to support the growth of EPEC. The test organisms were a lactose-negative and a lactose-positive strain of E. coli type 0124:B17 and a nonpathogenic strain of E. coli. The two 0124:B17 strains came from samples of cheese known to have caused diarrhea, and it is highly likely that they were in fact pathogenic. The three strains were grown for 24 h in heart infusion broth, and 0.01 ml of each culture was inoculated into tubes contain-ing 10 ml of the test media. The tubes were incubated for 18 h at 37 C and viable cells were estimated by plate counts on standard methods agar. The results in Table 1 show no significant difference in the levels of growth achieved by the nonpathogenic strain of E. coli in any of the media tested. Some inhibition of the lactosepositive pathogenic strain was noted in EC broth at 45.5 C, possibly because of inhibition by bile salts and the elevated temperature. All other media yielded comparable amounts of growth with this strain. The differences in growth levels for the pathogenic lactose-negative strain were much more pronounced. Growth was substantially less in the broths having a lactose base; however, cell numbers in GN broth and the modified GN broth were higher and of about the same value. Evidently, with the pathogenic lactose-negative strain the amount of growth in the modified GN broth was not affected by the fact that only the arabinose and not the lactose present in that medium could be utilized. In another experiment, a modified MacConkey agar was tested as a plating medium. Although lactose-negative EPEC will grow on MacConkey agar with the normal formulation, colonies do not appear as typical E. coli since acid is not produced from the lactose present. Normally these colonies would not be picked for routine screening. The medium was therefore modified by deleting half the lactose and substituting an equivalent amount of arabinose. This modification eliminated the need to pick lactose-negative isolates on MacConkey agar. This modified medium was tested by streaking cultures representing 20 different serogroups of E. coli on the plates and noting colonial morphol- ogy and color reaction. Strains were tested both singly and in combinations of a lactose-negative with a lactose-positive type. All strains gave typical colonial morphology and color reaction. No differences in selectivity were noted on plates streaked with the mixed cultures. When typical E. coli colonies were randomly picked from such plates and transferred to phenol red lactose broth, approximately equal numbers of lactose-positive and lactose-negative cultures were recovered. On the basis of these observations, the cultural procedure as outlined in Fig. 1 was devised for the screening of EPEC in soft cheese. On day 1, a slurry consisting of 50 g of cheese in 50 ml of modified GN broth is prepared. After 1 h of incubation, a loopful of the slurry is streaked onto modified MacConkey agar plates. One hundred and fifty milliliters of modified GN broth is then added and the slurry is incubated for 18 to 24 h at 37 C. On day 2, a loopful of the slurry is streaked onto a second set of modified MacConkey agar plates. The first plates are examined, and if typical colonies are present a minimum of 10 are picked. Each colony is used to inoculate a slant of Simmons citrate agar, a slant of heart infusion agar, and a tube of KCN broth. On day 3, if the citrate and KCN tubes are negative, the heart infusion slant is used as a source of cells for serological screening. The second set of modified MacConkey agar plates is used as above in cases where the initial direct plating does not yield presumptive E. coli based upon negative reactions in citrate and KCN. Isolates that are both KCN and citrate negative and give a positive reaction with E. coli OB antisera before and after boiling are considered presumptive positives. These cultures are then identified biochemically and serologically as outlined in the CDC Laboratory Manual (4). The above procedure has been used successfully during the last 2 years for routine screening of EPEC at the New York District FDA laboratories. Over 2,000 cheese samples were examined, and approximately 10% of the samples were found to contain E. coli belonging to serogroups associated with diarrheal diseases. The majority of these isolates were detected shortly after the initial food outbreak and belonged to the following serogroups: 0124:B17 (lactose negative); 0124:B17 (lactose positive); 0112:B11; 0124:B15; 0128:B12; and 0127:B8. Since not all strains in a given serogroup are virulent, actual pathogenicity of an isolated strain should be confirmed by a test such as the ileal loop procedure. After the initial samples generated by the outbreak had been analyzed, attempts were also . coli in soft ripened cheese. GN broth was modified by substitution of lactose and arabinose for glucose and D-mannitol. MacConkey agar was modified by substitutions of arabinose for half the lactose. Cells from slants of heart infusion agar were used for serological screening. made to screen cheese samples for EPEC by using immunofluorescent techniques. We felt this would be feasible since the conjugates available did cover serogroup 0124:B17, the organism implicated in the food poisonings. A slurry of cheese was prepared in modified GN broth and incubated for 18 to 24 h at 37 C as previously described (Fig. 1). After incubation, 0.5 ml was inoculated into 5 ml of modified GN broth and incubated for 4 h at 37 C. This second enrichment was found necessary to reduce the carry-over of product that fluoresced brightly under ultraviolet light. Three smears were prepared and stained with poly A, B, or C E. coli FA conjugate as described in Materials and Methods. Several hundred cheese samples were screened by this procedure. A number of positive enrichment cultures were detected, and these were identified as EPEC types by the cultural, serological, and biochemical procedure outlined in Fig. 1. No false FA negatives were found, but a false-positive rate of approximately 20% was encountered. These results indicate that the FA method might be developed as a routine screening procedure. If the 4-h enrichment culture is FA negative, then the analysis could be terminated; those cultures giving a positive reading would be streaked on modified MacConkey agar for subsequent confirmation. In some of the first cheese samples analyzed at the time of the food poisoning outbreak, counts in excess of 3 x 107 E. coli were detected. Studies were undertaken to determine the significance of these high levels. Fifty-five imported cheese samples (approximately 1 lb [454 g] each) were collected at the port of entry and brought to the laboratory under ice. Although the cheese was refrigerated during shipment, the actual temperature and any previous storage history was not known. On arrival in the laboratory, the samples were placed in a refrigerator at 4 C equipped with a continuous recording thermometer. Samples were individually removed from the refrigerator and cut into two sections. One section was returned immediately to the refrigerator. A portion of the other section was used to determine the initial level of E. coli using the MPN procedure. The remainder of this section was stored at ambient room temperature. Although the actual temperature of the refrigerated cheese sections was not known, it was assumed that they reached 4 C very rapidly since they were relatively small in size and were stacked to allow maximum air circulation. After 2, 4, 8, and 10 days of storage, the levels of E. coli in both sections were determined again by the MPN procedure. Precautions were taken to insure that there was no significant increase in temperature of the cheeses when they were removed from the refrigerator for enumeration of E. coli. The initial MPN values varied from less than 10 to 105 E. coli/g. The numbers of E. coli in all 55 samples increased upon storage. Data representing three typical cheese samples with high, medium, and low initial levels are shown in Fig. 2. The rate of growth for E. coli in the portions maintained at room temperature was significantly greater than for those kept refrigerated. However, given a significant period of time, sections of cheese maintained under refrigeration often achieved the same levels as their counterparts kept at room temperature. Apparently, the initial level of E. coli did not influence the ability of the organisms to eventually propagate. Samples that had 10 or less E. coli per g and even some with levels initially undetectable demonstrated an increase in E. coli upon holding. After 10 days of storage, samples held at room temperature had more than 105 E. coli/g. The refrigerated counterpart had more than 103 E. coli/g. Each type of symbol refers to a separate cheese sample that was divided in half and stored at two different temperatures. The data shown represents typical results for cheese samples with high (0), medium (A), and low (0) initial levels of E. coli. DISCUSSION The cheese samples collected as a result of the outbreak in 1971 and analyzed by our laboratory were found to contain as many as 3 x 107 EPEC/g. In the F.R.I. Annual Report of The University of Wisconsin, Trenk and Deibel also found E. coli in soft ripened cheese at levels greater than 100,000/g. Due to the high numbers present in the cheese, more than 90% of the EPEC detected in the original outbreak could be isolated by direct plating of the product. The strains isolated by direct plating were frequently not of the same type as encountered after incubation of the product in an enrichment broth. Selective agars streaked from enrichment broths, such as lauryl tryptose, brilliant green lactose bile, and EC, were predominantly populated with lactose-positive organisms in serogroups not associated with disease, and in many instances the negative or late lactose-fermenting E. coli, 0124:B17, generally found by direct plating, was not present. Studies conducted by Sakazaki et al. (9) further substantiate the need to look for lactose-negative E. coli. Over an 8-year period, of the 764 types of E. coli that these workers found associated with disease in man, 393 were negative lactose-fermenting strains. The need to pick at least 10 colonies per plate must be emphasized. The methods used for isolation do not select for only pathogenic types but for all E. coli that might be present. Furthermore, many isolates may autoagglutinate even in saline solution, making serological identification impossible. From samples consisting of 10 packages of cheese in which 10 colonies were picked per cheese, as few as three isolates were identified as EPEC. Therefore, initially we chose a method using a direct plating of the cheese slurry on MacConkey agar and enrichment in GN broth with subsequent streaking on MacConkey agar. In both instances, colonies appearing lactose negative or positive were picked for biochemical and serological screening. Later this procedure was changed to use of the modified MacConkey agar and modified GN broth. On the modified Mac-Conkey agar it was assumed that all or almost all E. coli strains would produce an acid reaction regardless of whether they were lactose positive or negative. Organisms belonging to the genera Edwardsiella, Providencia, Serratia, and Proteus would not give acid reactions, and if still present after enrichment in modified GN broth they would be readily distinguishable from E. coli. One disadvantage to the use of modified MacConkey, however, is that if lactose-negative E. coli are present in low numbers, they may be missed by random picking of 10 typical colonies. At this point it is not known which is the most advantageous, exclusion of the above four genera or the ability to distinguish lactose-positive and -negative strains of E. coli early in the analysis. If one were attempting to specifically isolate a lactose-negative strain, the nonmodified agar would be the obvious choice. For routine screening we prefer the modified agar medium; however, perhaps both should be used. Analysis of cheese manufactured by the firm implicated in the original food poisoning outbreak revealed that the problem was not confined to only one type of product. EPEC was found in multiple lots of Camembert, Brie, and Coulommiers cheese. At that time all products manufactured by the firm were removed from the market and a survey of all soft cheeses entering the Port of New York was instituted. During a 1-month period more than 10% of the soft ripened cheeses tested were found to contain EPEC types and the problem was not limited to one manufacturer. Once again these organisms were often encountered in very high numbers. The curing process required for soft cheese production and the physical properties of the final product are significant in explaining how high levels of E. coli can develop. Soft ripened cheese has a moisture content generally greater than 50%. After the formation of the curd, the curing or ripening is caused by decomposition of protein due to the activity of molds. This activity reduces the acidity of the curd and the pH rises from about 4.9 to 7.5. Thus, the acidic products produced originally by the growth of bacteria are neutralized and an ideal environment is created for the growth of E. coli. Those cheeses found to contain over 6 x 107 E. coli/g had pH values in the range of 7.2 to 7.4. A neutral pH after this much growth is normally not encountered in food products, and acids produced by bacteria usually cause inhibition and eventually reduction in cell numbers. It is well known that E. coli can propagate in dairy products. For example, Olson et al. (8) and Watrous et al. (11) demonstrated that coliform levels can increase in milk upon holding at 45 F after pasteurization. These studies were conducted on milk that had no detectable coliforms immediately after pasteurization, and precautions were taken to prevent post-processing contamination. Presumably, very low levels of E. coli were actually present after pasteurization but were not detected by the cultural methods, and these were capable of subsequent growth. The MPN of E. coli in cheeses stored at 4 C and at room temperature was determined in the studies reported here by ability to produce gas in EC broth at 45.5 C. Although this procedure is not a confirmatory one, it is frequently used as an indication of the presence of E. coli. Evidently, E. coli is capable of growing in soft ripened cheeses when low levels are present initially, even under controlled refrigeration. Another possibility is that the increase in numbers does not indicate growth but represents injured E. coli cells that were able to repair themselves at 4 C and ultimately grow in EC broth at 45.5 C. In any event, our studies emphasize the need for methods sensitive enough to detect extremely low levels of EPEC. At this point it is impossible to determine the relative significance of pasteurization and subsequent sanitation as regards the initial numbers of E. coli detected in the finished product. The FA procedure tested here has been successful in detection of E. coli 0124:B17 in enrichment cultures inoculated with cheese samples. The average cheese sample as received by FDA consists of 10 individual packages. Assuming 10 colonies are picked for each package tested, a sample would generate 100 cultures for identification. If the FA procedure could be developed as a routine screening method, the need for costly and time-consuming biochemical and serological testing of hun-dreds of negative isolates could be eliminated. This is not possible at the present time because the conjugates now available commercially are limited in their coverage of the EPEC types and would need to be expanded for use in a broad screening application. In the United States, food-borne strains of E. coli capable of causing serious disease in adults have so far only been associated with soft ripened cheese. Nevertheless, other foods might harbor and transmit these organisms, and testing laboratories must consider routinely that E. coli is a possible food-borne pathogen. In considering testing methods to be used, it must be emphasized that serotyping simply identifies an organism as a member of a group previously associated with the disease and does not establish that it is actually pathogenic. All organisms belonging to that group may not be pathogenic. Furthermore, it is known that factors associated with virulence can be transmitted from donor strains to nonpathogenic strains. For example, Smith and Halls (10) have shown that a genetic factor, presumably a plasmid, responsible for enterotoxin production in certain strains of E. coli can be transmitted to other non-enterotoxigenic strains of E. coli. The recipient strains were capable of causing diarrhea in pigs. Therefore, it is possible that new pathogenic strains can develop that have serotypes not previously associated with disease. At present, pathogenicity can only be established by testing in animals by a procedure such as the ileal loop technique. Unfortunately, such a procedure is too timeconsuming and expensive to use for routine analysis of all suspected isolates. Thus, existing procedures for detection of E. coli must be reevaluated and perhaps new and simpler methods devised for identification of the pathogenic strains.

v3-fos

2020-12-10T09:04:20.569Z

1975-08-01T00:00:00.000Z

237230225

s2

Aspergillus flavus Infection and Aflatoxin Production in Fig Fruits Immature fig fruits did not support colonization and aflatoxin production by Aspergillus flavus Lk. but became susceptible when ripe. While sun-drying on the tree, fruits were particularly vulnerable to fungal infection and colonization. Aflatoxin accumulation equaled levels frequently reported for such seeds as peanuts and cereal grains. Dried figs imported into the United States are sometimes rejected because of contamination with aflatoxin (3). Its presence in foreign-produced figs suggests that aflatoxin could, under certain conditions, be present in domestic figs and pose a threat to domestic production. The Food and Drug Administration therefore instituted a sampling program for 13 domestic food products including figs (2). Since aflatoxin is a potent carcinogen, it is imperative that the toxin be avoided (8), but effective avoidance must presumably be based on knowledge of the conditions that favor aflatoxin production and accumulation. In particular, it is important to know the time and circumstances of fruit invasion by the causal fungus Aspergillus flavus Lk. Aflatoxin contamination has been prevalent in plant seeds where A. flavus is a facultative parasite often preferentially colonizing embryos (7). First reported in 1961 in peanuts (4), aflatoxin has been commonly associated with peanuts, cereal grains, cottonseed meal, and milk from animals that have consumed such seeds. Fewer studies have been concerned with the invasion by A. flavus of living fruits destined for drying, and it is not completely clear whether the fungus is a pathogen of fruit as well as of seed. Whether A. flavus can parasitize living fruit tissue is a critical question. If A. flavus is a fruit pathogen, infection, colonization, and aflatoxin production would be expected in the orchard. If it is a strict saprophyte, one must look for faulty or inadequate methods of handling, drying, or storage, permitting fungus colonization of the dried commodity. This study was done to determine: (i) whether A. flavus is pathogenic to fig fruits and, if so, at what stages are they susceptible; (ii) whether fig fruits are a good substrate for aflatoxin production and accumulation; (iii) the effect of drying speed on aflatoxin accumula-tion; and (iv) the role of dried-fruit beetles and/or other insects in establishment of the fruit infections. MATERIALS AND METHODS The culture of A. flavus Lk. used in these tests was obtained from Eli Crisan, Department of Food Science and Technology, University of California, Davis. Growth in rice medium verified its abundant production of aflatoxins B, and G,. Stock cultures were maintained on potato-dextrose agar slants held at about 7 C. Spores used for inoculations were produced by colonies growing on 75 ml of potatodextrose agar (Difco) in 300-ml Erlenmeyer flasks incubated for 2 to 3 weeks in an air-conditioned laboratory (22 to 24 C). Spores were harvested from cultures by adding about 45 ml of Tween solution (2 to 3 drops of Tween 80 in 200 ml of sterile distilled water) to the culture flask. After the flask was swirled to wet and dislodge conidia, the spore suspension was transferred to a 50-ml centrifuge tube and centrifuged to form a pellet of spores. The spores were rinsed by resuspending in Tween solution, centrifuging, and decanting. After resuspension in fresh Tween solution, the concentration of spores was determined by comparing absorbance in a Bausch & Lomb Spectronic-20 colorimeter against a standard curve previously established for spore concentrations determined with a hemocytometer. Most inoculations were made with a 1-ml syringe. One-tenth milliliter of a suspension containing 105 conidia was injected through the ostiole into the central cavity of the fig fruit (syconium). Used in inoculations in a few experiments were dry spores. Spores were harvested by vacuum from the surface of cultures by using a miniature cyclone spore collector (a description will be supplied upon request) containing sufficient talc to dry the spore surfaces. Spores were applied with a camel's-hair brush to the fruit surface or to the interior of bags containing fruits and insects. Fresh fruit samples were divided and weighed. One portion was used for moisture determinations. The other portion was frozen for subsequent analysis for aflatoxin. The frozen sample was thawed and extracted by a procedure commonly used with peanuts, peanut meal, and peanut butter (1). Briefly, the sample was blended in a Waring blender for 3 min in a 4% aqueous KCI-methanol solution. The concentration of the methanol was adjusted to 55% after taking into account the moisture in the sample. The volume in milliliters of the extracting solution was adjusted to be at least three times the weight in grams of the sample. The blended sample was centrifuged, and the supernatant (volume noted for calculations) was transferred to an Erlenmeyer flask containing 10 ml of 20% lead acetate solution. Approximately 50 ml of filter aid was added. The sample was allowed to stand for 2 to 3 h before being filtered through a filter-aid pad in a Buchner funnel with use of vacuum. The filtered sample was transferred to a separatory funnel, and a volume of hexane was added equal to approximately one-third the volume of the sample. After shaking for 1 min, the phases were allowed to separate. The hexane was discarded and the sample was extracted three times with 30-ml portions of chloroform. The combined chloroform portions were washed with 30 ml of water by shaking the chloroform-water mixture in a separatory funnel for 1 min. The phases were allowed to separate and the chloroform was removed and evaporated to approximately 5 ml under N. in a 55 C water bath. The chloroform extract was transferred to a vial and evaporated to dryness under N., after which the vials were sealed and stored at about -10 C until analyzed. Volumes were measured where losses were possible throughout the extraction, and corrections were applied to obtain an accurate sample weight. Initial analyses were by thin-layer chromatography, essentially as suggested in Method III, Official Methods of Analysis (1), except that precoated thinlayer chromatography plates (EM Laboratories, Inc.) were developed with water-saturated chloroform-acetone firm-ripe; soft-ripe; and shriveled-ripe. Fruits classified as green had only partially changed from green to purple ("Mission" fruits) and green to yellow ("Kadota" and "Calimyrna"). Firm-ripe fruits were about fresh-market maturity and could be "eaten out of hand." Soft-ripe fruits had softened completely but without visible wilting, and shriveled-ripe fruits were still hanging on the tree but had developed a stretched, limber neck and were obviously wilted. In some cases, ripeness was further characterized by soluble-solids content of the fruits. The Conidia (10w in 0.1 ml of water) were injected through the ostiole into the fruit cavity. Subsequent inoculations were on 21 May, 4 June, and 19 June. On the final date, many of the fruits were fully devdoped and had reached the "green" category. When harvested, on 2 July, fruits inoculated at each date were segregated into various ripeness classes. A portion of the fruits judged shriveled-ripe were allowed to dry in sunlight. All other fruits were frozen immediately after harvest and stored until analyses were performed. In a second experiment, 200 fruits each of secondcrop Mission, Kadota, and Calimyrna were inoculated through the ostiole into the fig cavity as in the previous experiment. The fruits were nearly full size but still green. Twelve days later, when harvested, the fruits were divided into the ripeness classifications "green," "firm-ripe," and "shriveled-ripe." Four replicate samples of fruits (250 to 300 g of Mission, 170 to 250 g of Kadota, and 300 to 400 g of Calimyrna) representing each ripeness classification were frozen and stored until analyzed. In a third series of experiments, to better assess the effect of the length of time between inoculation and drying on aflatoxin levels, firm-ripe Mission fruits were inoculated while on the tree and were variously treated: (i) allowed to dry in place on the tree in a manner similar to commercial practice; (ii) inoculated and harvested immediately; (iii) inoculated and harvested after 24 h; (iv) inoculated and harvested after 72 h. Artificial drying was started immediately after harvest by holding fruits in a low-humidity constant-temperature room at 35 C with air movement provided by fans. A fourth experiment was devised to determine the (6) and may be associated with transmission of A. flavus. The inoculation methods used were: (i) injection of spore suspension (0.1 ml containing 10' conidia) into the fruit cavity with a syringe; (ii) dusting a dry conidia-talc mixture onto the surface of fruits with a camel's-hair brush; and (iii) dusting dry conidia on fruits subjected to the foraging of dried-fruit beetles. Control fruits were not inoculated. The treatments were replicated three times. After inoculations, the bagged fruits were placed in a constant-temperature room (35 C). Aflatoxin accumulation was used as a measure of the extent of colonizing activity. Table 1 shows the susceptibility of fig fruits to invasion by A. flavus. Green Mission fruits remained free of aflatoxin, presumably because they resisted invasion by A. flavus. As the fruits became firm-ripe and started to soften, they lost their resistance. Aflatoxin accumulation presumably continued until drying had progressed to the point that fungal growth ceased. RESULTS Results shown in Table 2, a summary of the second Experiment, confirm the previous results in that green fruits contained little aflatoxin. Firm-ripe fruits contained high levels, and the shriveled-ripe fruits were highest of all in aflatoxin. Evidently, the shriveled-ripe fruits generally had the highest levels of aflatoxin because they had reached the susceptible firmripe stage the earliest and had been colonized by the fungus for the longest period. Fruits allowed to dry on the tree in the experiment assessing the effect of the length of time between inoculation and drying on aflatoxin levels showed the highest level of ,sg/g. Fruits inoculated but harvested immediately had only 0.05 i 0.02 gg/g, presumably indicating that little fungus colonization had occurred before growth was arrested by drying. Table 3 shows that in the experiment to determine the influence of beetles on relative aflatoxin accumulation after various methods of inoculation, all inoculated fruits accumulated aflatoxin. It is therefore evident that the foraging of dried-fruit beetles was not required to infect these very susceptible ripe fruits, since dry spores Although no evidence could be detected of fungus colonization or aflatoxin accumulation when fruits were green, they became very susceptible when ripe enough to be eaten out of hand. Rapid fungal colonization and aflatoxin accumulation presumably continued until fungal growth was stopped by lack of moisture in the dried fruit. Further, fig fruits accumulated, on a dry-weight basis, aflatoxin levels comparable to those found in peanuts, soybeans, sorghum, corn, wheat, rice, and cottonseed (5). Spores dusted on the surface of highly susceptible fruits were able to infect and colonize the fruits, suggesting that conidia of A. flavus are capable of penetrating the fruit skin rather than requiring a wound for entry. Several alternative possibilities exist, however, which might have permitted a wound pathogen to enter fruit readily. Minute wounds might have been present in the fruit skin. The foraging of microscopic animals, such as insects or mites, might have caused wounds not readily visible to the naked eye. Or, sufficient fruit juice may have been present on the fruit surface to permit some fungal colonization before penetration, an event often facilitating penetration. It is evident that aflatoxin is a problem in the orchard. In fact, sun-drying in the field, traditional with many dried fruits, provides conditions highly conducive to aflatoxin accumulation if A. flavus infection occurs. The fruits are very susceptible to infection by conidia of A. flavus on exterior fruit surfaces as well as by conidia carried into the interior of the fruit by insects. Further, temperature conditions generally prevailing during fruit harvest would seemingly be near ideal for growth of the fungus. Despite this high susceptibility of the fruit and the favorable conditions for infection and colonization, aflatoxin incidence in figs is generally believed to be low. The reason for a low incidence is not readily evident. Possibly the fruit infection level is associated primarily with spore levels in and adjacent to orchards. The relatively low incidence possibly reflects poor conditions for fun-gus sporulation and/or inefficient spore dispersal.

v3-fos

2018-04-03T01:12:10.308Z

1975-09-01T00:00:00.000Z

23860665

s2

Feed Refusal Factors in Pure Cultures of Fusarium roseum ”graminearum” Isolations from 1972 Wisconsin feed refusal corn yielded predominantly cultures of Fusarium roseum ”graminearum.” With one possible exception, none of the selected isolates of this fungus induced emesis in pigeons, whereas six of nine isolates produced feed refusal responses in all test animals. A single isolate of F. roseum ”equiseti” also induced a severe refusal response and possibly slight emesis. None of the other fungi isolated from this corn (F. moniliforme, Acremoniella atra) or controls caused either emesis or feed refusal. Zearalenone was detected in all isolates and was shown to be partially responsible for refusal activity. The remaining activity was ascribed to one or more nonvolatile, neutral, relatively polar molecules. T-2 toxin, although not detected in these isolates, was shown to have dramatic refusal activity in rats. The 1972 growing season was particularly favorable for the development of Gibberella stalk and ear rot (Gibberella roseum 'graminearum' = G. zeae = Fusarium roseum 'graminearum') on corn (Zea mays) in the north central United States (6). In most areas temperatures were generally cool, rainfall frequent and often heavy during much of the late summer and autumn. At crop maturity ears on a high percentage of field plants (as much as 25% in some Wisconsin fields) were invaded by F. roseum. The extent of invasion varied from minor tip rot to almost complete decay of the ear. The situation was further aggravated by a delayed harvest due to wet conditions and by a shortage ofnatural gas for artificial drying ofthe corn. Heavily infested lots of this corn when fed to swine resulted in feed refusal and occasionally emesis (5). Refusal was evident with this corn even when it was later artificially dried or fermented as high moisture corn in sealed silos. Symptoms of vulvo-vaginitis (9) were generally absent. Cattle, horses, and poultry failed to develop any obvious problems after ingestion of similarly infested corn. Later development of other potentially toxic fungi in crib-stored, high-moisture corn made the overall field problem even more complex. Metabolites responsible for swine feed refusal have not been identified, nor has really definitive evidence been presented to suggest that feed refusal is a separate phenomenon from either emesis or uterine hypertrophy as was suggested by Curtin and Tuite (3). In their feeding trials using refused feed samples naturally infested with F. roseum, however, uterine hypertrophy was not observed in test animals. Recently Roine et al. (10) observed typical refusal accompanied by a decrease in weight gain in rats fed a diet of pure cultures of F. graminearum (=F. roseum) grown on moist, sterilized oat-barley-wheat grains. This diet was shown to contain 9.55 ,ug of zearalenone per g. In these studies, F. tricinctum cultures similar to those known to produce trichothecenes, failed to cause either feed refusal or weight loss when compared to controls. We report here the results of investigations which demonstrate the production of specific feed refusal factors by pure cultures of F. ro--seum 'graminearum' isolated from 1972-crop moldy corn. In addition, a practical laboratory 362 bioassay for feed refusal was developed and used to initiate purification and chemical characterization of these factors. MATERIALS AND METHODS Cultures. Representatives of the major components of the fungal flora in refusal corn were isolated in pure culture from Wisconsin field samples which were selected in November 1972 at the height of the feed refusal period (Table 1). Kernels from rotted ears of both field corn and sweet corn were surface disinfected for 10 min in a solution of 0.32% sodium hypochlorite, dried on clean paper towels, and incubated at room temperature on either potato dextrose agar or 2% water agar. Single conidial cultures were derived from pure cultures obtained as hyphal transfers from the original isolation plates. Mature perithecia present on corn husks of rotted ears were fastened to petri dish lids with masking tape, allowed to discharge ascospores onto water agar plates, and the resulting single germinating ascospores were transferred aseptically to potato dextrose agar tube slants. For long-term storage, fungus cultures were held on sterile soil in glass vials (12). From several hundred fungal isolates derived from these samples, 12 strains representing the predominant fungi were selected for detailed animal feeding trials. Pure cultures of each were grown in 500-ml Erlenmeyer flasks on moistened sterilized corn for one week at room temperature followed by four weeks at 12 C, oven dried at 50 C for 2 days, ground in a Wiley mill, and then stored frozen. In addition, isolate no. 3 was also grown at room temperature for 5 weeks. Feed refusal and weight change in pigs. Ten Chester White and 16 Hampshire pigs (average weight = 22.7 kg) in pens (1.2 by 2.4 m), two animals per pen, were fed a standard commercial swine ration for 2 days. After this, pigs in each pen received different ground, dry, fermented corn cultures (3.6 kg/day per pen) as their sole diet. The two control animals received similar amounts of unfermented ground corn from the same source of corn used to prepare the original cultures. The feed remaining each morning was weighed before the introduction of fresh feed. Animals were weighed immediately before confinement and every other day thereafter. The study was concluded on the 6th day after a 96-h exposure to the test diets ( Table 2). Feed refusal, weight change, and estrogenism in rats. Young (21 day old) female Sprague-Dawley rats (Holtzman strain), caged individually, were fed dried, ground corn cultures (five rats per isolate) ad libitum as their sole diet. An equal number of control rats were fed dried, ground, unfermented corn. The feeding period lasted 3 days (72 h) with the day length adjusted for 12 h. Trials were begun at 4:00 p.m. Friday and concluded at 10:00 p.m. Monday (66 h). Both rats and test diets were weighed initially and at the end of the study (Table 3). In all cases, the rats were starved for 8 h before initiating the assay. To determine possible estrogenic effects of fer-mented corn cultures rats were killed using chloroform, and both the uterus and ovaries were removed, carefully separated from surrounding fatty tissue, and weighed (Table 3). Statistical treatment of rat bioassays. Analysis of covariance on the control (C) and test (T) rat groups were performed in all assays. In each case, due to the relatively small in-group variances, a mean difference (D = T -C) of 1.5 and 2.0 was found to be sufficient to reject the null hypothesis (Ho: T = C) at the 0.05 level of significance (using a two-tailed test) for percentage of body-weight change and feed consumption (g), respectively. Emesis testing. All isolates were examined for emetic activity using an earlier procedure (5). Ground corn cultures (200 g/culture) were extracted with ethyl acetate (600 ml) by continuous stirring for 24 h. The solvent was removed in vacuo and the oily, yellow residue was force-fed (0.5 ml/dose) to adult pigeons. Control pigeons were fed similar extracts from unfermented corn. A positive response consisted of a distinct vomiting episode occurring within 35 min followed by further episodes over a 1-h period. Emetic activity in pigs, if any, was noted during refusal tests with the corn cultures. Chemical fractionation. Ground corn (500 g) inoculated with isolate number 3 (the most active refusal isolate) was extracted with ether (200 ml) in a soxhlet apparatus (30 extractions per h) for 3 h. The resulting solution was washed two times with 100ml portions of 5% sodium bicarbonate. The aqueous layer was washed twice with 100-ml portions of ether and the ether extracts combined to give fraction A. The aqueous layer was acidified with 1 M hydrochloric acid (125 ml) and extracted three times with ether as before. The combined ether layers were washed with two 100-ml portions of saturated sodium chloride and dried over sodium sulfate to yield fraction B (strong acids). The aqueous layers were discarded. Fraction A was shaken with two 100-ml portions of 0.1 M sodium hydroxide. The aqueous layers were backwashed with ether as before and the organic extracts were combined to yield fraction C. The aqueous phase was made acidic with 1 M hydrochloric acid and extracted with ether. The ether layers were backwashed with saturated sodium chloride to yield fraction D (weak acids). Fraction C was shaken twice with 100-ml portions of 1 M hydrochloric acid, the aqueous phase was backwashed with ether, and the combined organic layers were washed with saturated sodium chloride to yield fraction E (neutral). In a separate experiment, isolate number 3 (250 g) was extracted as above and the resulting ether solution was shaken with two 100-ml portions of 1 M hydrochloric acid. After washing with ether (100 ml) the aqueous layer was made basic with 1 M sodium hydroxide and extracted three times with 100-ml portions of ether to give fraction F (bases). For feeding studies, the above ether solutions were added to ground, dried, unfermented corn, and the ether was allowed to evaporate until no trace of solvent could be detected. This corn was used as feed for rats in the test groups. Rats used as controls against each test fraction were fed corn impregnated with the equivalent fraction from normal, unfermented corn which had been subjected to the same fractionation scheme. This procedure was adhered to in all subsequent assays. Isolation of zearalenone. To isolate zearalenone from corn cultures of F. roseum, 200 g of ground, dried culture was extracted for 3 h with ethyl acetate using a soxhlet apparatus (30 extractions/h). Evaporation of the solvent yielded an oil (5.1 g) which was dissolved in Skelly B (500 ml) and extracted twice with 300 ml of 5% aqueous sodium bicarbonate-saturated sodium chloride (2:1, vol/vol). The aqueous layer was discarded and the organic layer was extracted three times with 5% aqueous sodium carbonate (200 ml). The aqueous layers were combined, adjusted to pH 6.0 with 1 M hydrochloric acid, and extracted five times with chloroform (200 ml). The combined extracts were dried over sodium sulfate and evaporated to yield a yellow residue (54 mg). The residue was purified by preparative thinlayer chromatography using chloroform-methanolwater (70:30:1, vol/vol. Zearalenone (8 mg) was recovered from a band having R. = 0.67 to 0.77 and an intense blue-green fluorescence under ultraviolet light. The compound was characterized by mixed TLC in 2 solvent systems and comparison of its proton magnetic resonance spectrum and mass spectrum with an authentic sample of zearalenone. Quantitation of zearalenone in cultures. A solution of zearlenone in chloroform was serially diluted and duplicate aliquots (2 ,d) of each concentration were spotted on TLC plates (Silplate F22) every 1 cm with 2-cm margins at plate edges. Plates were developed to 12 cm with Skelly-B-chloroform-methanolwater (50:35:15:0.5, vol/vol) and examined under ultraviolet light (365 nm). The minimum detectable amount of zearalenone (0.25 0.005 ,ug) was used to determine the concentration in the moldy corn samples. For this purpose, each feed sample (100 g) was soxhlet extracted with ethyl acetate as above. Twomicroliter aliquots of serially diluted chloroform so-lutions of these extracts were spotted on plates alongside standards and the zearalenone concentration was determined by comparison (Table 3). Swine feed refusal. Of the nine isolates of F. roseum 'graminearum' from ascospore and conidial isolations tested for refusal activity, measured by either feed consumption or weight loss, all but one produced physiologically active quantities of the refusal factor(s) ( Table 2). The isolate of F. roseum 'equiseti' (no. 9) was also highly active and produced a refusal response equal to the F. roseum 'graminearum' strains. F. moniliforme and Acremoniella atra were inactive. With the exception of F. roseum 'graminearum' strains no. 10 and 11 which were fed only to Hampshires, each strain was fed to one Hampshire and one Chester White in the same pen. The Hampshires appeared more sensitive to the refusal factor. For purposes of statistical analysis, however, values obtained were tabu- lated as averages per pair. Body weight changes were expressed as percent change based on initial weights taken at the beginning of the test diet and strongly correlated (r, = 0.81, L.S. < 0.01) (11) with corresponding reluctance to eat as measured by total feed consumed (Table 2). Two female Chester White showed distinct signs of hyperestrogenism induced by corn cultures of F. roseum 'graminearum' strains no. 3 and 4. Rat feed refusal. The potential of rats for use as a more convenient assay animal was examined in a 3-day study. Groups of five rats were fed diets consisting of the same corn cultures used in the pig study. Feed refusal activity was measured as the percent body weight change and feed consumption as compared to controls. All test feeds in this trial resulted in weight loss, feed refusal, and, with the exception of strain no. 7 (F. moniliforme), increased fresh weight of the uterus plus ovaries. Emesis. Using an earlier procedure (4), strains 1 to 12 were examined for emetic activity by feeding evaporated residues of ethyl acetate culture extracts to pigeons. None of the samples proved active with the possible exception of strains 8 (F. roseum 'graminearum') and 9 (F. roseum 'equiseti'), which induced a limited, barely detectable vomiting response. Verification of these two isolates was complicated by the fact that the pigeons also vigorously refused these samples by expectoration. Emesis was never observed, however, in pigs during free-choice feeding studies. Chemical fractionation. Feeding studies using the rat assay showed that although refusal activity was extracted by ethyl acetate, substantial activity still remained in the corn residue. Later work showed that improved yields of activity could be obtained by extraction with methanol, suggesting that the factor might be somewhat polar. Activity was retained in the methanol solution even after repeated washing with Skelly B. Evaporation of ethyl acetate yielded an oil which was active in both rat and pig assays. The refusal factor was presumed relatively nonvolatile after exposure of the oil to high vacuum distillation (0.2 ,um, 2 h, 50 to 60 C) yielded an inactive distillate and an active residue in the rat assay. When the oil was heated at 110 to 115 C for 2.25 h under a nitrogen atmosphere, most activity was lost, suggesting that the refusal factor(s) may be thermally labile. The gross chemical nature of the refusal factor(s) was explored by fractionation into base, neutral, strong, and weak acid fraction by conventional liquid-liquid partition. No activity could be found in either the base or strong acid fraction. Activity was found consistently in the weak acid (e.g., phenols) fraction although it was not sufficient to account for all of the activity of the feed. Activity was also found in the neutral fraction and it later became evident that the extent of activity depended upon the degree to which the aqueous phase was backwashed with ether during the extraction. The weak acid fraction consisted predominently of one active compound which was isolated by preparative TLC and shown to be zearalenone by comparison with an authentic sample. Zearalenone content. Samples of ground corn cultures of F. roseum 'graminearum' (no. 2, 3, 5, and 11), F. roseum 'equiseti' (no. 9) and F. moniliforme (no. 7) were extracted with ethyl acetate and the concentration of zearalenone was determined by dilution to extinction on TLC plates. All samples examined contained zearalenone with concentrations ranging from 4.0 + 1.6 to 42.8 + 10.6 pug/g (Table 3). TLC analysis of a second extraction of the corn residue showed that complete extraction of the zearalenone was effected by the initial ethyl acetate procedure. Comparison of increased uterine and ovary weight over controls for each test rat gave a near perfect correlation (r8 = 0.96, level of significance [L. S.] < 0.01) with determined increased zearalenone consumption (vide infra). Zearalenone and trichothecene feeding. Zearalenone and/or T-2 toxin (3-hydroxy-4,15diacetoxy-8-isovaleroxy-12,13-epoxy-A9-trichothecene) were examined in a rat feeding trial Weight change (%) calculated from initial and final weight of rats. d A Weight change (%) = percent test weight changepercent control weight change. A Feed consumption = control feed consumptiontest feed consumption. f Standard deviations in brackets. 9 Rat control for no. 3 was 8.9%. h Zearalenone concentrations determined by dilution to extinction with errors propagated. using a diet of normal ground corn impregnated with 50 or 200 ,ug of zearalenone per g alone, 5 or 50 Mg of T-2 toxin per g alone, or in combinations. With zearalenone alone, both concentrations resulted in small but significant feed refusal activity (Table 4). Whereas no relative decrease in body weight was observed at 50 Mug/g, at 200 ug/g test rats lost weight slightly as compared to controls. Both T-2 toxin concentrations resulted in considerable feed refusal and weight loss when compared to controls. In the presence of 50 pug of zearalenone per g (the approximate concentration present in corn cultures of isolate no. 3), 50 jig of T-2 toxin per g caused an enhancement of feed refusal and weight loss when compared to 50 ,ug of T-2 toxin per g alone. In the presence of 50 Mug of zearalenone per g, 5 Mg of T-2 toxin per g caused no significant change in feed refusal but a significantly smaller weight loss when compared to 5 ,Mg of T-2 toxin per g alone. The smaller weight loss was presumably due to the anabolic activity of zearalenone. T-2 toxin (5 ,ug/g) plus zearalenone (50 gg/g) produced refusal activity comparably to isolate no. 3 in rats. DISCUSSION Rejection of moldy corn can now be definitively associated with the low temperature growth of F. roseum 'graminearum' on parasitized corn. In pure corn culture studies, eight of nine isolates of this organism induced active feed refusal by pigs in ad libitum feeding studies. The strain of F. roseum 'equiseti' was also active, but none of the other fungi commonly present in the field samples induced refusal. These results are consistent with the reports of Roine et al. (10) who noted refusal by rats of pure cultures of F. roseum 'graminearum' grown in pure culture on moistened small grains. Refusal has previously been assumed to be associated with this organism (3), but corresponding feeding studies were only conducted with field-infested corn and thus failed to offer definitive proof-of-cause. The fact that emesis was not evident in most of our F. roseum 'graminearum' corn cultures which clearly caused feed refusal provides evidence that these two phenomena are caused either by different metabolites, or by different concentrations of the same metabolite. The former possibility was suggested, but not clearly established, by earlier studies (3). The fact that slight emetic activity may have been associated with one strain of F. roseum 'graminearum' (no. 8) and one strain of F. roseum 'scirpi' (no. 9) does not alter these conclusions. Burmeister et al. (2) reported that certain strains of F. rosum 'graminearum' in the NRRL culture collection produced moderate quantities of the emetic trichothecene T-2 toxin. Closely related diacetoxyscirpenol has been isolated from strains of F. roseum 'scirpi' (1). Careful analysis of crude extracts of our most active refusal isolates failed to detect known emetic trichothecenes such as T-2 toxin, HT-2 toxin, or 4-deoxynivalenol (3,7,15-trihydroxy-12,13-epoxy-trichothec-9-ene-8-one), although the presence of other trichothecenes is not excluded. For practical purposes, in the chemical purification of refusal factors, laboratory rats were examined as possible assay animals. A 3-day feeding trial during the quiet weekend period provided the most consistent and least variable results. Under such conditions, although individual test diets showed statistically significant refusal activity relative to controls (Table 3), a weak correlation (r, = 0.32, L.S. > 0.05) was obtained when comparing feed refusal activity against corresponding weight gain. Overall correlations between pig and rat data, either on the basis of feed refusal activity (r, = 0.54, L.S. > 0.05) or weight gain (r, = 0.43, L.S. > 0.05) was not strong. The presence of varying concentrations of zearalenone with known anabolic activity appeared to be a factor in the overall variability of the feeding trials. The actual zearalenone consumption was determined from known concentrations in the feeds (Table 1) coupled with the actual feed consumption ( Table 3). Comparison of zearalenone consumption with uterine weights (including ovaries) or rats fed these diets showed near perfect correlation (r, = 0.96, L.S. < 0.01) in spite of possible errors in the fresh weight determinations (Table 3). Such concentrations of zearalenone are also within the range reported by Mirocha et al. (7) to result in whole body weight gain in rats. Thus it seems highly probable that the varying amounts ofzearalenone consumed in the Fusarium corn culture diets contributed to the lack of good correlation between feed consumption and weight gain. However, in spite of the lack of strong correlation between the results of rat and pig feeding studies the rat assay remained a convenient assay tool for chemical fractionation studies when feed refusal was measured. However, periodic confirmation feeding studies using pigs were necessary, and for this purpose feed refusal (as distinct from body weight) was considered the most appropriate parameter for FEED REFUSAL FACTORS 367 measurement. Thus, the stability of the refusal factor to the initial workup was verified by feeding the crude oil as a dietary component to both rats and pigs and noting significant refusal in each animal. The isolation and characterization of zearalenone as an active fraction stimulated our interest in its role in feed refusal. By dilution to extinction, the concentration (,uglug) of zearalenone was determined in six isolates (Table 3), and a strong correlation with refusal was found in rats (r. = 0.93, L.S. < 0.01) and a moderate correlation in pigs (r, = 0.68, L.S. = 0.05). However, further examination with pure zearalenone demonstrated that the magnitude of the refusal activity with pure corn cultures could not be accounted for by zearalenone alone. Compare, for example, the activity of isolate no. 3 (Table 3) with pure zearalenone (50 ,ug/g) ( Table 4). In fact, additional factors, either through additive or synergistic effects, must have been present in order to explain the refusal syndrome. Since preliminary chemical fractionations indicated the presence of active nonvolatile, relatively polar, neutral molecule(s), like trichothecenes, and since various trichothecenes have been associated in one way or another with Fusarium infested corn intoxication, the possible role of trichothecenes in the refusal syndrome was investigated. To investigate the potential of toxic trichothecenes, readily available T-2 toxin was evaluated in the rat assay. A diet of as little as 5 jig of T-2 toxin per g was found sufficient to account for all of the refusal activity of infected feed. In addition, at higher T-2 toxin concentrations (50 ,ug/g), zearalenone was found to enhance its activity. These results suggest that refusal activity may well be due to a combination of zearalenone and one or more of the trichothecenes. Further work to clarify this possibility is presently in progress.

v3-fos

2020-12-10T09:04:13.112Z

1975-10-01T00:00:00.000Z

237231485

s2

Toxigenic Aspergillus and Penicillium Isolates from Weevil-Damaged Chestnuts Aspergillus and Penicillium were among the most common genera of fungi isolated on malt-salt agar from weevil-damaged Chinese chestnut kernels (16.8 and 40.7% occurrence, respectively). Chloroform extracts of 21 of 50 Aspergillus isolates and 18 of 50 representative Penicillium isolates, grown for 4 weeks at 21.1 C on artificial medium, were toxic to day-old cockerels. Twelve of the toxic Aspergillus isolates were identified as A. wentii, eight as A. flavus, and one as A. flavus var. columnaris. Nine of the toxic Penicillium isolates were identified as P. terrestre, three as P. steckii, two each as P. citrinum and P. funiculosum, and one each as P. herquei (Series) and P. roqueforti (Series). Acute diarrhea was associated with the toxicity of A. wentii and muscular tremors with the toxicity of P. terrestre, one isolate of P. steckii, and one of P. funiculosum. Because of its resistance to Endothia blight (1), the Chinese chestnut (Castanea mollisima Blume) was introduced into the United States for hybridizing with the nearly extinct American chestnut (C. dentata [Marsh.] Borkle). Annual chestnut production in the state of Georgia is about 60,000 to 70,000 lb (ca. 27,215.52 to 31,751.44 kg) (personal communications, R. L. Livingston, University of Georgia) and is marketed principally in the Appalachian area of the eastern United States-roughly, the center of the original range of the American chestnut. Production estimates for other states are unavailable. Chestnuts are a perishable commodity, easily spoiled by fungi and insects. Mature nuts are allowed to drop from trees and may lie for several days or weeks until gathered. Decay development may begin while they are on the ground (5). Commercially, chestnuts may be held in refrigerated storage for several months before marketing. Losses due to fungi frequently occur, particularly at the consumer level (15). In experimental storage studies Hammar (6) found that spoilage ranged from 5 to 10% after 1 month and 15 to 60% after 7 months at 2 C. Wright (16) reported that 62% of kernels examined soon after harvest contained visible fungus infections. The most common fungi isolated from decayed tissues were Phoma castaneae Oud. and Pestalotia spp. Of minor importance were species of Phompsis, Penicillium, Alternaria, Fusarium, Rhizopus, and others. Researchers in Italy and France have found that the most common genera of decay fungi isolated from European chestnut (Castanea sativa Mill.) kernels in storage were Penicillium, Fusarium, Phoma, Aspergillus (A. niger), and Rhizopus (2,8,12). There have been no reports, to our knowledge, on the mycoflora of weevil-damaged chestnuts. The chestnut weevil (Curculio sayi Gyllenhal), commonly called the small chestnut weevil, is a major threat to chestnut production because it attacks the nut kernels (9). Adults infest trees from April to late June and deposit eggs in nearly mature nuts during August and September. The larvae feed on kernel tissues, then emerge by cutting through the shell. Infested nuts may contain several weevil larvae, or, if larvae have already emerged, may contain weevil burrows filled with excrement. Weevil-damaged nuts are likely to harbor a wide variety of mycoflora and be subject to spoilage. Nuts from which weevils have emerged are generally culled from the packing operations by flotation. Nuts containing weevils, however, are not separated by the flotation process. Weevils then emerge while chestnuts are in storage or transit, and damaged nuts enter the market channels. Although such nuts are generally discarded by the consumer, some might be incorporated into processed chestnut products or food combinations. The potential for consumption of spoiled chestnuts is increased by the absence of visible mold on many kernels with incipient fungal infections. Fungi ofthe generaPenicillium andAspergillus have been associated with toxin production on many agricultural commodities (14,17). Other fungi, especially those of the genera Alternaria and Fusarium, have also been re-ported as toxin producers (3,4). This study was to determine the incidence of some mycotoxinproducing fungi on weevil-damaged chestnuts and was limited to Penicillium, the most frequently isolated genus, and to Aspergillus, the genus most often associated with mycotoxin contamination of foodstuffs. MATERIALS AND METHODS Isolation of fungi. Freshly gathered chestnuts were obtained in October 1972 and 1973 from orchards in central Georgia. Chestnuts with weevilemergence holes were selected and stored for 1 week at 3 C. Kernel pieces containing sections of weevil burrows were surface sterilized for 3 min in 0.5% sodium hypochlorite solution containing 3% ethyl alcohol, rinsed in sterile water, and plated on maltsalt agar. Fungus colonies developing from kernel pieces after 3 weeks at 21 C were classified by genera. Those not readily indentifiable were placed in a miscellaneous category. All Aspergillus and Penicillium colonies were transferred to potato-dextrose agar slants by mass transfer, allowed to grow at 21 C for 2 weeks, and stored at 3 C. Bioassay for toxicity. Cultures for bioassay were grown on shredded wheat (7) or on fresh or on autoclaved medium. Autoclaved chestnut medium, prepared in a 500-ml flask, consisted of 50 g of quartered, fresh chestnuts and 10 ml of water, which were autoclaved at 15 lblin2 and 121 C. Fresh chestnut medium was prepared by adding 50 g of surfacesterilized quartered kernels to an autoclaved flask containing 10 ml of water. Media were inoculated by mass transfer of spores from the potato-dextrose agar slants, and cultures were grown for 4 weeks at 21 C. Cultures were extracted for bioassay by the method based on that of Kirksey and Cole (7). Cultures were blended with 200 ml of chloroform in a Waring blender for 45 s, and the homogenates were filtered through a 1-cm pad of sodium sulfate on a 90-mm Buchner funnel. Chloroform filtrates were transferred to 150-ml beakers containing 5.5 ml of corn oil and placed on a steam plate for 3 h to completely evaporate the chloroform. Five 1-day-old DeKalb 151 cockerels were dosed by crop intubation with 1 ml of corn oil which contained extract. Checks were dosed with corn oil to which only pure chloroform had been added and then evaporated. Cockerel mortality, expressed as survival ratios, and any clinical symptoms were recorded over a 5day observation period. If mortality was over 50% or if survivors exhibited unusual clinical symptoms one or two additional bioassays were conducted. If confirmatory tests were also positive, the extracts were considered toxic. Cultures were rated for degree of toxicity: less than 50% mortality but with debilitated survivors equals low toxicity; more than 50% but less than 90% mortality equals moderate toxicity; and over 90% mortality equals high toxicity. Identification of toxic isolates. Cultures which produced toxic extracts were transferred to diagnostic media (10) for taxonomic identification. All taxo-nomic identities at the species level were considered definitive if major cultural and microscopy characteristics of an isolate agreed with published descriptions (10,11). When one or more characteristics of an isolate were at variance with descriptions, identification was at series level only. All Aspergillus isolates and only those Penicillium isolates shown to be toxic were identified at species level. RESULTS Penicillium spp. were the fungi most frequently isolated (40.7% occurrence) from weevil-damaged chestnuts (Table 1). Next, in order of frequency of occurrence, were Rhizopus, Alternaria, and Aspergillus, each comprising about 17% ofthe total mycoflora isolated. Fusarium constituted 6.4% of the colonies isolated, and fungi of unidentified and miscellaneous genera constituted 1.3%. Twenty-one of the 50 Aspergillus cultures isolated from chestnuts were toxic to day-old cockerels ( Table 2). Most of the isolates were A. A. wentii caused acute diarrhea, loss of appeflavus isolates and one of the fourA. flavus var. tite, and general debilitation or mortality. The columnaris isolates were toxic ( ity, each causing a cumulative average mortalcaused over 90% mortality, and isolates CA 14, ity of less than 50% ( Table 3). The remainder of CA 22, CA 48, and CA 52 were moderately toxic the A. wentii isolates (CA 9, CA 15, CA 19, CA (Table 3). 43, CA 45, and CA 51) were moderately toxic, None of the A. oryzae or A. niger cultures causing over 50% but less than 90% mortality. isolates from chestnuts were toxic ( Table 2). The remaining toxic Aspergillus cultures be-Eighteen of the 50 bioassayed Penicillium isolates were toxic ( Table 2). Twelve of these 18 were associated with sustained muscular tremors and, in some cases, convulsions before death. Of the toxic Penicillium isolates, nine identified as P. terrestre (Series) were tremorgenic and of varying toxicity ( Table 3). Three of the toxic Penicillium isolates were P. steckii and were moderately to highly toxic. Two were P. citrinum and two were P. funiculosum, all but one were highly toxic. The P. funiculosum isolates were tremorgenic. One isolate of P. herquei (low toxicity) and one of P. roqueforti (moderately toxic) were also identified. Selected isolates of each major group of toxic fungi were cultured on autoclaved and on surface-disinfected fresh chestnuts. Of the three A. wentii cultures tested, CA 8 extracts from either autoclaved or fresh chestnuts were not toxic, CA 10 extracts from autoclaved but not from fresh chestnuts were toxic, and CA 13 extracts from both media were toxic ( Table 4). Six of seven A. flavus isolates tested (including A. flavus var. columnaris) produced toxin on autoclaved and on fresh chestnuts, and one (CA 46) was toxic on autoclaved chestnuts only. With the exception of P. herquei (CP 35), all extracts of Penicillium isolates grown on autoclaved chestnuts were toxic to day-old cockerels. On fresh chestnut medium, one isolate each (of two tested) of P. terrestre (CP 39), P. steckii (CP 18), and P. citrinum (CP 41) was toxic. The one isolate tested ofP. funiculosum (CP 25) was of low toxicity, and P. herquei (CP 35) and P. roqueforti (CP 14) were not toxic when grown on fresh chestnuts. DISCUSSION A high percentage ofPenicillium and Aspergillus isolates from weevil-damaged Chinese chestnuts were capable of producing mycotoxins. Forty-two percent of all aspergilli were toxic, and seven of 10 representative isolates tested produced toxin on inoculated, fresh chestnuts. Similarly, 36% of the penicillia bioassayed produced toxin on artificial media, and four of nine tested produced the toxins on fresh chestnuts. The organisms studied were fungi established in dehydrated or discolored tissues adjoining insect-damaged areas. No mycotoxins have been found on market chestnuts; however, a potential exists for toxin production in the event fungal development occurs on kernel tissues. The presence of surface contaminants also presents a potential problem if chestnut quality deteriorates in the market or in storage. The potential presence of mycotoxins in weevil-damaged chestnut kernels suggest the need for effective weevil-eradication programs in the orchards and for fastidious, quality control measures after harvest. Most A. wentii isolates lost a degree of toxicity during the course of this study. Initial subcultures of original isolates were highly toxic. Subcultures taken from original isolates in storage for 6 to 8 months were less toxic although diarrheagenic symptoms were strong. Prolonged storage of these toxigenic fungi on artificial medium or repeated subculturing may have resulted in mutations or in metabolic changes affecting toxicity. Further research is needed to test the capability offungi other than the aspergilli and penicillia present on weevil-damaged chestnuts. Genera such as Alternaria, Fusarium, and others have been associated with mycotoxicity. Research is now in progress to identify the toxins produced by the fungi reported in this study. The A. wentii toxin has been isolated and identified as emodin (13). Preliminary analyses suggest that aflatoxins and citrinin are the toxins responsible for the activity of A. flavus and P. citrinum isolates, respectively (unpublished data).

v3-fos

2018-04-03T04:21:25.517Z

1975-02-01T00:00:00.000Z

38679539

s2

Detection and Growth of Enteropathogenic Escherichia coli in Soft Ripened Cheese The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore, a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 105 at room temperature and over 103 at 4 C within 10 days. With higher initial counts, levels up to 109 were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination. In the United States, enteropathogenic types of Escherichia coli (EPEC) are usually associated with cases of infantile diarrhea. Studies from India and Vietnam by Gorbach et al. (6) and Japan by Sakazaki et al. (9) showed that EPEC cause disease not only in children but in adults as well. Dupont et al. (2) performed studies with several types of EPEC to determine the effects of these organisms on animals and human volunteers. Their work demonstrated that EPEC can cause disease in man by at least two mechanisms: by production of a choleralike enterotoxin and by an invasion of intestinal epithelial lining. Before November 1971, there were reports of water-borne outbreaks of EPEC in the United States, but no confirmed outbreaks were associated with foods (1). However, from 30 October to 10 December 1971, over 200 persons in more than 90 separate outbreaks suffered acute food poisoning symptoms after eating imported Camembert or Brie cheese. Our laboratory investigated several complaints of food poisoning in which Camembert cheese imported from France was implicated. None of the common food poisoning organisms were detected; however, high levels of E. coli were encountered and, with the methods used, were frequently the only organism found. This led to the serotyping of these organisms and their subsequent identification as EPEC. However, no in vivo tests were performed to confirm pathogenicity. Therefore, the term EPEC as used in this paper refers only to those strains belonging to serogroups having known association with diarrheal disease as outlined by Ewing (4). The most frequently detected type was a strain of 0124:B17 E. coli that did not ferment lactose within 48 h. This strain was found usually by direct plating of the cheese on agar media and was also recovered from individuals who had contracted the disease (7). These isolates all failed to ferment lactose broth within 48 h; however, after that time fermentation did occur with some strains. Since not all isolates were tested after 48 h, the term 179 "lactose negative" will be used throughout the remainder of this text. Methods for analysis of E. coli normally used fermentation of lactose as a selective factor. Therefore, slow or nonfermenting strains could remain undetected particularly when present in low numbers. With this in mind, existing methods for analysis of E. coli were evaluated and a tentative procedure for the detection of EPEC was developed that would not exclude lactose-negative strains. In addition, studies were initiated to determine whether the high levels of E. coli in the cheese were due to improper processing or simply to the ability of these organisms to grow in the product under normal conditions of handling. MATERIALS Media. Lauryl tryptose broth, brilliant green lactose bile broth, EC broth, GN broth (Hajna) and MacConkey agar were obtained from Difco and prepared according to the manufacturer's instructions. A modification of MacConkey agar was also used. In this, 5 g of lactose and 5 g of arabinose were substituted for the 10 g of lactose per liter normally present. A modification of GN broth was also used in which 1 g of lactose and 1 g of arabinose per liter were substituted for the glucose and D-mannitol. Antisera. Isolates were screened as presumptive EPEC by typing with E. coli OB poly A and poly B and with E. coli OK poly C antisera, all from Difco. Monovalent E. coli 0, OB, and OK antisera (Difco) were used in the identification of the specific serogroups present. Difco polyvalent fluorescein-labeled anti-E. coli globulin (poly A, B, and C) was used for the screening of cheese samples by the fluorescent antibody (FA) method. A 1:4 dilution of these conjugates was determined to be an appropriate working dilution by the method of Goldman (5). Identification. E. coli isolates were identified as possible enteropathogenic types on the basis of biochemical characteristics and serological titrations as outlined by Ewing (4). Enumeration of E. coli. Samples of imported soft ripened cheese collected at the time of entry at the Port of New York were analyzed for the numbers of E. coli present immediately upon receipt in the laboratory. Serial dilutions out to 10-7 were prepared in potassium phosphate buffer at pH 7.2. The same enumeration procedure was used in studies on the growth of E. coli in stored cheese samples. Each dilution was inoculated into three tubes of lauryl tryptose broth containing inverted durham tubes. Cultures producing gas in 24 and 48 h at 37 C were then subcultured into EC broth and incubated in a constant-temperature water bath at 45.5 C. The presence of gas was recorded after 24 and 48 h. The most probable number (MPN) of E. coli was then calculated per gram of original cheese. FA procedure. Three smears were prepared from GN broth cultures by using a 2-mm loop onto multiwell Teflon-coated slides (5). Smears were air dried; the slides immersed in xylene for 3 min, drained and placed in Kirkpatrick fixative solution (ethyl alcoholchloroform-formalin [60:30:10]) for 3 min, and air dried. Treatment of the slides with xylene removed lipids that interfered with fixation. The slides were then rinsed in 95% ethyl alcohol and air dried. The three smears were then treated separately, each with one drop of poly A, B, or C E. coli conjugate. The slides were placed into moist chambers and incubated for 30 min. Excess conjugate was drained and the slides were rinsed in phosphate-buffered saline (pH 7.5). The slides were washed twice in a phosphate-buffered saline bath for 5 min and rinsed in distilled water. After air drying, a drop of buffered glycerol saline at pH 7.5 was placed on the slide and covered with a no. 1 cover slip. Slides were examined with an American Optical Series 20 Microstar microscope equipped with a 50-W mercury vapor lamp. A cardioid dark-field condenser, BG 12 exciter filter, and an OG 1 barrier filter completed the system. Fluorescent cells were rated on a scale of 1 + to 4+. Slides with cells rated 3+ or 4+ (bright cell outline with clear lumen) were considered positive. RESULTS In preliminary studies it was found that strains of EPEC grew well in GN broth. It was thought that selectivity for EPEC might be improved by substituting lactose and arabinose for the glucose and D-mannitol normally present in this medium as carbon sources. According to Edwards and Ewing (3), Edwardsiella, Providencia, Serratia, and Proteus for the most part will not ferment either lactose or arabinose. However, 91% of E. coli ferment lactose and 99% use arabinose. Thus, chances of encountering strains of E. coli incapable of growth on one or the other of these sugars should be minimal. GN broth, the modified GN broth, and several other media normally used for selection of E. coli were compared for ability to support the growth of EPEC. The test organisms were a lactose-negative and a lactose-positive strain of E. coli type 0124:B17 and a nonpathogenic strain of E. coli. The two 0124:B17 strains came from samples of cheese known to have caused diarrhea, and it is highly likely that they were in fact pathogenic. The three strains were grown for 24 h in heart infusion broth, and 0.01 ml of each culture was inoculated into tubes contain-ing 10 ml of the test media. The tubes were incubated for 18 h at 37 C and viable cells were estimated by plate counts on standard methods agar. The results in Table 1 show no significant difference in the levels of growth achieved by the nonpathogenic strain of E. coli in any of the media tested. Some inhibition of the lactosepositive pathogenic strain was noted in EC broth at 45.5 C, possibly because of inhibition by bile salts and the elevated temperature. All other media yielded comparable amounts of growth with this strain. The differences in growth levels for the pathogenic lactose-negative strain were much more pronounced. Growth was substantially less in the broths having a lactose base; however, cell numbers in GN broth and the modified GN broth were higher and of about the same value. Evidently, with the pathogenic lactose-negative strain the amount of growth in the modified GN broth was not affected by the fact that only the arabinose and not the lactose present in that medium could be utilized. In another experiment, a modified MacConkey agar was tested as a plating medium. Although lactose-negative EPEC will grow on MacConkey agar with the normal formulation, colonies do not appear as typical E. coli since acid is not produced from the lactose present. Normally these colonies would not be picked for routine screening. The medium was therefore modified by deleting half the lactose and substituting an equivalent amount of arabinose. This modification eliminated the need to pick lactose-negative isolates on MacConkey agar. This modified medium was tested by streaking cultures representing 20 different serogroups of E. coli on the plates and noting colonial morphol- ogy and color reaction. Strains were tested both singly and in combinations of a lactose-negative with a lactose-positive type. All strains gave typical colonial morphology and color reaction. No differences in selectivity were noted on plates streaked with the mixed cultures. When typical E. coli colonies were randomly picked from such plates and transferred to phenol red lactose broth, approximately equal numbers of lactose-positive and lactose-negative cultures were recovered. On the basis of these observations, the cultural procedure as outlined in Fig. 1 was devised for the screening of EPEC in soft cheese. On day 1, a slurry consisting of 50 g of cheese in 50 ml of modified GN broth is prepared. After 1 h of incubation, a loopful of the slurry is streaked onto modified MacConkey agar plates. One hundred and fifty milliliters of modified GN broth is then added and the slurry is incubated for 18 to 24 h at 37 C. On day 2, a loopful of the slurry is streaked onto a second set of modified MacConkey agar plates. The first plates are examined, and if typical colonies are present a minimum of 10 are picked. Each colony is used to inoculate a slant of Simmons citrate agar, a slant of heart infusion agar, and a tube of KCN broth. On day 3, if the citrate and KCN tubes are negative, the heart infusion slant is used as a source of cells for serological screening. The second set of modified MacConkey agar plates is used as above in cases where the initial direct plating does not yield presumptive E. coli based upon negative reactions in citrate and KCN. Isolates that are both KCN and citrate negative and give a positive reaction with E. coli OB antisera before and after boiling are considered presumptive positives. These cultures are then identified biochemically and serologically as outlined in the CDC Laboratory Manual (4). The above procedure has been used successfully during the last 2 years for routine screening of EPEC at the New York District FDA laboratories. Over 2,000 cheese samples were examined, and approximately 10% of the samples were found to contain E. coli belonging to serogroups associated with diarrheal diseases. The majority of these isolates were detected shortly after the initial food outbreak and belonged to the following serogroups: 0124:B17 (lactose negative); 0124:B17 (lactose positive); 0112:B11; 0124:B15; 0128:B12; and 0127:B8. Since not all strains in a given serogroup are virulent, actual pathogenicity of an isolated strain should be confirmed by a test such as the ileal loop procedure. After the initial samples generated by the outbreak had been analyzed, attempts were also E. coli in soft ripened cheese. GN broth was modified by substitution of lactose and arabinose for glucose and D-mannitol. MacConkey agar was modified by substitutions of arabinose for half the lactose. Cells from slants of heart infusion agar were used for serological screening. made to screen cheese samples for EPEC by using immunofluorescent techniques. We felt this would be feasible since the conjugates available did cover serogroup 0124:B17, the organism implicated in the food poisonings. A slurry of cheese was prepared in modified GN broth and incubated for 18 to 24 h at 37 C as previously described (Fig. 1). After incubation, 0.5 ml was inoculated into 5 ml of modified GN broth and incubated for 4 h at 37 C. This second enrichment was found necessary to reduce the carry-over of product that fluoresced brightly under ultraviolet light. Three smears were prepared and stained with poly A, B, or C E. coli FA conjugate as described in Materials and Methods. Several hundred cheese samples were screened by this procedure. A number of positive enrichment cultures were detected, and these were identified as EPEC types by the cultural, serological, and biochemical procedure outlined in Fig. 1. No false FA negatives were found, but a false-positive rate of approximately 20% was encountered. These results indicate that the FA method might be developed as a routine screening procedure. If the 4-h enrichment culture is FA negative, then the analysis could be terminated; those cultures giving a positive reading would be streaked on modified MacConkey agar for subsequent confirmation. In some of the first cheese samples analyzed at the time of the food poisoning outbreak, counts in excess of 3 x 107 E. coli were detected. Studies were undertaken to determine the significance of these high levels. Fifty-five imported cheese samples (approximately 1 lb [454 g] each) were collected at the port of entry and brought to the laboratory under ice. Although the cheese was refrigerated during shipment, the actual temperature and any previous storage history was not known. On arrival in the laboratory, the samples were placed in a refrigerator at 4 C equipped with a continuous recording thermometer. Samples were individually removed from the refrigerator and cut into two sections. One section was returned immediately to the refrigerator. A portion of the other section was used to determine the initial level of E. coli using the MPN procedure. The remainder of this section was stored at ambient room temperature. Although the actual temperature of the refrigerated cheese sections was not known, it was assumed that they reached 4 C very rapidly since they were relatively small in size and were stacked to allow maximum air circulation. After 2, 4, 8, and 10 days of storage, the levels of E. coli in both sections were determined again by the MPN procedure. Precautions were taken to insure that there was no significant increase in temperature of the cheeses when they were removed from the refrigerator for enumeration of E. coli. The initial MPN values varied from less than 10 to 105 E. coli/g. The numbers of E. coli in all 55 samples increased upon storage. Data representing three typical cheese samples with high, medium, and low initial levels are shown in Fig. 2. The rate of growth for E. coli in the portions maintained at room temperature was significantly greater than for those kept refrigerated. However, given a significant period of time, sections of cheese maintained under refrigeration often achieved the same levels as their counterparts kept at room temperature. Apparently, the initial level of E. coli did not influence the ability of the organisms to eventually propagate. Samples that had 10 or less E. coli per g and even some with levels initially undetectable demonstrated an increase in E. coli upon holding. After 10 days of storage, samples held at room temperature had more than 105 E. coli/g. The refrigerated counterpart had more than 103 E. coli/g. Each type of symbol refers to a separate cheese sample that was divided in half and stored at two different temperatures. The data shown represents typical results for cheese samples with high (0), medium (A), and low (0) initial levels of E. coli. DISCUSSION The cheese samples collected as a result of the outbreak in 1971 and analyzed by our laboratory were found to contain as many as 3 x 107 EPEC/g. In the F.R.I. Annual Report of The University of Wisconsin, Trenk and Deibel also found E. coli in soft ripened cheese at levels greater than 100,000/g. Due to the high numbers present in the cheese, more than 90% of the EPEC detected in the original outbreak could be isolated by direct plating of the product. The strains isolated by direct plating were frequently not of the same type as encountered after incubation of the product in an enrichment broth. Selective agars streaked from enrichment broths, such as lauryl tryptose, brilliant green lactose bile, and EC, were predominantly populated with lactose-positive organisms in serogroups not associated with disease, and in many instances the negative or late lactose-fermenting E. coli, 0124:B17, generally found by direct plating, was not present. Studies conducted by Sakazaki et al. (9) further substantiate the need to look for lactose-negative E. coli. Over an 8-year period, of the 764 types of E. coli that these workers found associated with disease in man, 393 were negative lactose-fermenting strains. The need to pick at least 10 colonies per plate must be emphasized. The methods used for isolation do not select for only pathogenic types but for all E. coli that might be present. Furthermore, many isolates may autoagglutinate even in saline solution, making serological identification impossible. From samples consisting of 10 packages of cheese in which 10 colonies were picked per cheese, as few as three isolates were identified as EPEC. Therefore, initially we chose a method using a direct plating of the cheese slurry on MacConkey agar and enrichment in GN broth with subsequent streaking on MacConkey agar. In both instances, colonies appearing lactose negative or positive were picked for biochemical and serological screening. Later this procedure was changed to use of the modified MacConkey agar and modified GN broth. On the modified Mac-Conkey agar it was assumed that all or almost all E. coli strains would produce an acid reaction regardless of whether they were lactose positive or negative. Organisms belonging to the genera Edwardsiella, Providencia, Serratia, and Proteus would not give acid reactions, and if still present after enrichment in modified GN broth they would be readily distinguishable from E. coli. One disadvantage to the use of modified MacConkey, however, is that if lactose-negative E. coli are present in low numbers, they may be missed by random picking of 10 typical colonies. At this point it is not known which is the most advantageous, exclusion of the above four genera or the ability to distinguish lactose-positive and -negative strains of E. coli early in the analysis. If one were attempting to specifically isolate a lactose-negative strain, the nonmodified agar would be the obvious choice. For routine screening we prefer the modified agar medium; however, perhaps both should be used. Analysis of cheese manufactured by the firm implicated in the original food poisoning outbreak revealed that the problem was not confined to only one type of product. EPEC was found in multiple lots of Camembert, Brie, and Coulommiers cheese. At that time all products manufactured by the firm were removed from the market and a survey of all soft cheeses entering the Port of New York was instituted. During a 1-month period more than 10% of the soft ripened cheeses tested were found to contain EPEC types and the problem was not limited to one manufacturer. Once again these organisms were often encountered in very high numbers. The curing process required for soft cheese production and the physical properties of the final product are significant in explaining how high levels of E. coli can develop. Soft ripened cheese has a moisture content generally greater than 50%. After the formation of the curd, the curing or ripening is caused by decomposition of protein due to the activity of molds. This activity reduces the acidity of the curd and the pH rises from about 4.9 to 7.5. Thus, the acidic products produced originally by the growth of bacteria are neutralized and an ideal environment is created for the growth of E. coli. Those cheeses found to contain over 6 x 107 E. coli/g had pH values in the range of 7.2 to 7.4. A neutral pH after this much growth is normally not encountered in food products, and acids produced by bacteria usually cause inhibition and eventually reduction in cell numbers. It is well known that E. coli can propagate in dairy products. For example, Olson et al. (8) and Watrous et al. (11) demonstrated that coliform levels can increase in milk upon holding at 45 F after pasteurization. These studies were conducted on milk that had no detectable coliforms immediately after pasteurization, and precautions were taken to prevent post-processing contamination. Presumably, very low levels of E. coli were actually present after pasteurization but were not detected by the cultural methods, and these were capable of subsequent growth. The MPN of E. coli in cheeses stored at 4 C and at room temperature was determined in the studies reported here by ability to produce gas in EC broth at 45.5 C. Although this procedure is not a confirmatory one, it is frequently used as an indication of the presence of E. coli. Evidently, E. coli is capable of growing in soft ripened cheeses when low levels are present initially, even under controlled refrigeration. Another possibility is that the increase in numbers does not indicate growth but represents injured E. coli cells that were able to repair themselves at 4 C and ultimately grow in EC broth at 45.5 C. In any event, our studies emphasize the need for methods sensitive enough to detect extremely low levels of EPEC. At this point it is impossible to determine the relative significance of pasteurization and subsequent sanitation as regards the initial numbers of E. coli detected in the finished product. The FA procedure tested here has been successful in detection of E. coli 0124:B17 in enrichment cultures inoculated with cheese samples. The average cheese sample as received by FDA consists of 10 individual packages. Assuming 10 colonies are picked for each package tested, a sample would generate 100 cultures for identification. If the FA procedure could be developed as a routine screening method, the need for costly and time-consuming biochemical and serological testing of hun-dreds of negative isolates could be eliminated. This is not possible at the present time because the conjugates now available commercially are limited in their coverage of the EPEC types and would need to be expanded for use in a broad screening application. In the United States, food-borne strains of E. coli capable of causing serious disease in adults have so far only been associated with soft ripened cheese. Nevertheless, other foods might harbor and transmit these organisms, and testing laboratories must consider routinely that E. coli is a possible food-borne pathogen. In considering testing methods to be used, it must be emphasized that serotyping simply identifies an organism as a member of a group previously associated with the disease and does not establish that it is actually pathogenic. All organisms belonging to that group may not be pathogenic. Furthermore, it is known that factors associated with virulence can be transmitted from donor strains to nonpathogenic strains. For example, Smith and Halls (10) have shown that a genetic factor, presumably a plasmid, responsible for enterotoxin production in certain strains of E. coli can be transmitted to other non-enterotoxigenic strains of E. coli. The recipient strains were capable of causing diarrhea in pigs. Therefore, it is possible that new pathogenic strains can develop that have serotypes not previously associated with disease. At present, pathogenicity can only be established by testing in animals by a procedure such as the ileal loop technique. Unfortunately, such a procedure is too timeconsuming and expensive to use for routine analysis of all suspected isolates. Thus, existing procedures for detection of E. coli must be reevaluated and perhaps new and simpler methods devised for identification of the pathogenic strains.

v3-fos

2020-12-10T09:04:13.082Z

1975-09-01T00:00:00.000Z

237233971

s2

Fungal Growth on C1 Compounds: a Study of the Amino Acid Composition of a Methanol-Utilizing Strain of Trichoderma lignorum The amino acid composition of the C1-utilizing fungus Trichoderma lignorum, growing at the expense of methanol as the sole source of carbon, was determined. With the exception of an insufficient content of methionine, the essential amino acid composition of this novel protein source appears adequate in terms of the Food and Agricultural Organization Reference Protein for both direct and indirect use in the human diet as a food or animal feed, respectively. The exploitation of Cl-utilizing bacteria as a potential source ofdietary protein for animals is under active development (2,3). As a consequence of this interest, the amino acid composition of the protein from several methanol-utilizing (2, 3) and methane-utilizing bacteria (4) has been determined: almost all of these prokaryotes contained an essential amino acid complement compatible with their successful adaption as a nutritionally beneficial dietary protein for animals. Particularly significant in this respect was the fact that several such prokaryotic protein sources contained a higher percentage of methionine than conventional dietary plant proteins, such as soya bean meal, or novel proteins, such as British Petroleum (BP) yeast concentrate. The recent isolation of a methanol-utilizing strain of the fungus Trichoderma lignorum (10; R. J. Tye and A. J. Willetts, J. Appl. Bacteriol., in press) presents the possibility of exploiting mycelial protein produced by this eukaryotic microorganism as a source of dietary protein for animals. Fungi offer several advantages over bacteria in this respect, including a significantly lower nucleic acid-protein ratio and comparatively lower harvesting costs from spent media (6). The present investigation, conducted to examine the protein and amino acid composition of the methanol-utilizing fungus T. lignorum, was a pertinent contribution towards a rational assessment of this possibility. MATERIALS AND METHODS Growth of the fungus. The isolation, maintenance, and growth of the fungus, identified as a strain of T. lignorum, were as previously described (10; Tye and Willetts, in press). 343 Analytical methods. Amino acid analyses were made using a modification of the method described by D'Mello (4): 10 mg of methanol-grown fungal mycelium was hydrolyzed by autoclaving for 15 h at 115 C in 5 ml of 6 N HC1 in a sealed glass ampoule under nitrogen. The hydrolysate was made up to 100 ml in a volumetric flask with deionized water and subsequently filtered twice through a sintered glass funnel. A 10-ml aliquot was evaporated to dryness in a rotary evaporator. The residue was taken up in 2 ml of 10% (wt/vol) sucrose in 0.1 N HCl and analyzed for amino acids using a Technicon autoanalyzer (Technicon Instruments Co. Ltd., Basingstoke, U.K.). 2,4,6-Trinitrobenzene sulphonic acid was used as the developing reagent (11). Cystine was determined by the method of Moore (8), and tryptophan was analyzed in alkaline hydrolysates (5). The nitrogen content of the fungal mycelium was established by the classical Kjeldahl method (7). Chitin (1) and nucleic acid (9) levels were assayed as previously described. RESULTS The nitrogen content of the mycelium of methanol-grown T. lignorum was 9.82% of N, which was equivalent to a protein content of 61.4% (N x 6.25). Growth conditions which promote a high mycelial protein content are currently under investigation. The content of all of the individual amino acids in the protein of the fungal mycelium after growth on methanol was remarkably consistent ( Table 1). The essential amino acid content compared favorably with the 1957 Food and Agricultural Organisation (FAO) standard Reference Protein in most aspects; the one notable exception was a deficiency with respect to methionine ( Table 2). The mycelial protein of methanol-grown T. lignorum also compared favorably with whole wheat protein, domestic fowl egg protein, and the proteins from several other microbial sources (Table 2). Over 80% of the total nitrogen compounds present in the mycelium of methanol-grown T. lignorum was recovered as amino acids during a quantitative analysis after acid hydrolysis. The chitin and nucleic acid contents of the mycelium of methanol-grown T. lignorum were 3.8 and 5.1%, respectively. DISCUSSION The results indicate that the fungus T. lignorum growing at the expense of methanol as the sole source of utilizable carbon produces mycelial protein with a spectrum of essential amino acids superior in all but one respect to the FAO Reference Protein: the one major deficit ofmethanol-grown fungal protein is a subminimal methionine content, which shows a 15% shortfall with respect to the FAO standard. However, as indicated in Table 2, the methionine content of the mycelial protein of methanol-grown T. lignorum (1.85 g/16 g of N), although below the level in FAO Reference Protein, is nevertheless in excess of the content of many other sources of dietary protein including conventional plant proteins such as whole wheat protein (1.5 g/16 g of N) and soya bean meal (1.2 to 1.4 g/16 g of N), as well as other novel proteins such as BP yeast concentrate (1.4 to 1.6 g/16 g of N) and gas oilgrown Candida lipolyticum (1.6 g/16 g of N). Any dietary deficiency resulting from this subminimal content of methionine could be avoided by using mycelial protein from methanol-grown T. lignorum as the basis for a balanced food or feed incorporating compensatory amounts of either pure methionine or methionine-rich proteins such as domestic chicken egg protein ( Table 2). The mycelial protein of methanol-grown T. lignorum has an acceptable complement of all other essential amino acids, including lysine, as assessed against the FAO standard, as well as a full complement of non-essential amino acids ( Table 1). The complement of arginine and histidine, two amino acids which are not catagorized as essential human dietary components, but supplementary sources of which are widely considered necessary for the normal growth of children, is similar to that of many conventional dietary proteins. The amino acid composition of the mycelial protein of methanol-grown T. lignorum is similar to that of several methane-grown bacteria (4); the comparatively high content of tryptophan is particularly significant in this respect. Both the dietary and commercial potential of mycelial protein from methanol-grown T. lignorum as a food or feed remain to be assessed directly.

v3-fos

2018-04-03T03:23:05.632Z

1975-06-01T00:00:00.000Z

2896368

s2

Microflora of maize prepared as tortillas. Very little is known of the microflora in tortillas, the major component in the diet of many Guatemalans and other Central Americans. Based in a Guatemalan highland Indian village, this study examined the types and amounts of bacteria, yeasts, and molds in tortillas and in their maize precursors. Coliforms, Bacillus cereus, two species of Staphylococcus, and many types of yeast were the main contaminants, but low concentrations of alpha-hemolytic Streptococcus, facultative Clostridium, and other bacterial types were also found. When tortillas were first cooked, the bacterial counts dropped to 1,000 or fewer organisms per g, a safe level for consumption. Under village conditions, bacterial counts regained precooking levels (about 10(8) organisms/g) within 24 h and rose even higher after 48 h. Reheating caused very little change; hence, bacterial levels remained very high in old tortillas kept for later consumption. A search for the sources of contamination showed that most came from water used in preparation and from the soiled hands of women preparing the tortillas. As an attempt to correct certain nutritional needs of the population, the Institute of Nutrition for Central America and Panama initiated a tortilla fortification project in the Guatemalan village. The bacterial counts in fortified tortillas did not differ significantly from those in oridinary tortillas. Furthermore, the level of contamination was constant among tortillas of different sizes and among tortillas made from different types of maize. The importance of tortillas as a food product, the high incidence of bacterial gastrointestinal diseases among Guatemalan Indians (5), and the lack of information concerning this food's microbiological content led to this study. The microbiological implications of fortification were examined by comparing differences in bacterial growth between fortified and unfortified tortillas. Studies were made for tortillas of different sizes and thicknesses and for those made with different strains of maize. The work was done in the Mayan Indian During the period of the study environmental temperatures ranged from 10 to 28 C, with an average rainfall of 3.5 mm/day (range, 0 to 28 mm/day; this rainy season extends from June to October). Indian women prepared all the tortillas in their houses; many village households supplied the samples, which were collected only up to 48 h after preparation because tortillas are not kept longer under ordinary circumstances. Tortillas are usually consumed within several hours after preparation; older tortillas are eaten only on trips. In an attempt to find the sources of bacteria in tortillas, we examined the maize precursors: dry maize, nixtamal (maize that has been cooked with limestone for 50 min and then soaked in water for 14 h [2 ]), masa (a wet, pasty flour that results from the grinding of nixtamal at the mill), and the water used in preparation. The masa is rolled and patted into a flat pancake and then cooked for 4 or 5 min on a comal, a large, flat, hardened clay plate placed over an open fire. Reheating is also done on the comal, but for a shorter time interval ( Samples. Samples, made in individual homes, were randomly selected from among the tortillas and maize sources being prepared for the family's daily consumption. The tortillas were left towel-wrapped in a basket in the same house where they were prepared, until time of culturing (24 or 48 h after preparation). The same women who had made the tortillas also reheated them for later stages of study. Samples of masa and the water used to moisten it were obtained during tortilla preparation. Maize was obtained the previous night so that it would be from the same batch used in the other samples; nixtamal was obtained at the mill just before being ground into masa. All samples were cultured 15 to 30 min after receipt, with special care taken to process fresh and reheated tortillas immediately. Dilutions. Tortillas were brought directly to the laboratory at the village health clinic. A 10-g portion of tortilla pieces was ground in a mortar with sterilized sand and placed in a bottle containing 90 ml of charcoal water (tap water that had previously been adsorbed with charcoal, filtered, and sterilized) (14). This 10-dilution was mixed and then agitated for 5 min; progressive dilutions to 10-9 were prepared in charcoal water as needed (5). Samples for maize, nixtamal, and masa were prepared as above; kernels were ground in a Waring blender when necessary. The original sample of water was used as 10°d ilution, with further dilutions to 10-' prepared. Media used. Table 1 shows the media and types of incubation used. Total bacterial count was determined by using Trypticase soy agar (supplied by Baltimore Biological Labs, Cockeysville, Md., as were GasPak generators and all other media, except for cereus-selective agar, which came from Merck & Co., Inc., Rahway, N.J.), pour-plate method; Tergitol-7 agar with 0.004% triphenyl-tetrazolium chloride, Salmonella-Shigella agar, and brilliant green agar were used to select for coliforms. One gram of minced tortilla was also placed in tetrathionate Selenite-F Enrichment broths and restreaked on Salmonella-Shigella and brilliant green agars after 24 h of incubation to select for pathogenic coliforms. Mannitol salt agar was used to select for staphylococci; cereus-selective agar with 10% egg yolk emulsion in physiological saline (1:1) solution was the medium used to select for Bacillus. Schaedler base medium (14) was modified by adding 1% Trypticase soy broth instead of Trypticase and was used in anaerobic culturing (10). Sabouraud dextrose agar and Mycosel agar were used for yeasts and molds. Media inoculation and incubation. In inoculating plates from various dilutions, we used a 0.01-ml calibrated platinum loop and placed four dilutions on each plate. The plates for anaerobic culture were inoculated with 1.0 and 0.1 ml of the 101-dilution by the spread-plate method. Anaerobic plating was done both before and after boiling the 10-dilution for 30 min. Anaerobiosis was achieved by means of GasPak disposable generators (3). Table 1 lists the incubation periods and temperatures. Enumeration and identification of bacteria. Enumeration was made directly from the plates; representative colonies from each plate were Gram stained (Kopeloff method) and further identified through biochemical tests (1,6,7,9,12,13). All colonies found in anaerobic culture were subcultured aerobically at 37 C for 24 h; almost all were found to be facultative. Microflora of tortillas. Coliform levels of 103 to 107 organisms/g were obtained in 24to 48-h-old tortillas; Alcaligenes faecalis, Klebsiella sp., and Escherichia coli were the most common of those encountered (Fig. 1), indicating a high level of fecal contamination during tortilla preparation. These same species were encountered in the water at levels of 104 to 10l organisms/ml. Cooking the masa killed most coliforms, but a sufficient number survived to return the concentrations to precooking levels within 24 to 48 h of storage at room temperature (Fig. 2). Neither Shigella nor Salmonella were observed, even with tetrathionate and Selenite-F Enrichment; however, this finding does not rule out tortillas as a possible vector for pathogenic coliforms. Such bacteria would probably be much overgrown by other coliforms. The high fecal contamination rates make tortillas a prime candidate for further study in this area. Furthermore, the high E. coli counts suggest the probable presence of enteropathogenic E. coli. Staphylococci. Staphylococci levels in tortillas reached 107 to 108 organisms/g (Fig. 2), with equal numbers of Staphylococcus aureus and S. epidermidis found both in tortillas and in their maize sources. The high counts of S. aureus in the older tortillas could have significant health implications, because certain strains may produce a heat-stable enterotoxin in foods and also because of the possibility of staphylococcal infection. Bacilli. Bacillus cereus, B. macerans, B. megaterium, B. polymyxa, and B. subtilis were found in the maize precursors, with no one species predominating. Most species of Bacillus were killed during cooking, leaving almost exclusively B. cereus. Certain strains of B. cereus, in high concentrations, can cause food poisoning by producing an enterotoxin (8). Hence, the high B. cereus levels observed in tortillas after 24 or 48 h of storage (up to 109 organisms/g) may make their consumption hazardous (Fig. 2). B. subtilis and B. megaterium are also occasionally observed in tortillas. Clostridia. All clostridia encountered in anaerobic culture were found to be facultative. In tortillas, they appeared in low concentrations (101 to 102 organisms/g). Further identification was not pursued because facultative clostridia are nonpathogenic. Streptococci. Streptococci were encountered only in anaerobic culture and never in concentrations greater than 102 organisms/g. The streptococci present were almost exclusively alpha-hemolytic, so further identification was not pursued. Other anaerobes. A variety of other species were encountered, including Sarcina, Lactobacillus, Coccobacillus, and Micrococcus, always in very low dilutions and almost always facultative. Because these species are not pathogenic to man, they were not further identified. Yeasts. Both tortillas and maize sources supported a great quantity and variety of yeasts. Because yeast identification is both difficult and complex, only precursory examination was done, although further work in this area would be of great interest. Examinations revealed species of a red-pigmented Rhodotorula, Candida, Trichosporon, Geotrichum, Torulopsis, Saccharomyces, Pytirosporum, and others. Molds. Molds were found in very high concentrations in tortilla precursors, but were almost completely absent in tortillas, even up to 4 days after preparation. Colony morphology showed species of Penicillium, Aspergillus, Neurospora, and Rhizopus in the maize precursors. No further identification was carried out because these species were not present in any of the tortillas. However, the possibility of mycotoxin production by Aspergillus and Penicillium during the growth of maize should be investigated. Some mycotoxins are thought to be carcinogenic after repeated consumption (4). Unless they are inactivated by the limestone treatment of maize kernels (2,15), their presence could have serious health implications. Microfloral population of tortilla precursors during and after preparation. During early stages of tortilla preparation, bacterial levels rose rapidly, with most initial bacterial contamination resulting from the water used in preparation (Fig. 3) and probably from the hands of the women preparing the tortillas. Most bacteria were killed by cooking (Fig. 2). Enough survived, however, to return bacterial concentrations to precooking levels after 24 h and to even higher levels after 48 h. Yeast contamination was found in maize and throughout preparation. Molds did not reappear after preparation of tortillas, indicating that cooking effectively killed them. After 7 to 10 days, molds grew again on tortillas because of air contamination during storage. Fortified tortillas supported slightly greater aerobic growth than unfortified tortillas, both after 24 and 48 h, but the high levels present make small variations relatively insignificant. A 2-logarithm range in B. cereus levels between fortified and unfortified tortillas was the most important disparity; the several-logarithm difference in nonpathogenic yeasts had little effect on the safety of tortillas. Therefore, the microbiological difference between fortified and unfortified tortillas is minimal with respect to the safe consumption of either kind. Fresh tortillas are safe for consumption, but stored tortillas have sufficiently high bacterial concentrations within 24 h to make them dangerous to health. Most significant in tortillas more than several hours old are the extremely high levels of S. aureus, B. cereus, and coliform bacteria. Tortillas of various sizes and from several different types of maize showed no significant difference in bacterial counts or species isolated. Effects of reheating tortillas. In 24-h-old tortillas, reheating dropped the bacterial count 2 to 3 logarithms, but the counts nevertheless remained very high (Fig. 4). After 48 h, the reheating decrease was slightly less, leaving extremely high bacterial counts (105 to 107 organisms/g). This indicated that the usual 1to 2-min reheating time for the older tortillas did not render the tortillas safe for consumption. Possible existence of enterotoxins from B. cereus and S. aureus further increased the danger. Because such enterotoxins are often heat stable and pathogenic, a study of their existence in tortillas 48 h old or more would be worthwhile. Toasting or longer periods of reheating would possibly lower bacterial concentrations to safe levels. This is currently unfeasible, however, because additional heating would alter taste and texture and also increase fuel consumption. DISCUSSION Tortillas offer a very rich medium for bacterial growth. With the Indians' relative disregard for sanitary procedures, plus the high levels of bacterial and fecal contamination found in the water used during tortilla preparation, a great amount of contamination occurs during the preparation process. Although cooking destroys many of these bacteria, a sufficient number survive to bring bacterial concentrations up to precooking levels within 24 to 48 h. Because of the possible production of enterotoxins and because of the possible contamination by other pathogens, the Indians should avoid consuming older tortillas that have been stored unrefrigerated. The results indicate that sanitation and the use of comparatively clean water should be stressed to those living in such villages. Some of the water samples from tortilla preparation arrived in our laboratory clouded with masa and containing dead flies. Water used in tortilla preparation should be changed daily instead of being used for an extended period. Adopting such simple sanitation procedures would do much to lower contamination in tortillas that are not consumed quickly after preparation, especially in tortillas prepared for trips. This study involved 46 women in one highland village where conditions were similar to those of other indigenous villages. At lower elevations in Central America, ambient temperatures are much higher, and the bacterial growth can be assumed to be even more rapid. Hence, the need for better sanitary methods in the preparation of tortillas must affect populations throughout Central America.

v3-fos

2020-12-10T09:04:22.833Z

1975-02-01T00:00:00.000Z

237230518

s2

Actinomycetales from Corn Mesophilic Actinomycetales were isolated from whole corn, brewer's grits, and break flour received from three different mills. In addition, strains were isolated from high-moisture (27%) field corn; high-moisture, silo-stored corn (untreated); and high-moisture corn treated with ammonia, ammonium isobutyrate, or propionic-acetic acid. According to standard techniques, 139 strains were extensively characterized and 207 additional strains were partially characterized. On the basis of these characterizations, the streptomycete strains were identified by both the systems of Pridham et al. and Hütter because these systems are rapid and accurate. In general, only Streptomyces griseus (Krainsky) Waksman and Henrici was isolated from high-moisture whole corn (treated or untreated) except from grain exposed to ammonium isobutyrate. Strains isolated from high-moisture corn subjected to that treatment represented both S. griseus and S. albus (Rossi Doria) Waksman and Henrici. The strains isolated from corn and corn products from the three mills were identified with a number of streptomycete species. Of all Actinomycetales isolated, only three were not streptomycetes—two from brewer's grits and one from break flour. Mesophilic Actinomycetales are present in agricultural grains, residues, and commodities. Streptomyces albus (Rossi Doria) Waksman and Henrici, the type strain for the genus Streptomyces Waksman and Henrici, reportedly was isolated from straw in Prague about 1900 (4). This species also was isolated by Graves et al. (2) along with S. griseus (Krainsky) Waksman and Henrici from wheat and wheat flour. Mehrotra (7) reported isolating more than a thousand streptomycetes per gram of wheat grains. He found that S. albus (probably S. griseus by the Pridham et al. [11 ] classification) was in about 47% of all samples tested. He also reported finding only a few Actinomycetales in other grains (rice, barley, and millet). So far as we know, there have been no reports on the identity of Actinomycetales isolated from corn. Bothast and co-workers (1) have studied the microbiology of corn products. Total aerobic bacterial, mold, and Actinomycetales populations were determined for samples of whole corn, brewer's grits, and break flour supplied by three different mills. During their study, 190 strains of mesophilic Actinomycetales were selected and were isolated by dilution plating with Difco plate count agar, containing 100 ppm of cycloheximide (Actidione), and then by incubating at 28 C for 14 days. In addition to these strains, one of us (R.F.R.) later isolated 155 more from high-moisture corn, Harvestore silo corn (untreated), and corn treated with ammonia, ammonium isobutyrate, or propionicacetic acid. Because corn is an economically important crop in the United States and its production, harvesting, storage, and processing involve many aspects of applied microbiology, it was believed that information on the identities of the 345 strains of Actinomycetales would be of interest. The purpose of this study, then, was to determine the identities of these isolates using the most recent acceptable criteria for characterization and identification. All 345 strains were characterized and identifications were made according to the Pridham et al. (10,11) and HUtter (3) systems and keys (Table 1), because these two keys are the most practical ones extant and can lead to rapid identifications. (This paper was presented at the Annual Meeting of the American Society for Microbiology, 12 to 17 May, Chicago, Illinois.) MATERIALS AND METHODS Strains. The Actinomycetales studied were isolated and purified from treated and untreated corn and corn fractions. All are maintained at the Northern Regional Research Laboratory as yeast extractagar slant cultures (8) and as lyophilized preparations. Liquid inocula for all media needed for charac- terizations were prepared with tryptone-glucose-liver extract-yeast extract broth as described by Lyons and Pridham (5). Characterization. Strains were characterized according to methods outlined by Pridham et al. (5,6,9,10). These methods allow use of both the Pridham et al. and Hitter keys. Strains isolated and purified from samples from mill I were extensively characterized by the following criteria: spore-wall ornamentation; spore-chain morphology; ability to produce melanin pigments and to darken peptone-iron agar; color of aerial mycelium; ability to utilize D-glucose, D-arabinose, and L-rhamnose; amount of growth on Czapek's sucrose agar; ability to produce antibiotics; and the identity of the isomer of diaminopimelic acid (DAP) in whole cell hydrolysates. Also, strains isolated from the 1972 high-moisture field corn crop (untreated), Harvestore silo corn, and ammonia-treated corn were characterized according to these same criteria. All strains other than those from mill I were characterized only to the extent that identities could be made using the Pridham et al. key and the HUtter key. Criteria for these characterizations included only the first five mentioned above. DAP analyses. Whole cell hydrolysates were analyzed for DAP isomer by the methods described by Pridham and Lyons (10), except that the 72-h broth cultures were autoclaved for 25 min at 121 C before the whole cells were separated by filtering through Whatman no. 54 filter paper with a Buchner funnel. The cells were washed on the funnel with distilled water and then dried with 95% ethanol. They then were scraped from the filter paper and air-dried on a watch glass. The resulting products were stored at -20 C until analyzed. Identification of streptomycetes. All strains having L-DAP were identified by both the Pridham et al. key (10) and the HUtter key (3). Strains having meso-DAP were not identified because they are not considered to be streptomycetes. RESULTS The 15 different species of streptomycetes identified in this study were: Streptomyces albus, S. collinus Lindenbein, S. diastaticus extent of their occurrence are given in Table 2. The identities and extent of occurrence of the About 35% of the 345 strains exhibited the strains isolated from corn (whole and milled) formation of curious bodies (small clumps of from the mills appear in Table 1. The identities cells) which we termed "soft sclerotia" in the- aerial mycelium formed on yeast extract agar and glycerol-asparagine agar (Fig. 1). The soft sclerotia also seem to combine as larger masses over the entire mycelium of more heavily growing areas and sometimes resemble the exudate that forms in hygroscopic cultures. Although this phenomenon usually was associated with Rectiflexibiles type cultures, we did find three strains of the Spiralis type that also exhibited this characteristic. Only 197 of the 345 strains were tested for antibiotic activity; 95% of these strains showed activity. Although some of this activity was only minimal, it was readily detected by a simple spectrum-dish test (6). Cross-antagonism studies on the basis of a similar technique and a streptomycin-producing strain, were carried out with 62 strains. The results suggest that none of the 62 produce streptomycin. Only four strains of the 345 tested darkened peptone-iron agar, i.e., positive chromogenicity. Of that many strains randomly selected, 80 to 90 would be expected to be chromogenic (25 to 30%) based on our experience. Of the 345 strains, only one had spiny sporewalls and one had hairy walls; the rest were smooth walled. Again, based on the random selection techniques used, at least 5% of the strains would be expected to be other than smooth. DISCUSSION A variety of species of streptomycetes were isolated from corn and corn fractions supplied by three different mills. In contrast, Streptomyces isolated from whole high-moisture field corn (not grain from the mills) stored treated and untreated varied little. The results suggest that floristic changes occur sometime after storage and during processing. Strains isolated from mill samples showed in common: nonchromogenicity, smooth spore-wall ornamentation, the soft sclerotia, and antibiotic activity. They differed in spore-chain morphologies and color of the aerial mycelium. Almost all the strains isolated from the various treatments of the high-moisture field corn, with the exception of the ammonium isobutyrate treatment, were identified as S. griseus with the Pridham et al. system (10,11). In addition to the strains of S. griseus, strains of S. albus also were isolated from the ammonium isobutyrate-treated, whole high-moisture field corn. These were not isolated, however, until 24 days after treatment. Only strains of S. griseus were isolated from this sample for the first 12 days; but then that species was no longer detected, possibly because of the ammonium isobutyrate treatment. After that time only strains of S. albus were detected. None of the other treatments had this effect on high-moisture whole corn samples. Although 95% of the 197 strains tested produced antibiotics in laboratory tests, we know of no studies wherein whole corn or milled fractions have been tested for the presence of antibiotics produced as a consequence of the metabolic activities of Actinomycetales in corn or its milled products. Our interest in antibiotic production is concerned with its use in more precise characterization of strains through identification of the antibiotics produced.

v3-fos

2014-10-01T00:00:00.000Z

1975-10-15T00:00:00.000Z

18401085

s2

Heritability estimates of hatching time in the fayoumi chickens *SUMMARY Pedigree Fayoumi eggs were collected from individual sire matings, and were used for heritability estimates of hatching time. The eggs were saved for a period of ten days before setting. It was noticed that the distribution of hatching time approached normality with a mean of zr days plus 1hours. Heritability estimates of hatching time were ha = 0ha = 0and h5 = o.r8. The correlation between the time of egg storage and hatching time was highly significant (The regression coefficient of hatching time on holding time was 0 hatching period (about one hour) per day of holding. The correlation between egg weight and hatching time INTRODUCTION Several factors, both genetical and environmental, have been proved to affect the required hatching time of the chick embryo (B OHREN et al.,ig6i). Consistent differences in hatching time, as observed among breeds and lines of chickens, supported the heritable nature of this trait. (M OR G AN and KOHI,M!Y!R, 1957 ; H A S SAN and N ORDSKOG , i96! ; and ICIDNOE, 1973). Several investigators reported that large eggs require a longer incubation time than smaller ones (W ILLIAMS et al.,ig5z ;B OHR E N et al., ig6i ; H ASSAN and N ORDSKOG , ig6 7 ), however, I CHINOE (rg 73 ) reported that larger eggs tended to hatch earlier than smaller ones in the R.I.R. male R.LW. female hybrid and vice versa in the case of the Shaver strain of W.L. H ASSAN and N ORD S KO G ( 19 6 7 ) suggested that selection for high body weight retarded hatching time more than could be accoun-ted for by the correlated response in egg size and thus hatching time may have a genetic basis independent of egg size. Storage period of hatching eggs was also reported to affect hatching time. Bo!>~N et al. (i 9 6i) and Z AWALSKY (i 9 62) observed that increased preincubation storage time delayed hatching time. M ORGAN and KoH!,l!!YEx ( 1957 ) reported that the expected « hatch-day » range was frequently more variable within inbred lines than that for the non-inbred purebred and that for crosses. , The aim of this experiment therefore, was to estimate the heritability coefhcients of hatching time of Fayoumi chickens. Egg weight and pre-incubation storage were also investigated in relation to hatching time. MATERIALS AND METHODS Individual sire matings of Fayoumi males with females were used for heritability estimates of hatching time (length of incubation period). Twelve sires and i 44 dams were randomly mated in family pens supplied with trap nests where each family was composed of one sire and twelve females. The pedigree eggs were collected for a period of ten days and the fresh egg weights were recorded daily. The temperature of holding room ranged from 55° to 6o o F. and all eggs were turned daily until six hours before setting. The eggs were incubated in forced draft machine automatically maintained at a temperature of I ooof. and wet bulb thermometer reading of 86°F. At the beginning of the 2 ist day of incubation the hatcher was opened every four hours to remove the hatched chicks which were completely free of their shells. The first four hours were assigned period number one and the removal of chicks continued through i2 periods ( 4 8 hours). For statistical analysis, the dams used in this experiment had at least 3 offspring per dam, while the sires used had at least 4 dams per sire. Analysis of variance was based on the model given by KING and H ENDERSON (i 954 ), using an analysis appropriate to unequal subclass numbers. Variance components were for sire (s), dam (d) and full-sibs (e). In a random mating population the variance arising from genetic differences between sire (a8) is expected to contain one-fourth of the additive genetic variance, one-sixteenth of the additive X additive epistatic variance and a smaller portion of the higher order interaction variances involving only additive effects. The dam component of variance (aa) is expected to contain one-fourth of the additive genetic variance, one-fourth of the dominance variance, three-sixteenth of the additive X additive epistatic, one-sixteenth of the dominance X dominance variance and a smaller fraction of the higher order interaction variances. Variance from maternal effects will also contribute to the dam component (L ERNER ,195 8). Phenotypic correlations between egg weight and hatching time and between holding days and hatching time were estimated. The variables to be analyzed then were age of storage (X l ) in days, egg weight (X 2 ) in grams and hatching time (Y) in periods. In order to observe the effect of inbreeding on hatching time, eggs were collected from different inbred lines of Fayoumi hens having the coefficients of 6. 25 , 12 . 5 , 25 . 0 , 37 . 5 and 50 . 0 p. 100 plus a control of outbred hens. Heritability estimates of hatching time were h,' = 0 . 2 8, ha = 0 . 0 8 and ha + g = 0 . 1 8. It was clear that higher dam than sire component estimates of heritability for hatching time were observed. With the statistical model employed and random mating being assumed, variance arising from non-additive genetic and maternal effects may be expected to be found in the dam component of variance. RESULTS AND DISCUSSION Moreover, the correlation between the age of storage and hatching time was highly significant ( 0 . 37 ). The coefficient for the regression of hatching time on holding time (days) was 0 . 25 hatching period (one hour) + 0 . 03 , d.f = 563, P < o.ooi. Similar results were reported by Boxx!rr et al. (ig6i). The correlation between egg weight and hatching time was not significant (r = 0 . 05 and buzz = 0 . 024 ). Boxx!rr et al. (ig6i) reported a small and not significant correlation between these two variables on individual basis while it was significant between dam means. It had been shown by W ILL IA MS et al. ( 1951 ) that larger eggs required a longer incubation time than smaller ones. On the contrary, ICHI N OU ( 1973 ) found that larger eggs tended to hatch earlier as mentioned in the introduction. The relationship between egg weight and hatching time in this experiment, is not clear (r = 0 . 05 ) and this may be due to the medium size of Fayoumi eggs. I CHINO E ( 1973 ) reported that eggs of medium size showed less difference in hatching time. The following multiple regression equation was set up in order to predict the hatching time (Y), in periods, expected from a given egg weight (X,), in grams, and holding days (X,) : The effect of inbreeding on hatching time is shown in table 2 . It was noticed that incubation period was increased by the increase of inbreeding coefficients. While most of the chicks (at least 50 p. 100 ) of the control group hatched during the first three periods, most of the inbred chicks hatched during the first five periods. Similar results were obtained by M ORGAN and K OHLMEYER ( 1957 Des oeufs pedigree de la race Fayoumi fécondés par différents pères ont été collectés en vue d'estimations d'héritabilité de la durée d'incubation. Ces oeufs étaient conservés pendant une période maximum de 10 jours avant la mise en incubation. Il a été observé que la distribution de la durée d'incubation était proche de la normalité, avec une valeur moyenne de 21 jours plus i,z8 heures. Les estimations de l'héritabilité de cette durée étaient h' = o,z8, Aj = 0 , 0 8 et hâ +8 = o,i8. La corrélation entre la durée de conservation des oeufs avant incubation et la durée d'incubation était hautement significative (0,3!). Le coefficient de régression de la durée d'incubation sur la durée de conservation était 0 , 25 « période d'éclosion (environ i h) par jour de conservation. La corrélation entre le poids des oeufs et la durée d'incubation n'était pas significative.

v3-fos

2020-12-10T09:04:13.045Z

1975-09-01T00:00:00.000Z

237231388

s2

Influence of Wine Composition on the Heat Resistance of Potential Spoilage Organisms Pasteurization studies were conducted on 29 yeasts and five lactic acid bacteria. In general the yeasts were more heat resistant in wine than were the bacteria. The one exception was a strain of Lactobacillus fructivorans that gave an average D-value of 1.7 min at 60 C. Alcohol was the wine constituent that had the greatest effect on resistance; D-values for all test species were inversely related to the ethanol concentration. The response of organisms to other factors such as pH, sugar, and sulfur dioxide varied with the species. Bottled wines that have been sweetened or which contain some residual fermentable carbohydrate are susceptible to spoilage by yeasts and lactic acid bacteria (7,8,12). Multiplication of these organisms often results in off-flavors as well as objectionable turbidity and sediment (1). One of the methods used to prevent spoilage is pasteurization. Although a very old procedure, surprisingly few quantitative data are available regarding the conditions necessary to impart biological stability to wine. As a result, the heat processes that are presently used differ markedly in severity. Some wineries, for example, flash pasteurize at 80 C or higher; others use hot bottling procedures in which the wine is filled into bottles at temperatures ranging from 45 to over 70 C. In addition to the need for minimum process data, impetus for this research was provided by the so-called "mod" or "pop" wines that have been introduced in recent years. It seemed that the different composition of these wines might have a significant effect on their pasteurization requirements. Media. Yeasts were propagated in 100 Brix Thompson seedless grape juice. Potato dextrose agar, pH 5.6, was the plating medium used for viable counts. The lactic acid bacteria were grown in tryptoneglucose-yeast extract-salts broth (11) supplemented with 10% tomato juice serum and 0.5% fructose. (The latter ingredients were needed for growth ofLeuconostoc oenos and Lactobacillus fructivorans.) For plating, 2% agar was added to the medium. Heat resistance. Heating was conducted in flame-sealed capillary tubes (4,10). In a typical trial, stationary-phase cells were centrifuged from the culture medium, then suspended in the test 36 solution, usually a wine. A Hamilton syringe with a 500-iul capacity was used to transfer 20-Al volumes of the cell suspension to capillary tubes (1.6 by 50 mm); a Hamilton repeating dispenser facilitated this operation. The tubes, held in Thomas pinch clamps, were submerged in a constant-temperature water bath. After heating for a given time period, the capillaries were cooled in 95% ethanol. (A dual function of the alcohol was to destroy many of the microorganisms that might be present on the surface of the tubes. Ethanol-resistant organisms apparently were eliminated by subsequent dilutions; contamination was not a problem.) Five tubes from a given treatment were pooled in a dilution bottle containing 5 ml of 0.1% peptone solution. After crushing the tubes with a sterile glass rod, appropriate decimal dilutions for plating were made in 0.1% peptone solution. The yeasts were incubated at 28 C, and the lactic acid bacteria were incubated at 32 C. The incubation period ranged from 2 to 7 days, depending upon the culture. An experimental wine prepared in our pilot plant from Niagara grapes served as our model in most experiments. This 1971 vintage wine contained 12.1% ethanol, total acid of 0.82% (expressed as tartaric), 20 mg of tannin per 100 ml, less than 5 mg of free SO2 per liter, and less than 0.1% reducing sugar. Its pH was 3.2. The methods for these analyses are described by Amerine and Ough (2). Modifications of the wine were made in such a way as to minimize changes in the concentration of the various constituents. Thus solutions used to adjust pH and the concentration of glucose were prepared by dissolving the reagents directly into samples of the wine. RESULTS AND DISCUSSION Species resistance. During the course of this study, the resistance of 29 yeasts heated in the Niagara wine was determined. Many of the cultures were strains ofSaccharomyces cerevisiae used in commerical wine making; others were flor yeasts, species of Saccharomyces involved in the sherry fermentation. The average surival figures are summarized in a histogram ( Fig. 1). It can be seen that most of the yeasts yielded D-values of under 0.6 min when heated at 49 C. The most heat-resistant yeast, a strain of S. cerevisiae, gave an average D-value in five experiments of 1.6 min. The single nonwine yeast examined, Candida utilis NRRL Y-900, gave a D-value of under 0.1 min at 49 C. The initial studies with lactic acid bacteria were conducted with the first four species listed in Table 1. Because they were rapidly killed at 45 C, a temperature 40 lower than that used for the yeasts, it first was concluded that the lactic acid bacteria as a group possessed considerably less resistance than yeasts. L. oenos ML 34, an important malo-lactic strain (6), was especially sensitive; 43 C was a lethal temperature for it. These results are in agreement with those of Murdock et al. (5) whose studies with orange juice also indicated leuconostocs to be most heat sensitive, followed by the lactobacilli and then the yeasts. Our hypothesis about the lactics had to be revised, however, when we later isolated a strain of L. fructivorans from a spoilage outbreak involving a premixed Bloody Mary cock- tail. This organism was considerably more heat resistant than any of the yeasts: it was difficult to detect destruction of it at temperatures below 58C. L. fructivorans is relatively alcohol tolerant and our isolate grew rapidly in broth containing over 12% by volume. This raised the question as to whether the low heat resistance of the other bacteria was related to an alcohol sensitivity. Perhaps the shock of being exposed to alcohol for the first time was partly responsible for their death. To test this hypothesis, an attempt was made to select for more heat-resistant strains by growing the lactic acid bacteria in broth containing ethanol. As the results with Lactobacillus plantarum illustrate (Table 2), we were not successful. Ethanol-grown cells did not exhibit significantly greater heat resistance than those propagated in the standard medium. Ethanol. Of the different wine constituents that were studied, alcohol had the greatest effect on heat resistance ( Table 3). As illustrated here, the resistance of both lactics and yeasts was reduced. The flor yeast was affected to a greater extent than L. fructivorans: the Dvalue for it was over 50-fold greater in the absence of ethanol than when heated in the presence of 12%; the lactic exhibited a difference of less than fourfold. Thermal death time curves have been plotted for our most resistant yeast and lactobacillus strains when heated in wine containing two levels of ethanol, 8 and 12% (Fig. 2). The parallel slopes show that these differences in concentration did not affect the organism's response to a change in temperature. In other words, the zvalues for these organisms were not affected by alcohol. Studies by other workers also have shown ethanol to reduce the heat resistance of yeasts (3,4,9). Schanderl (9) observed that differences in alcohol concentration of only 3% had a marked effect on survival, which is in agreement with our findings. Carroll and Lopez (3), on the other hand, obtained little difference in thermal destruction of S. cerevisiae when heated in buffers containing 6 and 10% ethanol. pH. The effect of pH on heat resistance was determined by adjusting the reaction of Niagara wine with NaOH or tartaric acid. The study was limited to the pH range of 3 to 4 since these are common values for wine. The results ( stituent to which individual species responded differently. Thus the addition of 10% to Niagara wine increased the D-value of L. fructivorans almost threefold but afforded only slight protection to L. plantarum and the flor yeast (Table 5). These results suggest, of course, that the pasteurization requirements for a dessert or a pop wine might be somewhat greater than for, a dry, table wine. Sulfur dioxide. In the studies with SO2, freshly prepared solutions of potassium metabisulfite were mixed with the wine just prior to filling the capillary tubes. It is assumed, however, that the actual amount of free S02 was lower than the calculated value since some would react with aldehydes and other wine constituents. The results ( Table 6) again showed a variable response depending upon the organism: SO2 appreciably reduced the resistance of L. fructivorans, whereas an effect on the yeasts could not be detected. We were not too sur- VOL. 30, 1975 prised by these results since yeasts generally are more resistant to this germicide than are bacteria. The unheated L. fructivorans controls showed no decrease in viable counts when suspended in wine containing 50 mg of SO2 per liter. Other constituents. In addition to the more obvious variables already described, it seemed that wines might contain other substances that would influence heat resistance of potential spoilage organisms. For example, certain tannins or other polyphenolic compounds, natural constituents of the grape, might either protect organisms or cause them to be more heat sensitive. One approach in the search for other factors was to compare heat resistance in various concentrations of the Niagara wine. In these studies the wine was diluted with a solution of 12.1% aqueous ethanol so that the concentration of all constituents except alcohol would be decreased. The results (Table 7) suggest that a small amount of protection was provided by some ingredient(s) of the wine. Thus the survivals ofS. cerevisiae and L. fructivorans, averages of two experiments, were slightly lower when heated in the straight aqueous ethanol solution. In other trials resistance was compared in wines fermented from different grape varieties. To eliminate the influence of ethanol, after determining the amount present, small quantities of water were added so that all of the wines ended up with the same concentration of ethanol, 11%. Again the results failed to reveal the presence of other wine constituents that had more than a modest effect on heat resistance (Table 8). It is suspected that the Delaware wine actually permitted somewhat higher survivals since the averages of two trials agreed very well. So far, however, it has not been possible to relate increased resistance to some intrinsic property of the Delaware wine such as pH, total acid, or free SO2. It is concluded from these studies that poten- tial spoilage organisms often possess little resistance when heated in a traditional table wine and that many wines may receive a more severe pasteurization than is actually required. The data also show that some formula modifications would increase the process requirements, in particular, the lowering of the alcohol content, the raising of the pH, and the addition of sugar. The single organism encountered in this study that possessed relatively high heat resistance, L. fructivorans, would not present a spoilage problem for many wines because we have found that it will not initiate growth in the tryptone-glucose-yeast extract salts broth when acidified to pH 3.8. Most wines have a pH lower than this. It is also possible that L. fructivorans would be inhibited by the levels of free S02 that generally are present in commercial wines.

v3-fos

2020-12-10T09:04:20.736Z

1975-04-01T00:00:00.000Z

237229316

s2

Microbial Fermentation of Rice Straw: Nutritive Composition and In Vitro Digestibility of the Fermentation Products Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis. Microbial cells and undigested residue, as well as chemically treated (NaOH or NH4OH) and untreated straws, were analyzed for nutrient composition and in vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was digested, and 18.6% of the total substrate weight that disappeared was recovered as microbial protein. The microbial cell fraction was 37% protein and 5% crude fiber; the residue was 12% protein and 45% crude fiber. The microbial protein amino acid profile was similar to alfalfa, except for less cysteine. The microbial cells had more thiamine and less niacin than Torula yeast. In vitro digestibility of the microbial protein was 41.2 to 55%; that of cellulose was 52%. Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis. Microbial cells and undigested residue, as well as chemically treated (NaOH or NH4OH) and untreated straws, were analyzed for nutrient composition and in vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was digested, and 18.6% of the total substrate weight that disappeared was recovered as microbial protein. The microbial cell fraction was 37% protein and 5% crude fiber; the residue was 12% protein and 45% crude fiber. The microbial protein amino acid profile was similar to alfalfa, except for less cysteine. The microbial cells had more thiamine and less niacin than Torula yeast. In vitro digestibility of the microbial protein was 41.2 to 55%; that of cellulose was 52%. The major limitations of straw as an animal feed are low digestibility and low protein content. Efforts have been made to increase the feed value of cereal straws by chemical and physical treatments, as well as nutrient supplementation. The digestibility of various crop straws can be increased by treating with NaOH or NH4OH, but the low protein still requires nitrogen supplementation. Additions of urea, molasses, branched-chain fatty acids, sulfur, and other minerals have met with varying success (2,4,5,6,12,17). In vitro digestibility. In vitro cellulose digestibility was determined by measuring disappearance of substrate insoluble dry matter upon treatment with cellulase ("Onozuka" SS, Kanematsu-Gosho, Ltd, New York) and protease ("Pronase" B grade, Calbiochem, Los Angeles, Calif.), and was expressed as total solubles after enzymes. In vitro protein digestibility was defined as the decrease in substrate nitrogen after treatment with fungal protease (Streptomyces griseus) and chick pancreas acetone powder (18). Rumen metabolism. Rumen microbes were obtained from a fistulated sheep that was maintained on alfalfa hay with free access to dried range grass. Susceptibility of substrates to rumen microbial attack was determined by measuring total gas and volatile fatty acid (VFA) production. VFA samples acidified with meta-phosphoric acid were analyzed on an Aerograph 204 gas liquid chromatograph. The rate of ruminal fermentation was measured by anaerobic Warburg procedures and described by Oh et al. (17). Vitamin, amino acids, and chemical analysis. 510 All vitamin aasays were microbiological: thiamine and niacin by the methods of Gyorgy and Pearson (8), folic acid by the method of Jukes (14), and pyridoxine by the Association of Official Analytical Chemists (1). Amino acids were analyzed by ion-exchange chromatography after 10 mg of protein was hydrolyzed with 10 ml of 6 N HC1 under vacuum at 110 C for 24 h. Cystine plus cysteine was determined after pretreatment with performic acid. Lignin was determined according to Van Soest (19). Protein was calculated by multiplying the difference between total nitrogen and NH8-nitrogen by 6.25. All other chemical analyses, unless otherwise specified, were carried out according to the Association of Official Analytical Chemists methods (1). RESULTS AND DISCUSSION When a mixed culture of Cellulomonas sp. and A. faecalis was grown on rice straw, substrate utilized depended on the initial substrate level, inoculum size, fermentation time, and substrate pretreatment. Figure 1 shows material balance of a typical fermentation of NaOHtreated straw. After 3 days, 75% of the initial substrate had been digested, 150 g of undigested fermentation residue remained, and 178 g of mi-crobial cell precipitate had been produced. The residue fraction (mainly undigested straw plus some microbial cells) was 12% protein and the microbial cell fraction (mainly microbial cells, a small amount of fine fibers, and precipitated minerals) was 37% protein. Thus, 84 g of protein (18 g in the residue and 66 g in the cells) was obtained by digesting 450 g of the initial 600 g of rice straw, a net protein yield of 18.6%. Table 1 lists the chemical composition of the rice straw substrates and products of their microbial fermentation. The untreated rice straw contained 0.67% nitrogen, 29.8% crude fiber, and 15.8% silica that accounted for most of the 18.6% ash. NaOH treatment removed about half of the silica, probably by solubilization of the amorphous silica. It also removed the reducing sugars. The ammonia treatment increased both total and NH3-nitrogen, possibly by formation of ammonium salts and amides of cellulose. Ammonia treatment, however, removed silica less than NaOH. The undigested fermentation residue was about 12% protein and 45% crude fiber. by urea (2.5%) supplementation of straw (6). The increased crude fiber in the residue may have been from preferential microbial utilization of readily digestible carbohydrates, leaving cellulose and lignin. Lignin in the residue increased from 4.3% to 9.2%. Thus, 53% of the initial lignin was recovered in the residue, and the rest was solubilized in the effluent. The digestibility lowering effect of lignin may be caused by its chemical complex formed with cellulose. Therefore, the lignin in the residue, which may be partially dissociated from the plant cell walls by mechanical and microbiological action during the fermentation, should not be as detrimental as the native lignin in the straw. Ca, P, and S in the undigested residue were 2, 4, and 7 times, respectively, those in the untreated rice straw. These minerals are especially low in rice straw, and their increase could be an additional benefit from fermentation. The microbial cell fraction was 38% crude protein, 5.7% crude fiber, 25% ash, and 0.9% crude fat. The high ash may be due to silica and salt accumulation during neutralization of alkali-treated substrates. Membrane filtration only reduced ash to 23.4% and silica to 15%. As a comparison, the same microbial species grown on Trypticase soy broth (BBL) contained 11.9% ash and 1% silica. Although silica is nutritionally inert, its abrasiveness may damage the digestive tract of animals. More than 50% of the silica in the filtrate of fermentation mixture could be separated from cells by allowing the mixture to stand for a few hours at room temperature. Table 2 compares the amino acid composition of microbial and alfalfa protein. The essential amino acid profiles of Cellulomonas sp. and A. faecalis were similar, except for more methio- nine in the latter. Organisms grown on Trypticase soy broth contained a higher essential amino acid content than those grown on the straw-mineral solution. The amino acid profile of the mixed culture grown on rice straw was similar to that of alfalfa, except for less cysteine. Eighty percent of total nitrogen in microbial cells was recovered as protein nitrogen; the rest was the nucleic acids and other nitrogenous compounds. Table 3 compares the vitamins in microbial cells grown on straw with Torula yeast (Candida utilis). The microbial cells contained more thiamine and less niacin than yeast. Folic acid and pyridoxin in the microbial cells were equivalent to Torula yeast. Thus, microbial cells from straw fermentation may provide vitamins as well as protein. The in vitro digestibility of protein in the fermentation products ranged from 41% to 55% (Table 4). This relatively low protein digestibility may be attributed to microbial cell walls, since Yang (21) has shown that sonication of microbial cells improved their protein utilization by animals. Crude fiber digestibility (total solubles after enzymes) was 30% for raw rice straw, 73% for NaOH-treated straw, and 57% for NH4OH-treated straw. Some of the digestible matter in straw was apparently used by microorganisms for protein production. However, enough digestible matter (52% total solubles after enzymes) was left in the fermentation residue to warrant its potential use as a ruminant feed. Total in vitro gas production for the fermentation residue was lower than that for untreated straw and alkali-treated straw ( Table 5). VFA production, however, paralleled in vitro digestibility; the fermentation residue produced more VFA than untreated rice straw but less than alkali-treated straw. None of the straw or products from straw fermentation produced as much gas or VFA as alfalfa. Thus, the laboratory data indicate that the microbial cell fraction, with its high protein content, may be used as a protein source for the nonruminants, and the fermentation residue for the ruminants.

v3-fos

2020-12-10T09:04:22.488Z

1975-02-01T00:00:00.000Z

237233040

s2

Characterization of Hydrogen Sulfide-Producing Bacteria Isolated from Meat and Poultry Plants A survey of the types of aerobic organisms able to produce H2S on peptone iron agar (Levin, 1968), and commonly occurring in meat and poultry plants, revealed that these could be divided into four distinct groups. The ability of representative strains of each type to grow at low temperatures and cause off-odors on chicken muscle was examined. The results are discussed in relation to the role of these organisms in the psychrophilic spoilage of meat and meat products. Recent work on the bacteriological spoilage of flesh foods has indicated that only a fraction of the total microflora is capable of producing the organoleptic changes associated with spoilage (2,11). Several workers have emphasized the importance of attack on low-molecular-weight compounds (1, 13) and others indicated that off-odors are evident before extensive proteolysis takes place (18). In particular, the association of sulfide-producing organisms with spoilage has been noted (7,11,15,19,21). There is no concise data in the literature about the genera and species of organisms that produce sulfides, and which commonly occur on meat and poultry. This study was undertaken to provide adequate characterization of these organisms as a basis for further detailed studies. MATERIALS AND METHODS Origin and isolation of strains. The sources of the strains are given in Table 1. Peptone iron agar (PIA; Difco) developed by Levin (19) was used as a direct diagnostic plating medium for the recovery of hydrogen sulfide-producing strains. These formed black colonies due to the formation of ferrous sulfide. Plant surfaces were sampled by the swab-rinse technique and poultry carcases by the cut and rinse method described by Patterson (23). Other product samples (fresh and aged beef) were homogenized in saline peptone solution using a Colworth Stomacher (A. J. Seward and Co. Ltd., London) as described by Sharpe and Jackson (25). Serial 10-l dilutions were prepared from this homogenate. Pour plates of various dilutions were made in PIA and incubated for 3 (30) and motility was determined by the hanging drop technique. Size, shape, and cellular arrangement of stained and living preparations were noted. Representative strains of each group of organisms were stained for flagella by the Leifson technique (22). Best results were obtained by allowing a contact time of 30 s after appearance of a fine precipitate on the slide. Colonial morphology and broth characteristics were described after 3 days at 22 C on NA or in NB (30). Biochemical tests. All tubes were inoculated with 0.1 ml of a 24-h NB culture, plates and slopes being streaked from a similar source. The temperature of incubation for all tests was 30 C, the period being varied according to the test. Details of the tests employed are shown in Table 2. Carbohydrate reactions. The ability of strains to produce acid and/or gas from glucose, maltose, sucrose, and lactose was examined in Andrade peptone water. The carbohydrate solutions were sterilized by filtration and added to the basal medium to give a final concentration of 1.0% (wt/vol). Sole sources of nitrogen. Sulfur-containing nitrogenous compounds known to occur in meats (methionine, cystine, glutathione, taurine) were tested as sole sources of nitrogen. Filter-sterilized solutions of each N source (0.01 M) were added to a basal medium chosen to simulate the carbohydrate and salts composition of meat (16 water, and 40 ml of solution C and 10 ml of solution B were added. Computation of results. The results which were useful for differentiation were resolved into 58 features and presented as a table of strains versus features. The data were analysed by the program CLASP by which pairs of strains in all possible combinations were compared in turn. Similarity was defined as nJ/(n, + nd) (n, = ++, nd = + -). Negative matches were not counted (29). Strain numbers (123) were included in the computation; the remaining 36 isolates had identical responses with other isolates. Excision of sterile muscle sections. Sections of sterile muscle were excised from chicken breast (supra coracoid, pectoral proper) using a modification of the technique described by Sharp (24) and Gardner and Carson (10). Breast skin was carefully removed using sterile instruments and the underlying tissue was painted with aged, saturated solutions of crystal violet and brilliant green which were allowed to dry for 2 h. The muscle was not flamed as this procedure was found to cook into the depth of the tissue. The painted portion was sliced away using sterile instruments and large sections of the underlying muscle were excised and placed in sterile petri dishes. The large sections were cut into smaller portions (2 g) using sterile scissors and were stored in sterile screw-capped bottles. Sections were kept at refrigeration temperatures for at least 14 days and were examined visually and olfactorily before use. Five-milliliter quantities of nutrient broth were added to 10% of the sections and incubated at 22 C as sterility controls. Ability of representative strains to produce offodors. The ability of 15 pure cultures representing the various groups of organisms (Table 3) to produce off-odors was tested as follows. Cultures were grown in NB for 2 days at 22 C, harvested by centrifugation, washed, and resuspended in sterile phosphate buffer (0.1 M, pH 6.8) to give a final concentration of approximately 5 x 104 cells/ml. Sterile muscle sections were inoculated with 0.1-ml quantities and incubated at 5 C for 14 days. Sections were examined sensorily at 7 and 14 days for evidence of spoilage. Growth rates at 5 C. The growth of representative organisms from the major groups at refrigeration temperatures was measured using a nephelometer (EEL, Evans Electroselemium Ltd., Halstead, Essex). Optical density measurements of NB cultures were recorded at inoculation and on alterate days for 14 days. RESULTS The results of the computer analysis (summarized in Table 3) show mean similarities within and between the groups formed. The organisms within each group were recognized as follows: group 1, Pseudomonas putrefaciens; group 2, Proteus sp. (Proteus mirabilis and Proteus vulgaris); group 3, Citrobacter freundii; group 4, coryneform types. Summary characterizations of the groups are given in Table 4. In this survey, 48 of the 159 cultures isolated were strains of P. putrefaciens. These formed a very distinct group (9 = 87.0). The distribution of the strains can be seen in Table 1. It is of interest to note that all 26 isolates from spoiling steak stored at 5 C were P. putrefaciens. Other sources included frozen and chilled eviscerated chickens, poultry plant personnel, and equipment. The P. putrefaciens strains all grew well at 5 C and much more quickly at this temperature than the other H2S-producing types isolated. All five representative strains caused a typically sulfide-like spoilage odor when grown on sterile chicken muscle. Off-odors were detectable organoleptically after 7 days storage at 5 C. The group 2 strains all produced the characteristic swarming colonies of Proteus species. The group (53 strains) was distinct with an intragroup mean similarity of 83.2. All but three of the strains were recognized as P. mirabilis. The remaining three correspond to the description of P. vulgaris (6) and were grouped together at one extreme of the Proteus group. Growth at 5 C was recorded for 29 of 53 Proteus strains and four representative isolates developed faster than the Citrobacter types at this temperature, although much slower than P. putrefaciens. Despite the relatively slow growth rate at low temperatures, all Proteus strains tested produced the same characteristic odor. This was not recognized as sulfide-like but was described as "caramel" or "burnt sugar." (These strains liberate H2S when cultured at 5C.) The strains contained in group 3 were recognized as C. freundii (36 organisms, 9 = 82.2). Two anaerogenic types were included. Again, in relation to P. putrefaciens, these strains grow slowly at 5 C and only slight sulfide-like odors were detectable with two of the four representative strains tested after storage of inoculated chicken muscle for 14 days at 5 C. The coryneform organisms formed the least I distinct group; 19 strains were included in a group with an intragroup mean similarity of 64.1% S. The strains grew only very slowly if at all at 5 C and representative types did not produce any detectable off-odor after incubation on sterile chicken muscle for 14 days at 5 C. Seven of the eight strains isolated from beefburgers were coryneform types. It was not, however, possible to test these for spoilage ability against beefburger as sterile sections of this product could not be obtained. In view of their slow growth rate at low temperatures, members of this group are unlikely to be important spoilage agents of refrigerated meat products. DISCUSSION The results obtained indicate that a restricted range of bacterial types capable of producing detectable amounts of hydrogen sulfide on PIA occurs in the environs of meat and poultry plants. Of these, P. putrefaciens is recognized as potentially the most important spoiler. Both C. freundii and coryneform types are discounted as troublesome at 5 C. At higher temperatures, however, it is likely that these might develop much more rapidly with the subsequent onset of sulfide-like odors. Proteus strains provide apparently anomalous results; although developing only slowly at refrigeration temperatures, they produce a characteristic spoilage of chicken muscle which was not recognized organoleptically as sulfide-like. The role of P. putrefaciens as a spoilage organism is well documented. A number of workers have noted the presence of this organism and its role in the spoilage of chicken carcases (4,5). Lea and co-workers (17), however, concluded that P. putrefaciens and a pigmented pseudomonad produced changes little greater than those observed during sterile autolysis. All P. putrefaciens strains examined in this study produced potent off-odors from chicken muscle. P. putrefaciens has also been widely implicated in the spoilage of fish and fishery products (7,11,19). Other Pseudomonas strains have been reported in the literature as producers of sulfides and sulfide-like odors. Nichol et al. (21) recorded the formation of sulfmyglobin in prepacked beef by P. mephitica, but showed that sulfides were only produced under conditions of low oxygen tension. P. perolens was shown to produce a number of volatile sulfides when grown on sterile fish muscle (20) whereas P. putida, Pseudomonas group 1 (26), P. fragi, Pseudomonas group II, and Pseudomonas group IIJAV types possibly similar to P. putrefaciens also caused sulfide-like odors in fish muscle (11). In this study, the only sulfide-producing pseudomonad recovered from sources within meat and poultry plants was P. putrefaciens. Members of the Enterobacteriaceae have been shown to constitute a considerable portion of the flora of poultry carcases when the temperature is allowed to rise to 10 to 15 C (5). This study confirms that normal refrigeration temperatures permit at most only slow growth of the Citrobacter and Proteus types isolated. However, the latter are able to cause a characteristic off-odor on chicken muscle at 5 C in pure culture. The inherently slow growth rate, however, probably limits development of Proteus species in competition with more psychrotolerant organisms. Studies are being carried out to determine the number of cells of Proteus species required to cause off-odors in pure culture. Their development and the nature of spoilage in association with the faster growing P. putrefaciens is also of interest. It is possible that the odiferous compounds produced by Proteus strains are detectable organoleptically at extremely low levels and that relatively few cells are required to attain these levels.

v3-fos

2017-06-23T07:54:56.466Z

1975-08-01T00:00:00.000Z

25966550

s2

Characterization of Radiation-Resistant Vegetative Bacteria in Beef Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized. Meats contain a broad spectrum of microorganisms arising from the carcass and the processing operations involving equipment and human contact. Fresh meat provides a favorable environment for microorganisms which constitute public health and organoleptic spoilage problems. To alleviate these problems, various processing techniques have been investigated. Gamma irradiation is an example of a potential commercial process to extend shelf life of meat (15) and aid in public health protection (8). Some members of the microflora of commercially processed red meat are sensitive to gamma radiation whereas others are quite resistant to comparable doses (13). The vegetative cells of bacteria most resistant to gamma radiation do not appear to be a part of the commonly recognized spoilage flora (14). Tiwari and Maxcy (14), working with radiation-resistant organisms from meat, made a general classification with minimal characterization of these bacteria as Moraxella-Acinetobacter (M-A). These, or very similar bacteria, have been I Published as paper no. 3992, journal series, Nebraska Agricultural Experiment Station. Research reported under project no. 16-23. shown to be widespread, occurring in various types of unprocessed foods, e.g., dairy products (6), fish (9), and vegetables (10). Microorganisms of the M-A type may have gone unnoticed by food microbiologists, since these bacteria have not been associated with problems and are present in relatively small numbers (13). However, irradiation processing with nonsterilizing, low-dose gamma radiation to reduce total numbers of organisms brings the M-A into prominence. Many members of the M-A group have an inherent resistance to gamma radiation at least twice that of most other vegetative bacteria. The mechanism of this resistance is unknown. The purpose of this investigation was to isolate the most radiation-resistant vegetative cells from meat and to study them in detail with the hope of obtaining a better understanding of their significance. MATERIALS AND METHODS Isolation procedures. Samples of ground beef were obtained from various sources. These involved animals from numerous feed lots and carcasses from various abattoirs and processing operations. These samples involved a wide geographical area, including imported beef and seasonal variations of summer and winter. Ground beef was irradiated in vacuum packages and in atmospheric packages at approximately -30 C at various dose levels so that a 1:10 dilution yielded approximately 10 colonies of bacteria when cultured on plate count agar. Incubation was carried out at 32 C under aerobic and anaerobic conditions. Individual colonies were subcultured, observed for morphology and colony characteristics, and then tested for oxidase, catalase, reaction in litmus milk, and proteolysis on skim milk agar. Approximately 60 isolates that appeared to be different on the basis of any one of these reactions were used for further study. To select the most radiation-resistant bacteria, isolates were grown in pure culture, then combined, and added to ground beef. The inoculated product was then irradiated as described above to obtain a second enrichment. Nineteen different organisms surviving the second enrichment process were maintained in pure cultures for detailed study. For comparative purposes, 10 isolates from a previous study (14) obtained from a single enrichment were included in this study and denoted by the prefix M-A. Characterization of isolates. Gram strains were prepared from cultures on plate count agar and plate count broth previously incubated for 16 to 24 h at 32 and 37 C and examined for microscopy morphology. Isolated colonies on plate count agar, Trypticase soy agar (TSA), TSA containing 5% defibrinated sheep blood, TSA with 5% bovine serum, chocolate blood agar with 5% sheep blood, and brain heart infusion agar containing 5% defibrinated rabbit blood were observed periodically from 16 h to 7 days. Presence or absence of growth on basal mineral medium, O-F without carbohydrate, MacConkey agar, eosin methylene blue agar, and Shigella-Salmonella agar and growth on TSA with 2.5 and 6.5% NaCl were observed. Penicillin susceptibility was tested by the Bauer-Kirby technique (1), using 10-U disks. Thermal limits of growth were determined by culturing bacteria on TSA and incubating for 24 to 48 h at temperatures ranging from 0 to 50 C. An anionic surfactant, Ultrawet (Atlantic Refining Co., Chicago, Illinois), was incorporated into TSA and Trypticase soy broth (TSB) at concentrations of 0.01, 0.1, and 1.0% to test susceptibility to surfactant. To determine radiation resistance, pure cultures were grown in plate count broth or TSB, quick-frozen in a dry ice-alcohol bath, and treated with various doses of gamma radiation to give a 7to 8-log cycle reduction in population. To determine radiation resistance in meat, ground beef was irradiated with 200 krad, after which enough cells of the cultures to be studied were added to provide approximately 10" cells per g of meat. A cobalt-60 source provided approximately 12 krad of gamma radiation per min. Operation of the equipment has been previously described (13). Heat resistance of pure cultures in broth was determined by heating cultures in the plate count broth or TSB in which they were grown for various temperature-time combinations. To determine heat resistance in meat, ground beef was irradiated with an essentially sterilizing dose (2 Mrad) of gamma radiation. Then approximately 106 cells per g were ground with the meat and formed into patties 3-mm thick in polyethylene bags, after which the patties were immersed in a water bath for various temperature-time combinations. A separate patty was used for each time interval. Comparative alteration of Moraxelia osloensis by irradiation. The reference culture of M. osloensis, which had not been previously exposed to radiation, was grown in TSB overnight on a rotary shaker at 37 C. Cultures were quick-frozen in a dry ice-alcohol bath and treated with various doses of gamma radiation to provide a 7-to 8-log cycle reduction in population. A colony surviving the highest dose was grown in broth and subjected to the same irradiation process. Colony selection and irradiation were repeated for the third time. Survivors of the repeated irradiations were grown in pure culture and observed as described below. RESULTS Nature of isolates. Isolates of bacteria obtained from aerobic plating of beef irradiated either in vacuum packages or under atmospheric conditions appeared to be the same. When irradiated beef was cultured anaerobically, total counts obtained were less than 1% of those obtained by aerobic plating. None of the isolates obtained by anaerobic incubation was an obligate anaerobe. Colony morphology was similar on various types of media. Colonies were mainly circular, convex, entire, smooth, opaque, dull or glistening, and butyrous. After 18 to 24 h of growth at 32 to 37 C, colonies were pinpoint and white. After 1 or 2 additional days of incubation, they were 1 to 3 mm in diameter and white, cream colored, light buff, or pale yellow. Four of the isolates produced a bright orange to red pigment and were considered to be Brevibacterium. The majority of the isolates had no effect on blood agar medium or produced only indeterminate reactions. One isolate, MA-27, produced a lavender-green discoloration of the erythrocytes in blood agar medium. No beta-like hemolysis was detected. Microscopy examination of Gram stains showed short, plump, gram-negative rods usually in pairs or short chains. A few isolates showed some resistance to decolorization. Those considered to be Brevibacterium were gram positive. All the isolates appeared to be coccobacilli but frequently appeared as diplococci suggestive of Neisseria. Some isolates had a marked tendency toward pleomorphism (Fig. 1). Glucose utilization was tested on several media, each of which has been used by various workers. Some isolates oxidized or fermented glucose in certain media, but not in others. In addition, some reactions were weak or delayed ( Table 1). None of the isolates produced H2S, indole, urease, or phenylalanine deaminase. Isolates were grouped on the basis of morphology, cultural characteristics, biochemical reactions (Table 2), and growth on selective media ( Table 3). All of the isolates grew at temperatures between 20 and 37 C and most had a much broader range of growth temperatures (Table 4). Radiation and heat resistance. Although all the isolates were relatively radiation resistant, the range was from D 0 of 273 to 2,039 krad ( used for irradiation of meat. The D10 value of the reference culture was determined to be 137 krad (lines located by the method least squares). A pure culture from a colony that had survived 1,000 krad had a D10 of 145. When a pure culture from a colony surviving the second irradiation treatment of 1,000 krad was observed, the D10 value was 146. When slopes of lines were determined by the method of least squares, it was apparent they were approximately the same (Fig. 2). Prior to irradiation the M. osloensis used as the reference culture were predominantly coccoid, 1 ,um in diameter, or diplobacilli, 0.8 by 2 ,um. Some larger diplococci, 2 by 3 tm, or even bacillary forms, 0.5 by 6,gm, were seen. Occasionally a long bacillus, up to 10 ,m in length, was noted (Fig. 3). When one colony surviving a 1,000-krad dose was Gram stained, long cells were predominant (Fig. 4). Many cells were 15 to 20 gm long and it was not unusual to find a single cell 26 to 30 ,m. Often cells appeared to be two bacteria lying end to end and these forms measured up to 55 Am. After the irradiated M. osloensis had been subcultured a few times on TSA the morphology appeared to be similar to the parent reference culture. When penicillin sensitivity of the reference M. osloensis was tested by the standard Bauer-Kirby technique, there was a 32-mm zone of inhibition around a 10-U disk and a 13-mm zone of inhibition around a 2-U disk. A pure culture from a colony surviving the second irradiation treatment of 1,000 krad showed a 20-mm zone around the 10-U disk and no zone of inhibition FIG. 1. Gram-negative bacteria isolated from irradiated ground beef. Coccobacillary forms were predominant, but some pleomorphic forms were present. Marker indicates 10 um. b Characteristics of these organisms did not fit with recognized groups. around a 2-U disk. After a few subcultures on TSA, penicillin sensitivity was essentially the same as that of the parent culture. DISCUSSION Gram-negative, aerobic, nonsporeforming coccobacilli are widely distributed in nature and have been implicated as being important in irradiated foods (5,7,14). Thornley (12), working with organisms surviving the irradiation of poultry, found them to have characteristics similar to the M-A group and classified them as Alcaligenes-Achromobacter. These organisms were described by Thornley as being "gram-negative or gram-variable, nonmotile coccobacilli which have a doubtful taxonomic position." It is quite possible that the author was working with the M-A group of microorganisms, since members of the Alcaligenes-Achromobacter group are now considered to be gram-negative, motile, nonfermentative bacteria. Tiwari and Maxcy (14) recognized that the M-A group of microorganisms was present in beef and was found in a greater proportion in irradiated beef. These examples serve to illustrate the lack of availability of an adequate, universally accepted classification. To our knowledge this is the first investiga- I I I NG I I I I I I NG I I I NG I K I I NG I NG NG K K I I I I I I I I I I I I I I I I I I I + I I I I I I I I I I I I I I I I I I I I A I I AC I p p A A I I I I a None of the isolates produced H2S, indole, urease, or phenylalanine deaminase. +, Reaction positive; -, reaction negative; I, Inactive (growth but no change); NG, no growth; K, alkaline; A, acid; C, curd; P, peptonization. " Characteristics of these organisms did not fit with recognized groups. tion specifically designed to obtain radiationresistant bacteria from beef. Various enrichment techniques were used. Irradiation was carried out under atmospheric and vacuumpacked conditions. In addition, aerobic and anaerobic incubation was used for isolation. Detailed characterization was sought for a more adequate classification based on recent literature (2,11). Various media have been used to study carbohydrate utilization. The results obtained are not always identical, due to the different chemical composition of the media, e.g.. amount of nitrogen and presence of more than one carbohydrate. Some investigators (11) suggest using triple sugar iron medium to obtain preliminary information and then differentiating nonfer-mentative and fermentative bacteria by use of open and sealed tubes of O-F medium containing 1% glucose. Even though the isolates could be placed in groups based on commonly used morphological and physiological characteristics, it can be noted from Tables 1, 2, and 3 that they were a heterogeneous group. It is recognized that bacteria belonging to this group have not been extensively studied and, to date, many are still unnamed. More attention has been given to organisms isolated from clinical specimens. The Manual of Clinical Microbiology (11) lists and characterizes a number of unnamed bacteria not recognized by Bergey's Manual of Determinative Bacteriology (2). However, Bergey's Manual (2) mentions "oxidase positive sac-charolytic strains showing Moraxella morphology," but Moraxella spp. are described as not utilizing carbohydrates. These strains, which conform to the generic descriptions of both Moraxella and Acinetobacter, have been found in cold-stored poultry and fish, as well as from human pathological specimens (2). Some of the isolates investigated in this study had a range of growth temperatures from 2 to 50 C. It is apparent that these organisms could grow in foods stored at refrigeration temperatures or ambient temperatures and represent a potential spoilage problem. Tiwari and Maxcy (14) reported that the M-A group decreased in importance during storage of ground beef at 5 and 25 C because of overgrowth by less radiation-resistant organisms. None was particularly resistant to heat in comparison to spores. Few would survive pasteurization at 63 C for 30 min. Although the method of isolation dictated that all isolates be radiation resistant, it is apparent from the data in Table 5 that there was considerable variation in D10 values. Certain isolates, however, were as much as four to five times more radiation resistant than Micrococcus radiodurans or any endospore (12). To determine if our isolates had acquired radiation resistance by exposure during the ation were the great increase in numbers of very elongated forms and some loss of sensitivity to penicillin. The elongated forms apparently resulted from a failure of the bacteria to divide, which correlated with reduction of sensitivity to penicillin. Reversion to the same morphological form as the parent culture and sensitivity to penicillin occurred after a few subcultures. Although these bacteria are prevalent in nature and are found in various food products, they have not been recognized as being significant in organoleptic spoilage or in food-borne diseases. On the other hand, an extensive review by Henriksen (3) lists over 150 references to pathological conditions caused by Moraxella and Acinetobacter. It would appear that additional work is necessary to delineate the role of these organisms in our environment.

v3-fos

2018-04-03T00:25:55.453Z

1975-01-01T00:00:00.000Z

11718007

s2

Medium-scale production of citrinin by Penicillium citrinum in a semisynthetic medium. A convenient method is described for the production of up to 1.75 g of citrinin per liter by Penicillium citrinum growing in stationary culture in a 5-gallon (18.925 liters) carboy containing 4 liters of 4% sucrose and 2% yeast extract medium. In 1951 P. citrinum was isolated from yellowed rice imported from Southeast Asia to Japan. Saito et al. (12) reported that in 3-week experiments with rats fed P. citrinum-contaminated rice Tsunoda (16) found enlarged kidneys to be the characteristic lesions caused by this fungus, which has been frequently isolated from peanuts (3), corn (9), rice (12), and other raw and processed agricultural commodities. Citrinin was first isolated by Hetherington and Raistrick (5) in 1931 from a culture filtrate of Penicillium citrinum Thom. It has since been produced by 10 or more species of Penicillium and Aspergillus, and is regarded as a potentially important mycotoxin that may be ingested by man and animals as a contaminant of wheat, barley, rye, and oats (4, 7, 13). Hetherington and Raistrick (5) obtained yields of 525 to 700 mg of citrinin per 350 ml of medium with P. citrinum Ad. 23 in 12 to 16 days at 28 to 32 C. Timonin and Rouatt (15) obtained 500 to 700 mg of citrinin per 200 ml of mineral salts medium with Aspergillus candidus incubated for 20 days at 26 C. Highest yields of citrinin were produced by A. candidus on media containing honey (15) or maple syrup (17) rather than glucose as the carbon source. In 1966 Rodig (11) produced the equivalent of 2.6 g of citrinin per liter in 14 to 16 days at 40 C with Aspergillus niveus and 1.2 g of citrinin in 35 to 40 days at room temperature with P. citrinum using Timonin's medium containing twice as much glucose. This paper reports production of citrinin in 5-gallon (18.925 liter) carboys containing 4 liters of 4% sucrose and 2% yeast extract nutrient solution. This medium was previously described for ochratoxin A production by A. ochraceus (1, 2). A strain of P. citrinum, isolated from corn associated with toxicity in horses in Alabama, proved to be an efficient producer of citrinin. Subsequently, we found that Timonin's isolate of A. candidus deposited in a culture collection had lost its ability to produce citrinin and he no longer had a viable culture (personal communication with M. I. Timonin). Increased demand for citrinin for mycotoxicological investigations made it desirable to investigate the conditions for production of the toxin by this high-yielding strain of P. citrinum. P. citrinum AUA 532, used throughout this investigation, was identified by D. I. Fennell of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., and accessioned there as NRRL 5927. After sporulation, cultures were stored at 4 to 5 C on slants of agar containing 2% dextrose, 0.7% yeast extract (Difco), 0.5% KH2PO4, and 2% agar, and also as lyophilized cultures stored at -30 C. Wide-mouthed, 5-gallon Pyrex carboys containing 4 liters of 4% sucrose and 2% yeast extract nutrient solution were stoppered with cotton plugs and autoclaved for 30 min at 121 C (2). These were inoculated with a suspension of spores from 10to 14-day-old cultures of P. citrinum and incubated at room temperature (30 4 3 C) as stationary cultures. Carboys were placed on their sides to give maximal surfacearea-to-volume ratio. Two experiments with seven carboys per experiment were conducted with yields being measured as crude citrinin produced after 8, 10, 12, 14, 16, 18, and 21 days of incubation. A third experiment involved 10 carboys with data being taken on the above days and also after 25, 27, and 31 days of incubation. Streptomycin sulfate (0.2 g/liter) was added to the medium in the latter experiment to protect against bacterial contamination. The procedure for extraction and isolation of citrinin was essentially as described by Hetherington and Raistrick (5). The mycelium and VOL. 29, 1975 culture solution were filtered through Whatman no. 1 filter paper and the filtrate was acidified to pH 1.5 by stirring in approximately 50 ml of concentrated HCl per 4 liters of medium to precipitate the crude citrinin. After precipitation was complete, the bright yellow mass of c rude citrinin was filtered onto Whatman no. 42 filter paper, dried in vacuo at 50 C, weighed, and reported as total crude citrinin per liter of medium. The crude citrinin was dissolved in a minimal amount (150 ml) of chloroform and filtered to remove nonchloroform soluble impurities. The chloroform solution was evaporated to dryness, and the residue was taken up in hot ethanol, filtered, and recrystalized three times by the hot alcohol method of Hetherington and Raistrick (5). Comparison of the chemical and physical characteristics of the purified material with the ultraviolet, infrared, mass, and nuclear magnetic resonance spectra, melting point, and optical rotary power of citrinin as reported in the literature (6, 8, 10, 12) confirmed identification of the compound. Production of crude citrinin by P. citrinum in sucrose-yeast extract medium for varying periods of incubation is illustrated by the regression curve in Fig. 1 (data for three experiments were averaged and the curve plotted by quadratic regression). Citrinin was detected on day 8 with the highest yield recorded on day 21. The addition of streptomycin sulfate to the medium in the third trial had no apparent effect on citrinin production. As much as 7.3 g of crude citrinin was produced per carboy (4 liters of medium). Yields seemed to plateau and did not decrease thereafter. Apparently citrinin is not reabsorbed and further metabolized. Thus, production runs may be terminated at the convenience of the researcher rather than according to a predetermined or rigid time schedule. Surprisingly, yields were comparable to those scaled up (by calculation) from smaller systems (5, 11,15,17) rather than diminished as often occurs when small systems are scaled up. About 45 to 50% of the crude citrinin was recoverable in pure form by the method used (5). However, Timonin and Rouatt (15) reported that they recovered 70 to 80% of the crude product as pure crystalline citrinin using the dioxane method of Tauber et al. (14). Timonin and Rouatt (15) conducted a very thorough study on factors influencing citrinin production by A. candidus. Results of their investigation on A. candidus seem directly applicable to citrinin production by our P. citrinum isolate AUA-532. This isolate is maintained by lyophil in our cultun collection and is available for distribution. Small amounts of citrinin are also available as qualitative standards, or in larger amounts where circumstances warrant.

v3-fos

2020-12-10T09:04:20.514Z

1975-02-01T00:00:00.000Z

237233869

s2

Protozoa as Agents Responsible for the Decline of Xanthomonas campestris in Soil A streptomycin-resistant mutant of Xanthomonas campestris was used to assess the persistence of the plant pathogen in soil and the changes in populations that might be important for its survival. In soil into which large numbers of the organism were introduced, a marked decline in its abundance occurred, but after about 1 week its population density reached a level of about 105 and did not continue to fall during the test period. No such marked decline was evident in sterile soil inoculated with X. campestris. The bacterium did not lose viability if starved for carbon or inorganic nitrogen. Although abundant in soil, the numbers of propagules capable of producing antibiotics or lytic enzymes active against X. campestris did not increase coincident with the pathogen's decline, and no increase in tartrate-extractable toxins was observed. Neither bdellovibrios nor bacteriophages active against the xanthomonad were found in the soil, but a marked increase in the frequency of protozoa paralleled the phase of rapid diminution in the X. campestris population. In actidione-treated soil, in which protozoan activity was severely limited, the high cell density of the pathogen was maintained. On the basis of these data, it is concluded that predation by protozoa is responsible for the abrupt fall in frequency of the bacterium in natural soil. Plant pathogenic bacteria enter the soil as free propagules or in association with dead and dying plant tissues, although the soil may be the natural habitat of a few species that have the ability to grow in soil saprophytically. After their introduction into soil, however, most species of plant pathogenic bacteria decline rapidly, but almost all of these populations endure for long periods in sterile soil (1,7,10); thus, the lack of survival in soil appears to be a function of the prevailing antagonistic microbial relationships. These deleterious interactions may be competition, amensalism, parasitism, or predation. That amensalism might be of some importance is supported by the findings of Patrick (12), who showed that members of all genera of plant pathogenic bacteria are susceptible to antibiotic activity of soil microorganisms in vitro. Other investigators believe that bacteriophages are of considerable importance in reducing the abundance of pathogens in soil, and the ability of phages to lyse the pathogens in vitro has been demonstrated (11,21). The possible role of bdellovibrios in the ecology of the pathogens in soil has yet to be studied, but Starr and Baigent (15) showed that Bdellovibrio bacteriovorus could parasitize species of Er-winia and Pseudomonas in culture, and Scherff (13) reported that inoculating infected soybean plants with B. bacteriovorus led to control of the blight caused by Pseudomonas glycinea. The present study was designed to investigate why the population of a particular pathogen, Xanthomonas campestris, declines rapidly in soil and to establish which type of microorganisms is responsible for the decline. MATERIALS AND METHODS A streptomycin-resistant mutant derived from a culture of X. campestris C-3 (5) was used, and the bacterium was grown in tryptic soy broth (Difco) with and without 1.0 mg of streptomycin per ml. To provide inocula for the various experiments, a 24-h culture was harvested aseptically by centrifugation, and the cells were washed three successive times in sterile, distilled water. A 1.0-ml portion of a dilution of the bacterial suspension in carbon-free solution (3) was usually used, and the cultures were incubated at 30C. The survival of X. campestris in nonsterile Valois silt loam, pH 6.5, was determined by inoculating 1.0 ml of a suspension containing about 1010 X. campestris cells into 150-ml capped dilution bottles containing 10.0 g of soil at a moisture content of 25%. The bottles were gently agitated to insure mixing of the inoculum with the soil, and the bottles were incu-bated at 30 C. Counts were made of three of the dilution bottles at regular intervals by diluting the soil samples in the carbon-free solution and plating on nutrient agar (Difco) containing 1.0 mg of streptomycin and 0.50 mg of actidione per ml. Counts were made after the plates were incubated for 6 days. The persistence of X. campestris in sterile soil was determined in the same manner, except that the water lost during autoclaving was replaced with sterile, distilled water before the pathogen was added. To sterilize the soil, 10-g portions of moist soil were autoclaved for 2 h, incubated at room temperature for 5 days, and then autoclaved again for 2 h. The response of X. campestris to carbon starvation was determined by employing the carbon-free solution, and this solution minus KNO, was used for investigating the response of the bacterium to the combined lack of carbon and nitrogen. To 100-ml portions of these sterilized solutions contained in 250-ml Erlenmeyer flasks were added 1.0-ml samples of X. campestris cells washed by centrifugation and suspended in distilled water. The flasks were incubated at 30 C on a rotary shaker operating at 120 rpm, and triplicate samples were withdrawn regularly for plate counts on nutrient agar containing 1.0 mg of streptomycin and 0.50 mg of actidione per ml. Dilution bottles containing 10-, 50-, or 30-g portions of soil, respectively, were used for: (i) counting of antibiotic producers, lytic microorganisms, and protozoa; (ii) enumeration of bdellovibrios and bacteriophages; and (iii) the measurement of toxin production. Washed X. campestris cells suspended in the carbon-free solution were introduced into the bottles, each receiving 1.0.ml of the inoculum per 10 g of soil. After the soil and bacteria were well mixed, the bottles were incubated at 30 C. Counts of antibiotic producers were made by using Patrick's (12) glucose-rich agar medium. Dilutions of the soil samples were placed in petri dishes, and the molten agar medium, containing 5% (vol/vol) tryptic soy broth in which X. campestris was grown for 24 h, was poured into the plates and mixed with the soil dilutions. Antibiotic-producing colonies were considered to be those having clearing zones around them after a 5-day incubation period. The numbers of bdellovibrios and bacteriophages were determined by employing the double-layer technique, using the procedures and media of Stolp and Starr (16) for bdellovibrios and the technique of Cook and Quadling (5) for bacteriophages. Soil fungi were enumerated on peptone-glucose-rose bengal agar (8) after an incubation period of 5 days. Lytic microorganisms were enumerated on agar containing live and heat-killed X. campestris. Cells for making xanthomonas agar were obtained by growing the bacterium for 48 h in tryptic soy broth containing 1.0 mg of streptomycin per ml and then washing the cells by centrifugation using sterile, distilled water. The washed cells were resuspended in distilled water to yield approximately 1012 cells/ml. Either 20 ml of live cells or a bacterial suspension that had been autoclaved for 30 min was added to each 200 ml of melted and cooled water agar just before it was poured into petri dishes containing dilutions of soil samples. The number of colonies of bacteria, fungi, and actinomycetes that were surrounded by clearing zones was recorded after a 3-day incubation. The procedure for counting protozoa was a modification of the Singh ring method (14). Melted wateragar (1.5%) was added to petri dishes, and five sterile glass rings (20 mm wide by 10 mm high) were placed in each plate immediately afterward. When the agar was cool and well set, 0.1 ml of a thick cell suspension (derived from a 24-h culture of X. campestris) in sterile, distilled water and 0.5 ml of a soil solution were added to each ring. The plates were incubated for 3 to 6 days, and then the growth in each ring was examined under a microscope. Quantification was achieved by means of the most-probable-number technique (9). To obtain the 95% confidence limits for the counts, the values obtained are multiplied by 0.30 and 3.3 (4). To ascertain whether toxins were present in the soil, 50 ml of a 0.1 M sodium tartrate solution was added to a 30-g soil sample. The mixture was shaken vigorously and subsequently allowed to stand for 24 h. The particulate matter was then removed by centrifuging the suspension for 10 min at 15,000 x g, and the supernatant fluid was passed through Whatman no. 1 filter paper. The resulting filtrate was sterilized by passing it through a 0.45-jim membrane filter (Millipore Corp.). To assess the toxicity of this extract, 30 ml of tryptic soy broth, 5.0 ml of the extract, and 0.2 ml of a 24-h-old X. campestris culture were added to a 300-ml Erlenmeyer flask fitted with a side arm. The flasks were incubated on a rotary shaker operating at 120 rpm, and the growth rate was assessed by recording regularly the change in optical density at a wavelength of 550 nm using a Bausch and Lomb Spectronic 20 spectrophotometer. RESULTS The changes occurring when X. campestris was added to natural and sterile soil are shown in Fig. 1. In sterile soil, the abundance of the pathogen did not change appreciably in the first 2 days and then declined to a level of 4.5 x 106 cells/g on day 6, after which the cell density remained essentially constant for the test period. In marked contrast, the abundance of the pathogen in nonsterile, inoculated soil fell rapidly after day 2, declining from an initial value of 2 x 1010/g to a level about five orders of magnitude lower, in the vicinity of 7 x 104/g, before the period of rapid decline terminated. The dramatic fall in numbers in the sample containing the natural soil community as compared with the slight change in the soil that had its indigenous populations eliminated strongly suggests that biological agencies are responsible for the significant elimination of X. campestris cells. The stabilization in numbers of survivors in nonsterile soil at about 105/g indicates that the agent or mechanism of destroying the pathogen was effective at high but not at low bacterial densities. To assess whether the extent of elimination was, in fact, dependent on pathogen abundance, three different X. campestris densities were introduced into nonsterile soil, and the survivors were enumerated at regular intervals. The pathogen died at a reasonably rapid rate, but the final density of survivors at the end of the test period was the same whether the inoculum was 3.9 x 106, 3.8 x 108, or 1.6 x 1010/g, about 10' X. campestris surviving per g of soil (Fig. 2). The death of the xanthomonad may have been the consequence of competition between it and soil heterotrophs, the former dying because it could not cope with the more vigorous utilization by other heterotrophs of some limiting resource in the environment. Inasmuch as carbon is usually the chief limiting nutrient in soil for heterotrophs, followed by nitrogen, a study was made of the ability of X. campestris to withstand carbon and nitrogen starvation, assuming that starvation for the nutrient in inadequate supply is the probable reason that an organism, initially present in large numbers, loses viability when the mechanism of elimination is competition. When 2.1 x 10' cells/ml were added to the carbon-free solution, the maximum degree of change observed in the 12-day test period was less than one order of magnitude, and the number of survivors was never less than 1.5 x 107/ml. A slightly greater decline in X. campestris numbers took place if 3.2 x 10' cells/ml were introduced into the carbon-and nitrogen-free solution, but at no time did the count fall much below 107/ml and, indeed, a subsequent rise occurred so that the density at 12 days was almost the same as in the original inoculum. These data support the view that the bacteria did not lose their viability in nonsterile soil owing to a competition for carbon or nitrogen. The possible significance of antibiotic producers during the pathogen's decline was assessed by counting the numbers of antibiotic formers, as well as X. campestris, in Valois silt loam inoculated with the xanthomonad. Plate counts made every second day revealed that the pathogen density fell from an initial value of 1.6 x 1010 to 2.0 x 10' at day 8 but was still 8.7 x 104/g on day 12. There were initially 6.7 x 103 antibiotic formers, but after 12 days their abundance was still only 5.3 x 103/g; moreover, the counts made every second day revealed no value above 6.7 and none below 3.7 x 103/g. In a parallel test of the frequency of antibiotic formers in uninoculated soil, the numbers ranged from 4.0 x 104 to 7.3 x 104, with a mean of 5.7 x 104/g. Hence, no response is evident among these potential toxin-synthesizing microorganisms. Similarly, sodium tartrate extracts made every other day of a soil inoculated with X. campestris, as well as from an uninoculated sample, failed to depress the rate of growth of the bacterium in tryptic soy broth. The extracts were taken for a 12-day incubation period, during which time the bacterial population had gone from 1.6 x 1010 to 8.3 x 10'/g. Admittedly, the inhibitory principle may not have been extracted by the tartrate, but the present study provides preliminary data suggesting that amensalism is not the cause of the loss of bacterial viability. The possible role of lytic inhabitants in inactivating the pathogen was assessed by enumerating the propagules possessing lytic activity. Counts were made every second day for 12 days in a soil receiving 4.9 x 101 X. campestris cells/g and in uninoculated soil. The xanthomonad had declined to 3.0 x 108 in 2 days, 4.0 x 106 in 4 days, 2.7 x 106 in 6 days, and 4.7 x 10'/g at 8 days, and later counts revealed no appreciable further decline. By contrast, the abundance of lytic bacteria did not change during the course of the incubation in either the inoculated or uninoculated soil, maintaining a level of 2.1 x 107 to 3.7 x 107 in the former and 2.2 x 107 to 3.7 x 107/g in the latter. Similarly, the density of lytic actinomycetes did not vary significantly during the 12 days, ranging from 4.3 x 107 to 5.7 x 107 in the inoculated and 4.0 x 107 to 6.0 x 107/g in the uninoculated soil. Thus, although the frequency of cells possessing the potential for lysing X. campestris is high, their abundance is not modified as a result of the introduction of this species. In addition to bacteria and actinomycetes, occasional colonies of lytic fungi developed on the plates. The occurrence of these fungi was so irregular that it was difficult to assess their importance. However, counts of total fungi were made to determine whether fungi in general responded to X. campestris inoculation. In this instance, the bacterial density fell from 3.8 x 1010 to 3.1 x 10'/g, whereas the fungi showed no appreciable change in abundance, the counts of the latter ranging from 0.96 to 4.7 x 101 and from 2.1 to 4.7 x 108/g of the inoculated and uninoculated soil, respectively. Many yellow colonies grew on the agar medium containing a soil dilution and heat-killed cells of X. campestris. These colonies, which caused lysis of the pathogen and clearing of the agar, were observed during the first 4 days after the soil was inoculated with viable X. campestris, and the number of such lysis-inducing colonies was comparable with the numbers of viable cells of the pathogen remaining in the soil. No such colonies were apparent when dilutions from uninoculated soil were plated on agar amended with heat-killed xanthomonads. These results suggest that viable X. campestris lysed heat-killed cells of the same species. In support of this hypothesis, it was found that dilutions of a pure culture of the pathogen plated on the same solid medium lysed the heat-killed cells. Repeated attempts to isolate bdellovibrios and bacteriophages from soils inoculated with X. campestris yielded negative results, even when large quantities of soil were used for the attempted isolations. Four bdellovibrio isolates which were able to parasitize members of the genus Rhizobium were without effect on X. campestris. The lytic counts discussed above were performed on media with dead X. campestris cells, but no lytic colonies were noted if soil dilutions were plated on agar containing viable cells. By contrast with the lack of changes in the abundance of the previous microbial groups, protozoa responded to inoculation of soil with X. campestris. A marked difference in the protozoan density was evident between inoculated and uninoculated soil (Fig. 3). The counts in uninoculated soil remained reasonably constant, with only minor fluctuations at a cell density level of about 5 x 103/g of soil. By contrast, the abundance of these organisms in inoculated soil increased from an initial value of 9.2 x 103 to 5.4 x 105 cells/g of soil in a period of 4 days. The corresponding decrease in the X. campestris population was from 1.3 x 10' to 8.4 x 101 cells/g of soil. The soil protozoa, which included ciliates and flagellates but only rarely included amebae, did not respond to a further diminution in the X. campestris population. These findings argue for the direct involvement of soil protozoa in the decline of X. campestris in soil. It was observed in a preliminary experiment that protozoa were suppressed in Valois silt loam receiving 2.0 mg of actidione per g. The ability of X. campestris to persist in such a treated soil would show whether protozoa were indeed regulating pathogen density. Therefore, one sample of soil was amended with the antibiotic at the stated concentration, and a second sample received no actidione. The bacterium was added in large numbers to the two samples, and counts were made at regular intervals. The data in Table 1 confirm the previously noted increase in protozoa and decrease in xanthomonads in the actidione-free sample, and they also are in agreement with the preliminary test that suggested that the antibiotic held the animals in check. Furthermore, only a slight fall, rather than the usual abrupt decline, in the X. campestris level was evident in the soil supplemented with the chemical. DISCUSSION The pattern of population changes of X. campestris in natural soil-a rapid initial decrease followed by a stabilization in the cell density-is not unique to this organism. Thus, a similar trend was observed by Van Donsel et al. (18), who reported that 90% of the Escherichia coli and Streptococcus faecalis cells added to soil died in from 3 days to 3 weeks, depending on the prevailing conditions, whereas low counts of the organisms were evident for as long as 142 days. No experimental evidence has yet been provided to explain why low but not high cell densities can be maintained, but, if predation by protozoa is the means by which the bacteria are eliminated, then it might be hypothesized that the prey may persist in the presence of its predators when the energy that could be gained by predation is equal to or less than the energy expended in seeking the few surviving prey individuals. Such an explanation also may Alexander, unpublished data). In addition to such a prey density-dependent feeding by the protozoa, the physical obstruction to predation imposed by soil particles and the possible diminished edibility of prey sorbed to soil colloids may allow for survival. The ability of an organism, like X. campestris, to withstand starvation conditions and persist in the absence of carbon and nitrogen sources could be a beneficial characteristic in soil where competition for nutrients, particularly for carbon, probably often takes place. The mechanisms by which bacteria maintain themselves under starvation conditions, which would prevail for a .poor competitor, are not well defined. In the absence of exogenous substrates and if the organisms do not enter a metabolically inactive stage, bacteria probably derive energy from intracellular constituents such as poly-,B-hydroxybutyrate, polyphosphates, and polysaccharides, and several studies (3,17) have demonstrated that internal poly-,8-hydroxybutyrate reserves may be important in the resistance of microorganisms to starvation. On the other hand, bacteria surviving in nutrientdeficient solutions may persist because nutrients released from dying cells allow the growth of survivors (2), but the significance of cryptic growth in an environment with many species is probably small inasmuch as neighboring species will compete for the nutrients so released and probably are often better adapted to make use of them. Because the number of antibiotic-producing microorganisms remained remarkably constant and the level of tartrate-extractable toxins did not increase, it seems plausible to believe that toxin production is not a significant factor in the decline of the pathogen. Similarly, although the number of lytic bacteria and actinomycetes was high, their abundance was unaffected by the inoculation and subsequent decline of the pathogen. Furthermore, no microorganisms were found in soil which lysed live X. campestris cells, at least by the methods and with the sensitivity of the procedures employed. Nevertheless, the soil may contain a steadystate level of lytic enzymes or nonextractable toxins that do indeed contribute to the demise of an introduced alien species, and no relationship may exist between organisms producing antibiotics in a particular agar medium and those generating antibacterial compounds in soil. The reason for the resistance of live cells to lysis as compared to autoclaved X. campestris cells is not clear. Similar findings have been made in studies of the lysis of other microorganisms by actinomycetes (20) and myxobacteria (19). Webley et al. (19) proposed that the resistance of living cells to lysis probably results from the excretion by these cells of compounds inhibitory to the lytic enzyme or from the synthesis of cell walls at a rate faster than the enzymatic destruction of the walls. The appreciable increase in protozoan abundance associated with the decline of X. campestris and the failure of the pathogen population to be decimated in soil treated so that protozoa were inactive argue for a key role of predators in regulating the bacterial numbers. This hypothesis is also supported by the finding that the pathogen density reached a reasonably constant value when the protozoan number reached its maximum level. Further investigation is required to ascertain whether the protozoa are the sole agents responsible for elimination of the aliens and why the prey is able to maintain a reasonably large number of propagules despite the presence of its predators.

v3-fos

2018-04-03T04:15:50.993Z

1975-01-01T00:00:00.000Z

38352006

s2

Effect of pH and sodium chloride on growth of Bacillus cereus in laboratory media and certain foods. The effects of NaCl concentration, pH, and water activity (aw) on the ability of vegetative cells of Bacillus cereus to initiate aerobic growth in brain heart infusion broth at 30 C were studied in a factorial design experiment. By using multiple regression techniques, equations were derived which related the decimal reduction of the bacterial population to the concentration of NaCl and pH of broth to which the population was exposed. From these equations, the percentage of inoculated cells capable of initiating growth could be calculated. The reliability of these equations in foods was tested in laboratory-processed meat and rice media. The foods were less inhibitory than the broths, so that accurate prediction of growth initiation in foods was not possible by using the developed formulas. The impact of this type of quantitative study on the development of specific microbial standards for foods is discussed. When the NaCl concentration is increased, the aw is decreased and, with increased deviation of pH from optimum, more concentrated inoculum of B. cereus cells is needed to assure initiation of growth in culture media and foods. Bacillus cereus is a sporeforming bacterium which is widely distributed in nature. It has been recognized as a causative agent of food poisoning for almost 25 years. The symptoms are relatively mild and the duration is short. All kinds of foods can serve as vehicles, but typical ones are meat and certain meat products, as well as different types of puddings (10). Most epidemics reported have happened in Europe. In the United States B. cereus is rarely reported as the cause of foodborne disease: between the years 1968 and 1973, only seven outbreaks were reported (23). It has been shown recently that one or more substances produced during the exponential and late exponential phases of growth can induce fluid accumulation in rabbit ileal loop (21), a necrotic reaction in guinea pig skin (8), and an altered vascular permeability in rabbit skin (9). These substances are produced also by strains of B. thuringiensis and B. mycoides (9), and they are different from lecithinase C and hemolysin and yet indistinguishable from B. cereus lethal toxin (8). Whether these substances are responsible for human food poisoning still has to be proven. To evaluate the microbiological safety of a particular food, it is very important to know the percentage of inoculated cells capable of initiating demonstrable growth in that food environment. By using different preservation methods, this fraction of cells can be decreased to maintain a certain standard limit, allowing us to consider the food environment as safe. The effect of heat treatment on decreasing bacterial populations has been studied extensively. On the basis of studies done mainly with Clostridium botulinum spores, the canning industry has adopted the 12-D concept for low acid foods (16). This 1/1012 probability of a spore surviving canning is considered as a minimal safety standard. Decimal reduction values for the effects of other preservation methods, except heating and radiation, are not generally found or used. Genigeorgis et al. (5) explored the possibility of applying derived equations to predict the probability that one staphylococcal cell will initiate growth in media at various pH values and NaCl concentrations. Information concerning the effects of NaCl concentration and pH on the growth of B. cereus is limited (10,22). The purpose of this study is to obtain additional information of the effect of these parameters on the aerobic growth of B. cereus in laboratory media. Equations were developed which predict the decimal reduction of populations of various B. cereus strains exposed to such laboratory media. Practical applications of the equations were tested eventually in processed meat and rice environments. MATERIALS AND METHODS Preparation of experimental broths. The broths based on brain heart infusion (BHI) broth (Difco) were prepared as described previously (5). Inoculation and incubation of broths. The following five B. cereus strains were used: ATCC 9139, ATCC 14579 and 2006 obtained from G. York, Department of Food Science and Technology, University of California, Davis, and 01552 and 5063 obtained from R. Wood and T. Midura of the California State Department of Public Health, Berkeley. The latter two strains had been isolated from food poisoning outbreaks. Lyophilized stock cultures of the test organisms on porcelain beads were prepared according to a slight modification of the method of Hunt et al. (11). In the experiments, overnight BHI broth cultures of the strains were inoculated into 25 ml of BHI broth containing 0.25% Tween 80 (Difco). The fresh cultures were incubated at 30 C on a reciprocal shaker for 4 h. The cultures were then centrifuged, the cells were washed once with saline containing 0.5% peptone (Difco), and the concentration of cells was adjusted to an optical density of 1.4 to 1.7 at 615 nm with a Spectronic-20 colorimeter (Bausch & Lomb). Nine tubes, each containing 9 ml of broth, were prepared from each type of experimental broth. A 1-ml amount of the cell suspension was added to the first tube, and nine 10-fold serial dilutions of the suspensions were prepared. Three portions of 2 ml each were transferred with a sterile syringe from each of the nine tubes to 2-ml screw-cap vials. The caps were put on loosely and the vials were placed in 3-lb (ca. 1.4-kg) coffee cans (15.5-cm diameter by 17-cm height). In each can a vessel containing an NaCl solution of the same strength as the broth was placed. The cans were closed with their plastic lids, and the broths were incubated at 30 C for 10 days. Every other day vials with growth (turbidity) were removed and recorded. From the presence or absence of growth in the 27 vials prepared for each NaCl-pH combination, the most probable number of cells which had initiated growth was calculated from the tables of Fisher and Yates (4). The number of B. cereus cells present in the cell suspension used as inoculum was determined by plating on BHI agar (Difco) in duplicate. This number was always between 1.5 x 106 and 1.8 x 10'. Statistical methods. The experiments, arranged in a factorial design (20), involved five B. cereus strains, four NaCl concentrations (0, 2.5, 5, and 7.5%), and four pH values (4.6, 6.1, 7.5, and 8.8). For the statistical evaluation of the effects of NaCl and pH and their interaction upon the growth of B. cereus, the logarithm (log) of the ratio R,-RG was calculated for each broth and strain combination, where R, was the number of cells in the inoculum and R, the number initiating growth. This log represented the number of decimal reductions of a bacterial population resulting from its exposure to a particular environment. The logs determined in the factorial experiments were evaluated by using the biomedical computer program (2) for multiple regression analysis. Equations representing the trend surface were constructed for each individual strain and for pooled data. Each equation related the effects of NaCl and pH levels on log decrease for that strain. Preparation of experimental foods. (i) Meat. Cooked meats with different pH values and brine concentrations were prepared as described previously (6) and kept in the refrigerator until use. (ii) Rice. Commercial enriched long grain rice (Town House) was washed with water and cooked for 15 min (until all cooking water had evaporated). The rice was allowed to cool to room temperature, and then it was divided into two lots, to one of which 5% (wt/wt) NaCl was added. The two lots of rice were homogenized in mortars. After overnight refrigeration, both lots were again homogenized and 0.2% (wt/wt) glucono-delta-lactone was added to decrease pH of certain samples. The rice media were packed, pasteurized, and stored in a manner similar to that of the experimental meats (6). Inoculation and incubation of experimental foods. Small food disks used as growth media in the experiments were made by means of 9-mm diameter sterile cork borers and were placed in a sterile standard plastic petri dish. B. cereus cell suspensions were prepared in a similar way as in the broth trials. Nine 10-fold serial microbial dilutions in sterile 0.1% (wt/vol) peptone were prepared. Portions of 0.01 ml from each dilution were inoculated on three food disks with a sterile microsyringe. The petri dishes containing the disks were then placed in 3-lb coffee cans. In each can there was a vessel containing a brine of the same NaCl concentration as that of the food sample. Incubation was similar to the broths. After the appropriate incubation time, impression smears from each inoculated disk and uninoculated control disks were prepared on standard microscopic slides. The smears were examined for B. cereus growth with a Carl Zeiss phase contrast microscope. The most probable number of cells initiating growth was calculated in a similar way as in the. broth experiments. Physicochemical analysis of the growth media. Water content, NaCl and brine concentration, and pH of the experimental media were determined by methods previously described (6). Water activities (a.) of the experimental broths as well as food samples were measured by a model electric hygrometer (Hydrodynamics, Inc., Silver Spring, Md.) equipped with a gray band hydrosensor (range a, 0.81 to 0.99). Recalibration of the sensors was based on the use of NaCl solutions of different known molalities and a. values at 25 C (17). A 30-g sample of material was put into a 200-ml Kerr mason jar that was allowed to stand at 25 C for 24 h for humidity equilibrium before the dial reading was taken. Two unused sensors were utilized. Water activities of the experimental broths were also determined by an equilibrium moisture absorption technique as described by Vos and Labuza (24), based on the use of avicel microcrystalline cellulose. RESULTS The raw data of the combined effects of pH and NaCl of the broths on the log (decimal) decrease of the populations of the five B. cereus strains exposed to various broth environments are presented in Fig. 1. From the multiple regression analysis of the data, the following five equations were derived for the strains used, When the data of all the strains were pooled, the summary equation was as follows: Ye = 44.26 -0.80 (salt) -11.88 (pH) + 0.03 (salt)2 + 0.79 (pH)2 + 0.21 (salt x pH). Statistical analysis of the data indicated significant effects of (salt), (pH), (pH)2, and (salt x pH) upon the magnitude of log decrease. Although the effect of the term (salt)2 on the log decrease was not statistically significant, it has been retained in the equations for symmetry. By using the equations given above, response curves were constructed for each strain and for the pooled data relating pH, NaCl, and log confidence contours for a specified log reduction can be obtained by using Ye ± standard error, and approximate 95% confidence contours by using Ye ± 2 standard errors. A response curve and the approximate 68 and 95% confidence limits for 4-log reduction of B. cereus strain ATCC 14579 are presented in Fig. 3. To test the above-developed equations in foods, the log decreases of bacteria populations caused by meat and rice environments with different pH values and NaCl concentrations were determined for B. cereus strains 5065 and 01552 ( Table 1). The a, of the BHI broths with 0, 2.5, 5.0, 7.5, and 10% brine were found to be 1. Response curve (0) and approximate 68 (0) and 95% (U) confidence limits for 4-log reduction of B. cereus strain ATCC 14579 exposed to different pH and NaCl combinations. DISCUSSION The range of pH permitting growth of B. cereus in laboratory media has been reported to be pH 4.9 to 9.3 (10). The few articles (13)(14)(15) dealing with growth in foods of different acidity give the minimal pH for growth varying from pH 4.5 to 5.15. According to existing data (12,19,22) B. cereus is able to grow in 7% NaCl but not in 10%. Minimal a, value allowing growth is reported to be 0.950 (18). Previous data agree generally with the findings of the present study. Initiation of B. cereus growth in a meat environment at a pH as low as 4.35 has been observed for the first time. However, a high inoculum (106 cells in 0.01 ml) was used, thus giving greater probability of initiating growth. The maximal pH used in the study was 8.8. Though this pH was found to be relatively noninhibitory to growth of B. cereus, its significance to food protection is minimized by the fact that very few foods have such a high pH. In this study the effects of NaCl and pH on the log reduction of B. cereus populations ex-posed to different environments were studied. The quantitative approach used permits prediction of the percentage of inoculated cells capable of initiating growth, when inoculum and environmental parameters (% NaCl, pH) are known. This predictive potential is important in the development of microbiological standards for different types of foods. Equations were derived relating NaCl concentration and pH of BHI broth medium to log reduction of a B. cereus population exposed to this environment. The probability that one cell can initiate growth can be calculated from these equations. For example, for a broth at pH 5.5 with 2% NaCl, the log reduction for strain 5065 is 3.88, the antilog is 7585, and the probability of initiating growth is that 1:7585 or 0.0134% of inoculated cells will be capable of initiating growth. If salt concentration is increased to 4%, then 0.0011% of inoculated cells will be capable of initiating growth. Similar calculations can be done to any NaCl-pH combination. Growth characteristics of B. cereus do not differ qualitatively from the general bacterial behavior pattern when exposed to different NaCl and pH conditions. These shapes of response curves resemble those found by Genigeorgis et al. (5) in their study on growth of Staphylococcus aureus. The general findings can be summarized as follows. (i) The effects of pH and NaCl on growth of B. cereus vary with strain and growth medium used. (ii) There is a decreased rate of growth of B. cereus when exposed to media with NaCl concentrations increasing from 0 to 10%. (iii) When we increase the NaCl concentration of a medium, more concentrated inoculum is needed to assure initiation of growth. The same is true for pH when the values drift away from pH 6.5 to 7. (iv) High NaCl concentrations and extreme pH values prevent growth. (v) Smaller concentrations of NaCl are required to inhibit initiation of growth at pH values remote from optimum. Food served as vehicles in epidemics of B. cereus food poisonings are usually highly contaminated. Microbiological examinations have regularly revealed contamination levels of 106 to 107 cells per g of food involved in food poisonings (10). The amount of contaminated food eaten by an individual before getting ill is, however, infrequently mentioned in reports concerning food poisoning epidemics caused by B. cereus. Generally, low attack rates for the sources of outbreaks as well as experimental feeding trials (10) indicate that a relatively large amount of a contaminated food must be consumed in order to produce symptoms. Because of the relatively low prevalence of reported food poisoning cases caused by B. cereus and the mild nature of the disease, no standards have generally been established for foods. To prevent B. cereus growth to as high levels as mentioned above, the growth environment should be inhibitory enough to reduce the probability of growth initiation at least by a factor of 104. The response curve for 4-log decrease for a population of B. cereus strain ATCC 14579 in BHI broth is presented in Fig. 3. Any NaCl-pH combination above the response curve causes the desired minimal inhibition. Figure 3 indicates also the approximate 68 and 95% confidence contours of the response curve. The curves are specific for this strain and growth environment and, as such, cannot be applied to foods. To test the reliability of the formulas developed for BHI broths in food environments, experiments were made in which laboratoryprocessed meats and rices served as growth media. These types of foods have frequently been reported as vehicles in B. cereus food poisoning epidemics (10,23). The data collected (Table 1) indicate that the food environments tested are remarkably less inhibitory than BHI broths. Thus, the equations will give too high log reduction values if applied directly in foods. For instance, strain 5065 inoculated into meat with 4.1% NaCl concentration in brine and pH 6.1 had a log decrease of 4.80 instead of 0.07 measured. Similarly, for the same strain, a meat environment of 4.6% brine concentration and pH 7.85 would have a log decrease of 3.98 instead of 1.76 measured. Rice also appears to be a better growth medium than BHI broth. Sample number two (Table 1) had 0% salt and pH 5.0. The equations for strains 01552 and 5065 and such a salt-pH combination produced "expected" log decreases of 5.03 and 4.79, respectively, instead of 1.02 and 3.24 log decreases obtained experimentally in rice. Genigeorgis et al. (7) have also obtained less inhibition of staphylococci inoculated in processed meats than the predicted level of inhibition based on formulas derived from studies on BHI broths. To make accurate predictions of the probability of growth initiation in a certain food, the formulas should be developed for that particular food item as bacterial growth media. Glucono-delta-lactone was used as an acidulant for the food samples and HCl was used for the broths. However, this fact cannot explain the differences between inhibitory capabilities of the environments. At least in the case of Salmonellae, HCl has been shown to permit growth at lower pH values than gluconic acid (1). Foodborne bacterial pathogens in general grow at a, levels of 0.83 to 0.999 (22). In this high range, measurements by the electric hygrometer having a moisture-sensitive, saltcoated probe are considered to be inaccurate, especially when the probe gets older (3). In these experiments two sensors were used, one new and the other several years old but unused. When calibrated before use, no significant differences could be found between the results. Shape of the standard curves was, however, different. The new sensor gave remarkably vaster ranges for dial readings at a, values over 0.9, thus giving better accuracy. New hygrometer probes are considered to be accurate within i 0.005 a, inside their specific ranges, that in this case (a gray band sensor) ends when a, = 0.99. Water activities greater than that were reported as a, = 1.000. The water activities measured by equilibrium moisture absorption of the microcrystalline cellulose method were generally 0.01 a, units lower than those given by electrical hygrometer. This trend does not agree with the results of Vos and Labuza (24), who got about equal values by both methods. The lowest limit of a, = 0.950 for growth of a vegetative B. cereus cell has been stated (18). The results of this study agree with the reported information. The lowest aw value that permitted growth was 0.955 when measured by an electric hygrometer.

v3-fos

2018-04-03T01:05:13.473Z

1975-09-01T00:00:00.000Z

22937204

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Effects of an abrupt change in ration from all roughage to high concentrate upon rumen microbial numbers in sheep. When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values. Previous reports have shown that the type and level of ration consumed by an animal affect the numbers and types of bacteria and protozoa in the rumen (1,4,15,17,22,23). When the animal is changed from one ration to another, a period of microbial adaptation occurs, which can be defined as that time interval required for the rumen microbial population to stabilize. However, very little information is available on the absolute microbial changes that occur during this adaptation period (9,18,23). Several investigators have estimated the length of this adaptation period by measuring digestibility data as a relative indicator of rumen microbial activity (10,16,19). In general, results of these studies have indicated that the length of time required for adaptation depends upon how radical a change is made in the ration. A recent study conducted in this laboratory with sheep, using daily dry-matter and cellulose digestibility as criteria, investigated the length of the adaptation period when animals were changed from an all-roughage ration to either a corn silage ration, a 60% cracked corn-40% orchardgrass hay ration, or a chopped alfalfa hay ration and back to the roughage ration (20). The most marked changes in daily digestibility coefficients occurred when the sheep were placed on the 60% cracked corn-40% orchardgrass hay ration. However, in almost all cases the digestibility coefficients for all rations showed little if any further change after 5 days. On the basis of these results, the present study was initiated to investigate the changes in bacterial and protozoan concentrations that occur in the rumen of sheep during this abrupt change from a ration of all orchardgrass hay to one containing 60% cracked corn-40% orchardgrass hay. In addition, concentrations of specific types of rumen bacteria, i.e., cellulose digesters and total volatile fatty acids (VFA), were also measured. Although the sheep were fed at approximately equal dry-matter intakes, the markedly different nature of the rations could affect both rumen volume and rate of passage. Thus, measurement of liquid and drymatter turnover rates and rumen volume was included as a possible help in interpreting any observed microbial changes. MATERIALS AND METHODS Animals. Three cross-bred wethers, weighing approximately 45 kg, were fed a ration of 800 g of chopped orchardgrass hay per day and then abruptly changed to a ration containing 800 g of 60% cracked corn and 40% chopped orchardgrass hay. The animals were fed once daily at 9:00 a.m. and had free access to water. All three sheep were surgically prepared with rumen fistulae, and composite samples of rumen contents for counting the microbial concentrations were collected from various locations within the rumen just before the daily feeding. Nine ewes, four fed orchardgrass hay and five fed 60% cracked corn-40% orchardgrass hay, were used to estimate dry-matter turnover. The rations were fed at least 3 weeks before slaughter. Rumen microbiology: general. The anaerobic culture techniques were similar to those described by Hungate (11). Methods of preparation of media and dilution of rumen ingesta have been described by Dehority (6). Total viable bacterial numbers were determined in roll tubes with 40%.rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA), similar to that described by Bryant and Burkey (3). Total substrate concentration in the RGCSA medium for experiments 1 and 2 was 0.3% carbohydrate, which was divided into 0.125% (wt/vol) each glucose and cellobiose and 0.05% (wt/vol) soluble starch. For experiment 3, a total substrate concentration of 0.1% carbohydrate was used, 0.025% (wt/vol) each glucose and cellobiose plus 0.05% (wt/vol) soluble starch. Colonies were counted with a binocular dissecting microscope after 7 days of incubation at 38 C. Bacterial counts were also determined with differential media, in which either 0.3% xylan, 0.3% pectin, 0.3% soluble starch, or 0.75% ball-milled cellulose replaced the glucose, cellobiose, and starch added to the basal medium. Methods used to solubilize the xylan and pectin were those described by Dehority (6,7). Total and molar percentages of rumen VFA were determined in all samples from sheep 2 and 3 by gas chromatography (8). Protozoan numbers per milliliter and percent generic distribution were determined according to the procedure of Purser and Moir (21). Experimental procedures. Before the start of the experimental periods, all sheep were fed the chopped orchardgrass hay for 6 weeks. Sheep 1 was sampled intermittently over a 29-day period from days designated as t_ to tL21. From tL to to the sheep was fed chopped orchardgrass hay. After a sample was taken on day t0, the animal was changed to the concentrate ration for the remainder of the experiment. With sheep 2, samples were only taken over a 22-day period from t-to t14. Sheep 3 was sampled over the 28-day period from t7 to t21. Other than the cellulose medium, the only differential counts attempted with sheep 3 were with a 1% soluble starch medium. The starch roll tubes were incubated at 38 C for 24 h, and after the colonies present were counted the rubber stoppers were carefully removed and the tubes were filled with Lugol iodine, which had been diluted 1:8 with distilled water. Within 10 to 15 min the agar medium turned a dark brown to purple color with resultant clear zones appearing where starch colonies had grown. Although considerably more colonies appeared with incubation up to 7 days, after 24 h the clear zones overlapped so much that accurate counts could not be made. Rumen volume, fluid, and dry-matter turnover rates. Rumen volume and fluid turnover rate were measured by using polyethylene glycol as a marker. Analytical procedures were similar to those of Hyden (14). Preliminary experiments, following a sampling schedule similar to that proposed by Hyden (14), gave abnormally high estimates of rumen volume. Further studies indicated that for animals fed once daily the magnitude of dilution effects upon concentration of a soluble marker was quite large, giving a very steep slope in the first few hours and a corresponding large rumen volume when the line was extrapolated back to zero time. This dilution effect has also been experimentally demonstrated by Warner and Stacy (24). In the present work, polyethylene glycol was added 1 h before feeding, and sambles for analysis were taken 1 and 24 h later. Dry-matter turnover in the ewes was determined at slaughter according to the procedures described by Hungate (12). Acid-detergent lignin (26) was used as a marker to estimate digestibility. RESULTS Bacterial colony counts. Anaerobic bacterial colony counts per gram of rumen contents for the three sheep during the period when they were abruptly changed from orchardgrass hay to the 60% corn-40% orchardgrass hay ration (day 0) are presented in Fig. 1 and Table 1. Rather marked differences in bacterial concentrations between the three sheep can be seen in Fig. 1. Bacterial concentrations were much lower in sheep 1, and aside from a slight drop on days 1 and 2 after the ration change concentrations gradually increased through day 21. In sheep 2, bacterial concentrations began to increase immediately after the ration change and increased at a much greater rate than for sheep 1. No values were obtained for day 21 in this animal, since it was inadvertently sheared on day 20 and went off feed. Concentrations also began to increase immediately after changing rations for sheep 3; however, a tremendously high peak occurred on day 5, followed by a decrease on days 7 and 14, with an indication of a gradual increase between days 14 and 21. Although the peak observed on day 5 could be the result of a sampling error, concentrations of starch-and cellulose-digesting bacteria (to be presented later) and protozoa all increased in a similar fashion. In the study reported by Potter gestibilities of these same rations were estimated over the same period, most of the change occurred between days 1 and 5. Values appeared to be relatively stable from days 6 through 21. On this basis, means of the bacterial concentrations were calculated for those days before the ration change (to to to), the socalled transition period (t1 to t5) and from t7 on. These data are shown in Table 1, and in all cases concentrations from t7 on were significantly higher (P < 0.01) than those on the orchardgrass ration (t8 to to). In sheep 2 and 3, where days t, to t5 were a period of rapid increase, mean values were not different from either of the other two means. In sheep 1, where most of the increase occurred after day 5, the transition period mean did not differ from the value on orchardgrass hay. Although there were considerable differences between the three sheep, bacterial concentrations increased in all animals after the ration change. Digestibility data (20) had suggested that the transition or change was complete within 5 days; however, this would be in contrast to the present data, where concentrations continued to increase through 21 days and may not even then have reached a plateau. Selective medium colony counts. In addition to total colony counts for sheep 1 and 2, an attempt was made to estimate the concentra-tions of bacteria capable of fermenting starch, xylan, pectin, and cellulose. A selective medium was used in which the substrate under study was the only added energy source. With either xylan, pectin, or starch as substrate, 7-day colony counts were made and results were expressed as a percentage of the colony count obtained with RGCSA medium. For the five samples from sheep 1 while on orchardgrass hay, values for the xylan medium ranged from 70 to 113%; for pectin, 70 to 106%; and for starch, 71 to 136%. Similar results were obtained at the other sampling times and also for sheep 2. The probable explanation for this discrepancy is that the 40% rumen fluid basal medium contains enough energy sources to support growth of considerable numbers of colonies (5). Using the procedure of staining with Lugol iodine, described earlier, the concentration of starch-digesting colonies at 24 h was estimated for sheep 3. The actual number of starch-digesting colonies appearing within 24 h was probably far less than would be obtained at 7 days and represented the faster-growing species. However, the numbers of starch-digesting colonies obtained on days t-5, tU3, t-1, and to were 13, 10, 10, and 18%, respectively, of the colonies observed with RGCSA medium. For days t1, t2, and t3, the percentages rose to 25, 40, and 36%, respectively. Too many colonies were present to count on days ti, and a value of 47% was obtained for the sample taken on day t7. by t14 and t2,, percentages had fallen to 9 and 16M, respectively. These data suggest a temporary marked increase in the starch-digesting species, which grow rapidly enough in artificial media to show colonies in 24 h, during the first week after the change to the high-concentrate ration. The concentration of cellulose-digesting bacteria can be estimated fairly specifically by counting the clear zones in cellulose-agar roll tubes after incubation for 28 days. Cellulolytic colony counts for the three sheep, over this period of ration change, are shown in Fig. 2. The most obvious difference between animals is that the concentration of cellulolytic bacteria in sheep 3 was about 20 times higher than in sheep 1 and 2. For all animals, decreased concentrations were observed immediately after the change in ration. For sheep 1, colony counts decreased up to 5 days and then increased back to nearly the same level as on the orchardgrass hay ration. In contrast, a marked peak was observed on day 5 in sheep 2 and 3, followed by a decrease to a level lower than the colony counts on all roughage. When mean values on all roughage were compared with mean values for day t7 on, the difference was significant only for sheep 3 (P < 0.01). Protozoan concentrations. The concentrations of rumen protozoa determined over the same experimental period are presented in Fig. 3 and Table 2. In general, concentrations were similar in all sheep on the roughage ration, and, as can be noted in Fig. 3, the response to changing from all roughage to concentrate was consistent among the three animals. Concentrations began to increase on day 1 and appeared to reach a plateau about day 5. These data were grouped and the means were calculated in the same manner as for bacterial colony counts ( Table 2). For all sheep, a significant increase in protozoan concentration was found between the all-roughage ration and from day 7 on after the change to concentrate. These data agree with the pattern of increased cellulose and drymatter digestibility reported by Potter and Dehority (20). With respect to percent generic composition of the protozoa, a significant decrease in Entodinium (94.5 to 88.9%) and increase in Diplodinium (1.6 to 5.7%) (P < 0.05) were observed in sheep 1. No changes were found with sheep 2, and the percentage of Entodinium increased significantly (P < 0.05) with sheep 3 (92.4 to 95.1%). These data do not suggest any obvious relationships between ration and percent generic composition. VFA. For sheep 2, a significant increase in total VFA (P < 0.05) was observed when the animal was changed to the concentrate ration; mean millimolar values were 158.5 for days t-8 to to compared with 234.4 for days t1 to t14. A significant decrease (P < 0.05) in the molar percentage of acetate and an increase in propionate also occurred. Although not significant, the molar percentage of n-butyrate showed an increase in the concentrate ration. Changes in total VFA and molar percentages for sheep 3 were almost identical to those for sheep 2; however, in this instance only the decrease in molar percentage of acetate was significant at P < 0.05. All other changes were significant at P < 0.1. No particular trends in either total VFA or proportions with time after the ration change were apparent. These results agree with the VFA changes reported by Latham et al. (15) when animals are changed to a concentrate ration. Rumen volume and fluid turnover rate. Using polyethylene glycol as a soluble marker, rumen volume and fluid turnover rates were estimated for the three sheep on both rations (Table 3). These determinations were made after completing the work on microbial changes, and at least 3 weeks were allowed for adjustment of the animal to the ration. A significant decrease (P < 0.05) was observed in ru-men volume for two of the three sheep; however, rather unexpectedly, fluid turnover rate was not significantly affected by ration. Table 4 presents total bacterial colony and protozoan counts based on rumen volume. Values for the transition period (days t1 to t5) have been omitted, since no estimates of rumen volume were made for that period. These data differ from those in Tables 1 and 2 in two respects: total bacterial numbers in sheep 1 were not significantly affected by the change in ration when rumen volume was considered, and there was a striking similarity between total bacterial and protozoan numbers in sheep 2 and 3. Possible reasons for the failure of total rumen bacterial numbers to increase in sheep 1 in response to an increased energy intake are not immediately obvious. As can be noted in Fig. 1, bacterial concentrations were still increasing on day 21, and possibly if this animal had been sampled at a later date higher concentrations would have been found. On the other hand, protozoan concentrations did increase, but the increase was less than in the other two sheep, and values were fairly stable from days 7 through 21. These data suggest that, for studies comparing rumen microbial numbers, colony counts and protozoan numbers based on unit volume APPL. MICROBIOL. on May 7, 2020 by guest http://aem.asm.org/ Downloaded from Dry-matter turnover rate. Since both the roughage and concentrate rations were fed at similar dry-matter intakes, it seemed possible that this might explain equal fluid turnover rates. However, because of the readily fermentable nature of the concentrate ration, it would as reported in Tables 1 and 2 can be misleading and more meaningful values can be obtained by using values based on total volume. Also, if marked differences occurred in fluid turnover rates between animals or rations or both, some adjustment of values would be required. 18 Mean and standard error of the mean. d For each parameter, means within a column followed by different superscripts are significantly different at P < 0.05. Tables 1 and 2 multiplied by rumen volume, Table 3. Mean and standard error of the mean are presented. AddFor each parameter, means within a column followed by different superscripts are significantly cantly different at P < 0.01; b, d means are significantly different at P < 0.02. appear that this ration should have a faster rate of passage. Dry-matter turnover was estimated in four ewes fed orchardgrass hay and five ewes fed 60% cracked corn-40% orchardgrass hay (Table 6). Dry-matter digestibility, as estimated from lignin ratios, was significantly higher (P < 0,01) in the concentrate-fed animals; however, turnover of the dry matter was slower. These data agree with the concept proposed by Hungate (12), that turnover rates based on indigestible feed components will be inversely related to digestibility when feed intake is held constant. Hungate further suggests that with such concentrate rations the fluid turnover rate is probably a better index of rumen function. Although of considerable interest, these values for dry-matter turnover in the rumen do not appear to offer information that can be used to better estimate total daily microbial production. DISCUSSION The variability between the three sheep used in these experiments agrees with the observations of Warner and Stacy (24) concerning animal differences; however, most of the responses have been noted in various studies by other workers. For example, the increase in bacterial colony counts in sheep 2 and 3 in response to an increased intake of available energy substantiates the results of Bryant and Burkey (4) and Maki and Foster (17). In contrast, Latham et al. (15) obtained results similar to those observed with sheep 1, in which bacterial concentrations did not increase when available energy intake increased. On the other hand, protozoan concentrations increased in all sheep when the available energy intake was raised, agreeing with previously reported results (1,18,23). Warner (23) has reported the changes in ru- He observed that about 10 days was required for microbial changes to occur, and concentrations were relatively stable after that time. These findings differ from the present results, in which bacterial concentrations appeared to still be increasing after 14 to 21 days and protozoan concentrations had stabilized after 5 days. When the present observations are considered with respect to the results of Potter and Dehority (20), where digestibility of these two rations had stabilized after 5 days, it would appear that the continuing changes observed in viable bacterial concentrations are not of major significance to overall digestion in the animal. Maki and Foster (17) have reported that viable counts of bacteria in the rumen contents of cows fed a high-roughage ration represented only 3 to 12% of the number determined by direct count, whereas 57 to 73% of the bacteria from cows fed a ration without roughage (grain plus alfalfa meal) could be cultured. A similar increase in the proportion of viable to direct bacterial count was noted in rumen contents from an all-concentrate-fed animal by Bryant and Burkey (4); however, they also observed that the percentage value of viable counts was similar to that of direct counts, about 8.4%, when animals were fed all roughage or a mixture of roughage and concentrate. Although direct counts were not made in the present study, the concentrate ration contained 40% orchardgrass hay, and based on the results of Bryant and Burkey (4) it appears probable that viable counts on the two rations represented a similar percentage of the total bacteria. A possible reason proposed for the discrepancy between direct and viable counts is that the direct count measures dead cells and those metabolizing cells which cannot be grown in the artificial medium (12). The differences between roughage-and concentrate-fed animals must then be attributed to a lower proportion of these two cell types in the concentrate-fed animal, a shift in the population to more organisms that can be grown, or possibly sequestration of the cells in the fibrous material in hay-fed animals that are not readily disrupted by mixing. One additional point to be considered is that in the studies of Bryant and Burkey (4) and Maki and Foster (17), viable colonies were counted after 3 and 4 days, respectively. More recently, Bryant and Robinson (5) have reported that their 3-day counts averaged only 67% of 7-day counts. Rumen contents for their study were obtained from a cow fed a 79%o alfalfa-21% grain ration. A similar increase in colonies has been observed in our laboratory between 5 and 7 days. If those organisms which appear after 3 to 5 days of incubation are specifically associated with roughage digestion, the differences observed between direct and viable counts on roughage-and concentrate-type rations may be less than previously reported. Adjustment of rumen microbial concentrations for rumen volume in sheep 2 and 3 revealed a striking similarity in the microbial protoplasm supported by a fixed energy intake. In sharp contrast, however, were the low concentrations in sheep 1. After adjustment for rumen volume, total bacterial numbers were the same on the two rations. Obviously other factors must be affecting bacterial concentrations in this animal. The decrease in rumen volume for two of the three sheep, when changed to equal dry-matter intake of the concentrate ration, probably reflects differences in salivary flow and digestibility. Any attempt to estimate total rumen microbial production per day must take into account the rate of passage of bacteria and protozoa from the rumen. Several investigators have studied this problem; however, because ofdifferences in methods and rations, rather marked differences in results were obtained (13,25). The primary questions that must be answered VOL. 30, 1975 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from are: what is the relationship between fluid flow and microbial passage, and how is this affected by type and amount of ration and frequency of feeding. Since fluid turnover rate did not change between the two rations, we have assumed microbial passage rates to be similar. Further studies may show this assumption to be unwarranted. Although rumen dry-matter turnover of the roughage and concentrate rations was found to differ, no obvious effect on microbial numbers was evident. A recent report by Akin et al. (2) has confirmed the attachment of rumen bacteria to plant tissue, suggesting that rate of passage for certain species may be influenced by quantity of rumen dry matter. In the present study, rumen dry matter decreased by 21% in animals maintained on the concentrate ration. Concentrations of cellulolytic bacteria were observed to decrease in sheep 1 by 22% and in sheep 2 by 28%, indicating a fairly close relationship between dry matter and cellulolytic bacteria. In contrast, cellulolytic bacterial concentrations in sheep 3 were almost 20 times higher on the roughage ration and decreased by about 77% with the ration change. LITERATURE CITED

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Possibility of reducing calving difficulties by selection. III. – A note on pelvic size in relation to body weight of cattle HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. POSSIBILITY OF REDUCING CALVING DIFFICULTIES BY SELECTION III.A NOTE ON PELVIC SIZE IN RELATION TO BODY WEIGHT OF CATTLE St. C. S. Taylor, L. S. Monteiro, B. Perreau INTRODUCTION The relationship between calving difficulties and internal pelvic size has been studied by M!LrISSI!It and V IS S A C ( 1971 ) and M!rWSSI!R, B IB A, and PE RR E AU (Ig73). In a study involving the factorial crossing of Limousin, Charolais and Maine Anjou cattle, they found that as a dam breed the Limousin had least difficult calvings. The superiority of the maternal component of ease of calving in the Limousin was attributed to the lower birth weight of the calves and to the larger pelvic area relative to their body weights. By contrast, the Charolais with lower birth weight than the Maine Anjou had the smaller pelvic area per unit weight and consequently had the poorest performance of the three breeds with respect to ease of calving. L AS TE R ( 1974 ) analysed the effect of pelvic area on calving difficulties in 14 different breed groups and found a very significant within-breed relationship between pelvic area and body weight among animals at the same age, but not a very strong within-breed association between pelvic area and calving difficulties. The corresponding inter-breed relationships were not subjected to a similar analysis, but the purebred and crossbred means showed that there was a tendency for calving difficulties to become more frequent as mean body weight and mean pelvic area increased. M ON TE IRO ( 19 6 9 ) used data from Friesians, Ayrshires and Je y seys and their crosses to develop an index of calving difficulty based on the relationship of calf birth weight to dam weight. This index was essentially the ratio of calf birth weight to dam weight raised to the power o.q., this being the power which gave the best discrimination between difficult and easy parturitions. MorrT!IRO suggested that (dam weight) 0 , 4 might well be an indirect measure of some critical dimension of length, width or area associated with pelvic size or calf size. This note presents some relevant data on the relationship of internal pelvic measurements to body weight at different stages of growth and also across breeds. MATERIAL AND METHODS Measurements of pelvic height and width were taken on 33 females from 6 different breeds and 4 different crosses, ranging from 8 months to 6 years of age. Most animals ( 27 ) had not had a calf even though some were 3 or 4 years old. About half the animals (r8) were reared on a conventional diet and treated in the normal traditional manner for dairy cattle. Of these i were Ayrshires and 5 were Herefords and they provided the main range of ages. Four Ay 'Y shires, one He r eford and all animals from the other 8 breeds or crosses had been fed ad libitum on standard complete diet A.A.6 (a nutritionally balanced all-pelleted ruminant diet containing 30 p. 100 of chopped straw), and these intensively reared animals ranged from 15 to 50 months of age. The comparison of conventional and complete diet was somewhat confounded with breed differences. The technique described by M ENISSIER and V ISSAC ( 1971 ) was used to take internal measurements of the height and width of the pelvic opening. All measurements were made by the same observer. The variables analysed were the natural logarithms (In) of pelvic height (y h cm), pelvic width (yw cm), body weight (W kg), and mean mature weight (M kg). Body weight was expressed in terms of degree of maturity in order to separate the growth and inter-breed components of the relationship. Only the breed mean for mature weight was used, and mature weight therefore had the same value for all individuals of the same breed. RussELL's ( 1970 ) least-squares a Compreg » program was used for the statistical analysis. Three different linear models were used and applied separately to pelvic height and pelvic width. These models were where a is the allometric or growth coefficient, {3 is the inter-breed regression coefficient, d k is the effect of k the diet, p, is the parturition effect, e is the regression constant, and the e jJk ;! are residual errors for the j th animal from the i t &dquo; breed. In model ( 2 ), a and {3 are assumed to be equal, and the b t are a set of breed constants with crosses regarded as different breeds. In model ( 3 ) the allometric and inter-breed regression coefficients are all set equal to o. 4 . In all models the limitations of the data precluded useful tests for linearity of regression and interactions between parturition, diet and breed, while in model (i) the data was insufficient to estimate separate weighting factors for breed deviations from the regression and individual deviations from the breed means. To this extent the standard errors and tests of significance are only approximations ; but since there was usually only one animal in each parturition X diet X breed X weight-interval sub-class, the conclusions are unlikely to be seriously affected. RESULTS Pelvic height ranged from 13 cm in an 8 month old Ayrshire up to 21 cm in a 6 year old biparous Ay y shire. The corresponding range for pelvic width was io to 1 8 cm. In nulliparous females over 3 years of age, pelvic height ranged from 17 cm in a Dexter up to 30 cm in a British White. The corresponding breed range for width was 12 to 1 8 cm. Body weight ranged from 19 8 to 710 kg. Mean mature body weight on the complete diet, which was about 30 p. 100 greater than mean mature weight on the conventional diet, ranged from q .q.o kg in the Dexter breed up to 750 kg in the British White. Animals ranged from being about 30 p. 100 mature in body weight to being almost fully mature. Model (i) The results obtained from the least squares analysis are shown in table i. There were highly significant allometric relationships between pelvic size and degree of maturity in body weight. There was an equally significant increase in pelvic size with mean mature weight of breed. For pelvic height the effect of stage of maturity was insignificantly greater than the effect of mean breed weight ( 0 . 35 and o. 4 6), while for pelvic width there was an insignificant difference in the reverse direction (o.q.o and 0 . 37 ). It was somewhat surprising that the non-genetic allometric relationship and the genetic inter-breed relationship should be almost the same. The growth coefficients for pelvic height and pelvic width were very similar, and suggest that both height and width were growing at about the same rate relative to body weight. Thus pelvic height appeared to be on average about 33 p. 100 greater than pelvic width. Differences probably exist at different stages of growth but they could not be determined in the present data. The fitted regressions accounted for 8 3 p. 100 and 7 8 p. 100 of the total observed variation in pelvic height and width respectively, while the residual standard deviations indicate that pelvic height and width might be predicted with errors of about ! 5 p. 100 and 7 L 7 p. 100 respectively. Parturition was estimated as permanently increasing pelvic size by(9.8 ! 2 .6) p. 100 in height and ( 3 . 2 ! 4 . 0 ) p. 100 in width. The significant and much greater increase in height than in width is somewhat unexpected. Animals grow about 30 to 40 p. 100 faster in body weight on the complete diet than on the conventional diet. The allometric relation of pelvic size to body weight may also be affected. With more data it would have been desirable to estimate different growth coefficients and constants for each diet. Only different constant were fitted. For animals on the complete diet pelvic size was smaller in relation to body weight than for animals on a conventional diet by ( 9 .8 ! a.6) p. 100 for height and ( 4 .8 ! 3 . 4 ) p. 100 for width although these estimates may to some extent be confounded with breed differences. Intensive rearing thus appeared to promote relatively more fattening than bone growth. Caution is therefore necessary where body weight of intensively reared females is used as a guide to their readiness for insemination. Model ( 2 ) In view of the similarity of the allometric and inter-breed coefficients, the data were re-analysed with body weight as the only c-ontinuous variable and with the mean mature weight of each breed replaced by a set of breed deviations or constants. The amount of variation accounted for by breed differences was highly significant. The Ay y shire breed had a pelvic opening ( 2 . 3 ! 1 .6) p. 100 higher and (6.5 zb 1 . 9 ) p. 100 wider relative to body weight than the average for all breeds. The corresponding values for the Herefo y d were (&mdash; 2 .g ! z. 3 ) p. 100 and (&mdash; i. 5 ± 2 .6) p. 100 , both less than average but neither significantly so. The only other breed with a significant deviation was the Dexter which was below the average for all breeds by (io.8 ! 3 .5) p. 100 in height and ( 9 -2 ! 4 . 0 ) p. 100 in width. The proportion of the total variation accounted for increased to 93 p. 100 and the accuracy with which pelvic size was estimated from body weight also increased, being ± 4 . 1 p. 100 for height and 4 .6 p. 100 for width. The regression on log body weight now appeared to be slightly greater for width than for height, more consistant with the evidence from external measurements that the whole pelvic structure matured somewhat more slowly in width than length or height. Estimates of the effect of parturition and diet were slightly greater and became significant for pelvic width (table 2 ). , Model (3) The allometric and inter-breed regression coefficient in model (i) and the allometric coefficient for pelvic height and width in both models (i) and ( 2 ) were very similar and all close to 0 . 4 . The analysis was therefore repeated with the regression coefficients fixed at o. 4 . Virtually the same set of results were obtained except that the regression constants were then very much more accurately determined (table 2 ). This model accounted for 92 -93 p. 100 of the total variation and gave estimation errors of ! 4 -5 p. 100 . The present limited data was therefore adequately summarised in terms of a single coefficient of o. 4 . Thus pelvic size could be roughly estimated from body weight at any stage of maturity and in any breed by the simplified equations : pelvic height (cm) = 1 .6 3 (body weight in kg) 0 . 4 &dquo; pelvic width (cm) = i.22 (body weight in kg) '!4 &dquo; in which the constants refer to nulliparous females on conventional diet. In figure I the plot of the data unadjusted for the effect of parturition, diet or breed shows the extent of gross deviations from these simplified regression equations. When parturition, diet and breed were ignored, these equations accounted for only 6 5 p. 100 and 7 6 p. 100 of the total variation in height and width respectively (instead of g 2 -g 3 p. 100 ) and gave estimation errors of ± 7 p. 100 for both height and width (instead of ± 4 -5 p. ioo). When comparing breed deviations, adjustments should therefore be made for parturition and also diet if possible. DISCUSSION The most interesting result is the approximate uniformity of the relationship of both pelvic height and width to degree of maturity and mean mature weight of breed. The best prediction of pelvic size from body weight both during growth and accross different breeds was given by an exponent of about o. 4 . In MONT!rFtO'S (ig6g) study of the relationship between calf weight, dam weight and calving difficulties, precisely the same value of 0 . 4 was found to give the best descrimination between dams with difficult and easy parturitions. Moreover, the effect of changing from nulliparous to parous females was to increase M ON TE IRO ' S index of calving difficulties by o.i. In non-logarithmic terms this is equivalent to an increase of about 10 p. 100 , which is again similar to the difference in pelvic size between parous and nulliparous females found in the present data ( 9 -15 p. 100 in pelvic height, and 3-12 p. 100 in pelvic width). The implication is that, in Morr2!nto's index of calving difficulties, dam weight to the power 0 . 4 was functioning as an estimator of pelvic size. Thus the ratio of calf weight to pelvic height or pelvic width or their geometric mean (but not their product) should also serve as an index of calving difficulties. Calving difficulties would then depend on the ratio of calf weight to a linear dimension which of necessity increases very much more slowly than body weight, so that calving difficulties must be expected to be greater for larger breeds. Selection to decrease calving difficulties might therefore aim to increase the ratio of pelvic size relative to calf birth weight. A more attractive alternative would be to use pelvic size relative to body weight in the form (pelvic size)/(body weight) 1 -4 &dquo; or with separate exponents for height and width. Such an index could be used irrespective of age and breed and with a 10 p. 100 correction for the effect of a first parturition. A restricted selection index may be necessary to prevent selection reducing body weight but no valid assessment can be made without information on heritabilities and genetic correlations both within and between breeds. Another conclusion also appears to be that a linear pelvic measurement is more relevant and critical in relation to calving difficulties than is the area of the pelvic opening. -All conclusions from the present data are very tentative, but they point to the potential value of an index based on pelvic size and body weight and hence to the need for more accurate estimates of both the allometric and interbreed relationships of pelvic size and body weight.

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Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 × 102 to 3.3 × 106 per g. The organisms most frequently isolated were Clostridium subterminale, C. sordellii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridum. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content. The obligately anaerobic bacteria have two principal habitats in nature, the alimentary tract of animals and man and the soil. Those found in the alimentary tract are primarily nonsporing rods and cocci (7,11,12), whereas those from soil are sporeformers. However, the obligate anaerobes of the soil have received little attention, except for special physiological groups. The nitrate and sulfate reducers, the flax retters, the nitrogen fixers, cellulose digesters, and methane formers have all been investigated (15,16) as well as the pathogenic clostridia (14), but the occurrence of obligate anaerobes not falling into these groups has largely been overlooked, although it seems generally recognized that they are an integral part of the soil microflora. Moreover, as Garcia and McKay (4) have pointed out, we have but little information on the relationship of soil factors and the occurrence of soil bacteria. In this investigation, we determined the relative numbers of facultative and obligately anaerobic bacteria in 21 soil samples, isolated and where possible identified to species the most commonly occurring anaerobes, and endeavored to correlate the properties of some of the soil samples with the presence of Clostridium botulinum and C. tetani. MATERIALS AND METHODS The source, pH, and organic content of the soil samples are shown in Table 1. They were obtained during the summer of 1973 by removing the top 2 to 5 cm and sampling the soil to about 15 cm and were transported in polyethylene bags to the laboratory, where they were transferred to parchment bags (2) for sufficient drying to allow seiving and sampling. Most probable number (MPN) counts were carried out by adding 1-g samples of each soil specimen to 9 ml of 1% gelatin solution, pH 6.9, dispersing with the aid of a Vortex mixer, and making decimal dilutions in the same solution. One-milliliter amounts of each dilution were inoculated, anaerobically under CO,, into five tubes of cooked meat-glucose medium (6) which were incubated anaerobically for 3 weeks. Growth was determined by microscopic examination. When growth first became evident in the MPN tubes, plates of Trypticase soy or of brain heart infusion agar were surface inoculated from tubes of the three higher dilution series showing growth and were incubated anaerobically in Brewer jars at 30 C to provide isolates for the determination of the proportion of facultative and obligate anaerobes in each specimen, as well as for the identification of the strains of obligate anaerobes. To obtain isolates of the slower growing bacteria, plates were also inoculated from the MPN tubes at the end of the 3-week period of incubation and incubated anaerobically. Isolated colonies were picked without selection from the isolation plates and deeply inoculated into tubes of meat infusion semi-solid medium (beef infusion with 1% Trypticase, 0.2% glucose, 0.3% agar, pH 6.8 to 7.0) which were incubated at 30 C aerobically. Those strains growing up to and on the surface of the semi-solid medium were designated as facultative anaerobes and those not growing up to the surface as obligate anaerobes. Occasionally, strains were en- countered that grew to the surface but not above it. Such strains were surface inoculated to blood agar plates that were incubated aerobically at 30 C. Growth on such plates was taken to indicate that the strain was a facultative anaerobe. Identification to species of the obligate anaerobes was carried out by the methods of Holdeman and Moore (6). In preliminary experiments, decimal dilutions of soil were made and aerobic and anaerobic plate counts were carried out on four soil specimens using two media and three plates per set and by incubating the cultures at 30 C until further incubation of the aerobic plates was rendered useless by the overgrowth of the agar by molds. This was usually 2 to 4 days, and incubation of the anaerobic plates was terminated at the same time, and the colonies were counted. Two media were used, brain heart infusion agar and a medium containing 0.1% peptonized milk and 1.5% agar. The latter was similar to the medium found (8) to give highest counts of aerobic bacteria from soil, except that we did not use the anti-fungal agent actidione because of lack of knowledge of its action on anaerobic bacteria. Demonstration of the presence of C. botulinum and C. tetani in these soil specimens was determined by inoculating 10 1-g samples of each specimen into tubes of cooked meat medium and incubating at 20 to 22 C for 5 to 7 days. Five tubes were inoculated instead of 10 for specimens A, B, and C. After incubation, the tubes were frozen and held in frozen condition overnight to reduce non-botulinic deaths (1). The contents were then thawed and centrifuged. Two mice were injected intraperitoneally with 0.3 ml of the supernatant fluid from each tube and were held for 3 days. If the mice died, the experiment was repeated using culture fluid to which had been added C. botulinum antitoxin, types A or B. Occasionally, the toxic material in the cultures could not be neutralized by types A and B antitoxins; in such cases, neutralization with other antitoxins, those to C. perfringens, C. septicum, C. tetani, and to C. botulinum C, IQ, E, F, and G, was attempted. Neutralization of the toxic supernatant fluid was achieved in each case. The statistical significance of the difference between two means was made by using a two-tailed Mann-Whitney U test, a nonparametric test. The hypothesis that two means were the same was rejected at the P = 0.05 level. RESULTS AND DISCUSSION The results of the experiment using nutritionally rich and nutritionally dilute media under aerobic and anaerobic conditions are given in Table 2. The short incubation time used in this experiment, 2 to 4 days, was necessitated by the overgrowth of the aerobic plates by filamentous fungi. Consequently, colonies of only the more rapidly growing organisms could be counted and the aerobic counts reported here are considerably below those of Larkin (8), who used a similar dilute medium with a fungus inhibitor and an incubation period of 10 days. Nevertheless, the results of the comparison of the two media show that the richer medium was definitely better for enumeration of anaerobes, probably a reflection of the relative inefficiency of anaerobic metabolism in obtaining energy from dilute nutrient. The MPN count and the proportions of facultative and obligate anaerobes isolated from each soil sample are given in Table 3. The MPN count, rather than the plate count, was used for estimating the anaerobe population because of the superiority of cooked meat medium for stimulating germination of anaerobe spores. The MPN count includes both facultative and obligate anaerobes and extends over a considerable range, from fewer than 700 per g for soil 23 to more than 6 x 106 for soil 9. Repetition of the MPN procedure for soils 15 and 23 yielded much the same results. Of the 576 strains isolated under anaerobic conditions in this study, 40.3% were obligate anaerobes. The proportion of obligate anaerobes isolated from the various soil samples varied considerably, ranging from <3 to 85%. The obligate anaerobe population (MPN x percentage of obligate anaerobes) varied from 0.00027 x 106 to 3.3 x 106 per g. This range is considerably wider than that reported by Gibbs and Freame (5), who determined the clostridial population of six soil samples, using "differential reinforced clostridial medium," and who found counts ranging from 0.035 x 106 to 1.2 x 106. Thayer (17) found slightly more than 105 clostridia per g in a Texas highplains shortgrass prairie soil, with only a slight drop in the clostridial count from the surface to 40 cm. The identity of the 232 strains of obligate anaerobes isolated in this study, and the soil specimens from which they were isolated, are given in Table 4. No effort was made to isolate and identify all the species of anaerobes in these soil samples, only those present in greatest numbers. Isolation from soil specimen 11 was carried out twice, resulting in a larger number of isolates and more species of clostridia than the other soil specimens. All strains of obligate anaerobes were clostridia; either spores were demonstrable microscopically or the organisms were large gram-positive rods withstanding heating at 70 C for 10 min. Clostridia that did not appear to fall into any of the species listed in the eighth edition of Bergey's Manual of Determinative Bacteriology were isolated from 17 soil specimens and made up about onethird of the isolates. Finding C. subterminale, C. sordellii, C. sporogenes, C. bifermentans, and C. perfringens among the anaerobes from the soil was not surprising, for these organisms are frequently encountered elsewhere, probably often from dust contamination. However, C. indolis and C. mangenoti were isolated more frequently than was expected. The few previous isolations of C. indolis have been mostly from clinical specimens. C. mangenoti is probably ubiquitious, for it has previously been reported from marshy soil from the Ivory Coast, from soil of Saigon, Sicily, and Indochina, as well as from a case of Madura foot in Tchad and the liver of a sheep in France (13). In general, the findings in this study are in some contrast to those of Matches and Liston (9) who investigated the mesophilic anaerobes of marine sediments of Puget Sound, for these workers found three-fourths of their isolates to fall into three species, C. perfringens, C. bifermentans, and C. novyi. Because the methods used for isolation and identification in that study and this were much alike, the difference in the organisms found probably reflects a real difference in the anaerobic flora of the two sites. Type A strains of C. botulinum as determined by antitoxin neutralization tests were demonstrated in 6 of 21 soil samples, a proteolytic strain of type B in one, and types E and F in one. All type A strains were from samples taken west of the Missouri River, a finding in accord with those of Meyer and Dubovsky (10) who found type A strains predominantly in soils from the western United States. All the soil samples in which C. botulinum types A and B were demonstrated in this study were neutral or alkaline in reaction (average pH 7.9 compared with an over-all average of 6.3), were low in organic matter (average 1.4% compared with 4.09), and had low anaerobic MPN counts (0.83 x 106 compared to 1.22 x 10" per g). This association between the occurrence of C. botulinum types A and proteolytic B and neutral to alkaline soils was statistically significant at the P = 0.001 level; the association of these organisms with soil of low organic matter content was also significant (P = 0.005), but that with low anaerobic MPN counts was not. C. botulinum types E and F were demonstrated only in one sample, an acid soil from the Olympic rain forest which had a pH of 4.5, 13.7% organic matter, and an anaerobic MPN count of 0.13 x 10". The two soil specimens in which C. tetani was demonstrated had intermediate pH values, 5.5 and 6.3, with organic matter contents of 7.5 and 5.3% and anaerobic MPN counts of 0.24 and 1.6 x 10. (Table 5). Dubovsky and Meyer (3) demonstrated C. tetani in 19 of 2,379 samples of soil and vegetables that they examined. They found this organism in 17 soil samples from the United States; 16 were from 397 specimens taken east of the Mississippi and one from 991 specimens from the western United States. They concluded that this organism was to be found in virgin and uncultivated forest soil where plant material is undergoing fermentation and decay. Neither C. botulinum nor C. tetani was demonstrated in or isolated from six soil specimens from Virginia or three from Costa Rica. Most of the other clostridial species did not seem to be regionally distributed, except that C. bifermentans and C. sordellii were isolated TABL 5. Demonstration of C much more frequently from eastern soils than from western, afid C. mangenoti was isolated only from soils of pH 5.7 or lower. The reasons for the association of certain clostridia, such as C. botulinum type A and C. mangenoti, with soil of certain characteristics are not known. Although the association with alkaline and acid soils, respectively, seems clear, this may be more than the simple effect of the level of hydrogen ion concentration. The complexity of soil structure allows the establishment of a multitude of different microenvironments in a small volume and, consequently, the existence of a complex microbial population. Nevertheless, the restriction of these two species to soils of different levels of acidity indicates that some simple parameters may be of importance in determining the make-up of the microbial population, at least for the anaerobes.

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Production of Aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus Isolated from Market Pecans One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus. The production of aflatoxin appears to be limited primarily, if not exclusively, to strains of Aspergillus flavus and Aspergillus parasiticus (4). Investigators have noted differences in both the types (B1, B2, G1, G2, etc.) and quantities of aflatoxins produced by A. flavus andA . parasiticus species. It has been reported (4) that 82% of A. parasiticus isolates produced both aflatoxins B1 and G1. Among the A. flavus strains, the largest group produced only aflatoxin B1, but a number also formed both B1 and G1. Several other reports have suggested that A. parasiticus are the most active aflatoxin producers (2,5). Numerous instances of isolations of both A. These investigators reported that A. flavus isolates out-numbered A. parasiticus by four to one, but they did not determine the aflatoxinproducing potential of the isolates. In this study, 148 isolates ofA. flavus and A. parasiticus from 5,608 pecans sampled in Georgia and Chicago markets were identified, and aflatoxin production was quantitatively determined. MATERIALS AND METHODS Isolation and identification. Pecan halves or pieces were disinfected before plating by immersion for 2 min in a solution consisting of 20 ml of 5% sodium hypochlorite and 20 ml of 95% ethanol in 60 ml of water. Georgia pecans were plated on rose bengal-streptomycin agar prepared as described by Tsao (8), except that streptomycin sulfate was added at the level of 0.06 g/liter. All of the other pecans were plated on the Botran modification of Martin's rose bengal-streptomycin medium devised by Bell and Crawford (1). All plates were incubated at room temperature and examined periodically for 2 to 3 weeks for the presence of members of the A. flavus group. Species of that group were transferred to malt extract agar slants for storage and subsequent identification. Assay for aflatoxin production. All isolates identified as A. flavus or A. parasiticus were assayed for ability to produce aflatoxins by inoculating 106 conidia of each isolate into 250-ml Erlenmeyer flasks containing 50 ml of yeast extract (2%)-sucrose (20%) (YES) media and incubating at 25 C for 7 days. Aflatoxin was extracted by adding 50 ml of chloroform to each flask and shaking for 15 min in a gyrorotary shaker. After separation in a 250-ml separatory funnel, the lower (chloroform) layer was drained through Whatman no. 1 filter paper into a 500-ml boiling flask, and the upper (media) layer was returned to the original 250-ml Erlenmeyer flask. This extraction procedure was repeated three times. The combined chloroform extracts were evaporated to a small volume on a rotary vacuum evaporator, quantitatively transferred to a volumetric flask, and adjusted to exactly 10 ml for quantitation by thin-layer chromatography. The concentrated extracts or appropriate dilutions were spotted on Adsorbosil-1 (0.25 mm thick; Applied Science Laboratories, State College, Pa.) thin-layer chromatographic plates along with standard aflatoxin solutions of known concentrations (Southern Utilization Research and Development Laboratories, U.S. Department of Agriculture, New Orleans, La.) and developed in chloroform-acetone (88:12). Aflatoxins were estimated by comparing the intensity of the fluorescence of sample spots with standards with a fluorodensitometer (365-nm excitation filter, 425-nm emission filter). RESULTS AND DISCUSSION Ninety percent of the 148 isolates ofA. flavus and A. parasiticus collected from market pe-581 KOEHLER, HANLIN. AND BERAHA cans were A. flavus, whereas only 10% were A. parasiticus. A total of 5,608 pecan halves were plated, 2,758 from Chicago markets and 2,850 from Georgia markets. Of the 148 isolates ofA. flavus and A. parasiticus found, 47 were from samples collected from Georgia markets, whereas 101 isolates were from Chicago market pecans. The percentage of internal invasion by these species was 7.3% in pecans from Chicago markets and 1.7% in those from Georgia markets. Growth and aflatoxin production of all 47 Georgia isolates and 101 Chicago isolates were determined in YES media. No significant differences in growth were observed ( Table 1). The average dry weight of the mycelia differed less than 0.1 g between the two species. In addition, the average weight of all the nonproducing isolates (1.53 g) was identical to the producing isolates (1.47 g). Of the 148 A. flavus and A. parasiticus isolated from pecans, 57% produced one or more aflatoxins when grown 7 days in YES media. All aflatoxin-producing isolates produced primarily aflatoxins B1 and G,, Aflatoxin G1 production was always associated with the production of aflatoxin B1 (Table 2). In no case did aflatoxin B2 or G2 contribute significantly to the total toxin produced. Overall, 57% of the isolates produced some aflatoxin on YES media. Over 93% ofA . parasiticus strains isolated from pecans were capable of aflatoxin production, whereas only 54% ofA. flavus isolates produced aflatoxin under the same conditions. Aflatoxin G, production occurred in a much higher percentage ofA. parasiticus isolates than A. flavus. Eighty-seven percent ofA. parasiticus isolates produced aflatoxin G,, whereas only 10% of A. flavus did so. The average amount of aflatoxin B1 produced by toxigenic A. parasiticus isolates was over twice that produced by aflatoxin-producing strains of A. flavus. A. parasiticus produced larger amounts of aflatoxin G,, but the difference (1.3x) was not as great as for aflatoxin B1. The average ratio of aflatoxin B1 to G1 production by A. flavus (0.3) and A. parasiticus (0.5) did not differ significantly due to the wide variation occurring in this ratio. The B%-G1 ratio in A. flavus species varied from a maximum of 2.8 to a low of 0.1, whereas the ratio in A. parasiticus ranged from 1.9 to 0.2. Although the origins of all of the pecans collected in the Chicago market area were known, the majority of the samples were from only three states, Georgia, Alabama, and Oklahoma. Insufficient numbers of isolates were available from other areas to make any valid comparisons. Pecans originating from Georgia APPL. MICROBIOL. yielded a considerably lower proportion of isolates capable of aflatoxin production than those from the other regions (Table 3). In addition, those Georgia isolates that were aflatoxigenic produced lower average quantities of aflatoxin B1 (1.7 mg/50 ml) than isolates from the other regions. Oklahoma pecans, whose toxigenic isolates had the highest overall yields of aflatoxin B, (3.4 mg/50 ml), included an unusually high percentage of A. parasiticus isolates. The 17% rate of isolation of A. parasiticus was over three to four times that of other regions. This in itself would tend to cause higher average aflatoxin production, since, generally, A. parasiticus isolates are stronger producers than A. t~avus (Table 1). Six isolates of Aspergillus tamarii, another member of the A. flavus group, were isolated from market pecans but none of these produced any aflatoxins when grown on YES media. An Aspergillus oryzae strain isolated from pecans also failed to produce any aflatoxins.

v3-fos

2019-03-30T13:07:07.130Z

1975-11-01T00:00:00.000Z

86327679

s2

Detection of Salmonellae in the Environment The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Mountain may serve as an ecological niche for the survival and transmission of salmonellae. The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Mountain may serve as an ecological niche for the survival and transmission of salmonellae. Recent studies (2,3) have shown that the salmonellae are widely distributed in the environment and pose a potential threat to the health of humans and other animals. Salmonellae have been recovered from rivers and streams in remote areas devoid of any apparent human fecal contamination, as well as from urban streams and recreational lakes subjected to fecal pollution from humans and other animals. Fair and Morrison (4) concluded that unpolluted, potable surface water sources do not exist. Cherry et al. (2) demonstrated the ease of recovering salmonellae from urban and rural streams and suggested that these organisms may be a better indication of a health hazard than the standard coliform count. It has also been suggested by Cherry et al. (3) that these organisms, whose habitat has been thought to be the intestinal tract, may in fact be freeliving in nature. The present study was conducted to determine the incidence of salmonellae in a harsh and unfavorable environment as compared with that of a lush hardwood forest characterized by abundant moisture and moderate temperatures. MATERIALS AND METHODS Sampling sites. Environmental samples were collected from two locations within the metropolitan Atlanta area. The first area sampled was Lullwater Estate, which is a mature hardwood forest located on the Emory University campus. It is well protected against casual human intervention. The forest provides an excellent habitat for diverse populations of both warm-and cold-blooded vertebrates, as well as many invertebrates. Samples were taken from three small streams, a spring, and a pond edge. The second sampling site was the top of Stone Mountain, which is the largest of a series of granite outcrops near Atlanta, Ga. The top of the mountain receives heavy human usage as the central attraction in a state park. The total number of feral animals which the environment will support is small in comparison to a forest. Samples were collected from 13 different weather pools, 10 of which were sampled several times over a 3-month period. Only one of the pools remained wet during the entire sampling period, and it was created from the runoff from a large air-conditioning unit. All other pools exhibited intermittent drying. Collection of samples. Environmental samples were collected in or near standing or running water and from weather pools. Samples consisted of water, algae, mosses, tree roots, grasses, and soil. These were collected in sterile, screw-capped, 35-mm film canisters which held either 30 ml of water, 50 g of soil, or 5 to 25 g of vegetation depending upon its type and moisture content. The samples were returned to the laboratory within 3 h of collection and placed in enrichment broth for isolation of salmonellae. Cultural methods. For culturing, the samples were placed in 0.5-liter refrigerator jars containing 100 ml of tetrathionate broth and a 1:10,000 concentration of brilliant green dye. They were incubated at 41.5 + 1.0 C for 48 h. The broths then were streaked on brilliant green agar and on bismuth sulfite (Difco) agar plates and incubated at 35 C. Colonies resembling salmonellae were fished from the plates after both 24 and 48 h of incubation and inoculated onto triple sugar iron agar, lysine iron agar, and urea agar slants (all from Difco). All salmonellae isolates were confirmed by further biochemical and serological tests. Representative isolates of each 0 group found at each collection site were definitively serotyped by the National Salmonellae Reference Center at the Center for Disease Control. 764 on March 22, 2020 by guest http://aem.asm.org/ Downloaded from FA methods. Smears of the tetrathionate broth cultures were made on multiwell slides and fixed and stained with Salmonella polyvalent OH conjugates as described previously (8). The enrichment broths were examined at the end of 24 h, and those found negative by fluorescent antibody (FA) were reexamined after 48 h of incubation. Representative colonies from specimens found positive by FA but negative by culture were suspended in sterile 0.85% NaCl and stained with the FA reagent to detect organisms that cross-reacted with the Salmonella conjugate. RESULTS Lullwater Estate. A total of 24 samples of soil and vegetation were collected. The vegetation consisted of mosses, leaves, emergent plants, and algae. Eleven of the 24 samples were positive for salmonellae by FA staining tests. Six of these samples, which were collected from three separate sites, were confirmed as Salmonella by cultural methods. Four Salmonella serotypes, S. typhimurium, S. give, S. infantis, and S. bern, were isolated. No samples were culture positive and FA negative. When the results obtained from examination of soil were compared with those derived from the testing of vegetation, neither showed any apparent advantage for harboring salmonellae (Table 1). However, when the samples were classified as to their visible moisture content and the results were compared, a decided difference between the wet and dry samples was noted ( Table 2). No salmonellae were isolated from the dry samples, whereas 10 of the 12 wet samples were positive for salmonellae by FA, and salmonellae were isolated from six of these. Stone Mountain. A total of 76 environmental samples were collected. Of these, 19 were positive for salmonellae by FA tests. All isolates but one were S. barielly and S. weslaco. S. barielly was isolated from one or more samples from 6 of the 10 sites sampled repeatedly. No samples were culture positive and FA negative. The Stone Mountain samples were classified into the categories of soil, vegetation, and water (Table 3). Of the 58 soil samples, 15 were positive by FA staining and eight of these also were culturally positive. Only one ofseven vegetation samples was positive by either test. Three of 11 water samples were positive by FA tests and one was positive by culture. Although the number of vegetation and water samples is small, there is no apparent difference in the isolation rate among the three types of samples. When the samples are classified according to their moisture content, the results are striking (Table 4). No FAor culture-positive results occurred among the 20 dry samples, but 19 of the 56 wet samples were positive by FA, and 10 of these were confirmed by cultural methods. When the wet samples were classified by type (Table 5), soil and vegetation samples were found to yield almost twice the percentage of positive culture results as did the samples of water, even though the difference was not significant by the chi-square test. The weather pools differed biologically by the presence or absence of macroinvertebrates. Three different types of aquatic microinvertebrates were observed. One was a clam shrimp, another, the endemic fairy shrimp (both in the order Eubranchiopoda), and the third, a single species of water mite of the order Hydracarenida. The isolation rate of salmonellae from specimens collected in pools containing macroinvertebrates was not significantly higher than the isolation rate from pools with no macroinvertebrates. Six of 16 samples collected from pools in which invertebrates were noted were positive for salmonellae by FA tests. Salmonellae were isolated from four of these six samples. Of 26 samples collected from pools with no macroinvertebrates, 11 were positive by FA, and salmonellae were isolated from four. DISCUSSION In previous studies (2, 3) of urban and mountain streams, salmonellae were associated with the water, stream vegetation, and stream bottom substrates. They were not found in other soil samples, forest litter, aquatic vertebrates or invertebrates, or terrestrial invertebrates. Cherry et al. (3) found that salmonellae survived on contaminated vegetation taken from Lullwater streams for at least 1 week, and they suggested that the surface of aquatic plants may provide a good substrate for salmonellae multiplication. In 1943, Zobell (9) demonstrated that certain aquatic bacteria adhere so tenaciously to solid surfaces that they are not dislodged by washing with running water or by staining procedures. Hendricks (5) suggested that the increased recovery rate of salmonellae from stream bottom sediments versus surface water may be due to adsorption of organisms to sand and clay particles. More recently, Hendricks (6) confirmed that significant numbers of enteric bacteria were adsorbed on the surface of collection bottles, thereby reducing the enteric population within the liquid. Absorption of organisms to soil particles could account for negative results on water samples taken from weather pools where salmonellae were isolated with ease from substrate samples. The two sampling sites were selected because of their dichotomous nature. At the Lullwater site, the forest canopy reduces the range of fluctuation of air movements, and of humidity, temperature, and photogenic effects. At Stone Mountain, the intensity and range of fluctuation of environmental effects on the bare rock are profound. Also, wildlife associated with the two sites is quite different, being much more numerous and diverse in the Lullwater forest than on Stone Mountain. Another difference is the abundance of flowing water within the Lullwater site, whereas the summit of Stone Mountain is characterized by pools of stagnant water which are subjected to intermittent drying. A major difference noted between the results obtained from the two sites was the diversity of salmonellae isolated from Lullwater as compared to Stone Mountain. Four different Salmonella serotypes were isolated from six culturally positive samples obtained during a single survey of the Lullwater area. A total of 76 samples were collected from the top of Stone Mountain on five different occasions over a 3month period. Only two Salmonella serotypes were isolated from the 10 positive samples. One of these, S. weslaco, was isolated only once from a single sample. All other isolations were S. barielly. Only two sample characteristics were noted at the time of collection: the predominant material of which the sample was composed and its moisture content. The composition of the sample material appeared to have no significant effect on detection or isolation of salmonellae in samples from Lullwater (Table 1) or Stone Mountain (Table 3). However, the presence or absence of water in the sample had a significant effect on the detection of salmonellae (Tables 2 and 4). Only one of 32 dry samples from both locations was positive by FA staining, whereas 25 of 68 wet samples were FA positive. Salmonellae could not be cultured from any of the dry samples, whereas these organisms were isolated from 18 of the 25 wet samples which were positive by FA staining. The appearance and disappearance of the salmonellae during the wet and dry periods provokes speculation on what occurs in these small weather pools. Do the salmonellae die off during the drying process and are they reintro-duced each time it rains, or do they somehow survive the desiccation and reappear once they are rehydrated? Recently, Mossel et al. (7) emphasized the importance of resuscitating stressed Enterobacteriaceae before inoculating them into a selective medium. Bissonnette et al. (1) have shown that Escherichia coli exposed to river water for varying periods of time acquired a nonlethal injury that prevented their growth on selective media. This injury, however, could be rapidly repaired by placing them in a nutritionally rich, nonselective medium. The absence of salmonellae in dry samples should be investigated further by rehydrating the samples and incubating them in nonselective pre-enrichment broth before placing them in the inhibitory tetrathionate broth. Generally, microbiologists have assumed that Salmonella-contaminated water supplies were the result of fecal contamination by either humans or other animals. It appears that most creeks, lakes, and remote mountain streams contain these organisms. Consumption of the contaminated water by wildlife, domestic animals, or by humans presumably completes one phase of the cyclic movement of these organisms in nature. Our studies suggest that this view may be a simplism, and our results indicate the need to define the role of aquatic vegetation, benthic sediments, and, perhaps, other factors in maintaining salmonellae in the environment. The studies reported here have shown that even a harsh environment such as that found in small weather pools at the summit of a granite outcropping may serve as an ecological niche for the survival and transmission of salmonellae.

v3-fos

2018-04-03T01:05:59.443Z

1975-02-01T00:00:00.000Z

28004670

s2

Characterization of Hydrogen Sulfide-Producing Bacteria Isolated from Meat and Poultry Plants A survey of the types of aerobic organisms able to produce H2S on peptone iron agar (Levin, 1968), and commonly occurring in meat and poultry plants, revealed that these could be divided into four distinct groups. The ability of representative strains of each type to grow at low temperatures and cause off-odors on chicken muscle was examined. The results are discussed in relation to the role of these organisms in the psychrophilic spoilage of meat and meat products. Recent work on the bacteriological spoilage of flesh foods has indicated that only a fraction of the total microflora is capable of producing the organoleptic changes associated with spoilage (2,11). Several workers have emphasized the importance of attack on low-molecular-weight compounds (1, 13) and others indicated that off-odors are evident before extensive proteolysis takes place (18). In particular, the association of sulfide-producing organisms with spoilage has been noted (7,11,15,19,21). There is no concise data in the literature about the genera and species of organisms that produce sulfides, and which commonly occur on meat and poultry. This study was undertaken to provide adequate characterization of these organisms as a basis for further detailed studies. MATERIALS AND METHODS Origin and isolation of strains. The sources of the strains are given in Table 1. Peptone iron agar (PIA; Difco) developed by Levin (19) was used as a direct diagnostic plating medium for the recovery of hydrogen sulfide-producing strains. These formed black colonies due to the formation of ferrous sulfide. Plant surfaces were sampled by the swab-rinse technique and poultry carcases by the cut and rinse method described by Patterson (23). Other product samples (fresh and aged beef) were homogenized in saline peptone solution using a Colworth Stomacher (A. J. Seward and Co. Ltd., London) as described by Sharpe and Jackson (25). Serial 10-l dilutions were prepared from this homogenate. Pour plates of various dilutions were made in PIA and incubated for 3 (30) and motility was determined by the hanging drop technique. Size, shape, and cellular arrangement of stained and living preparations were noted. Representative strains of each group of organisms were stained for flagella by the Leifson technique (22). Best results were obtained by allowing a contact time of 30 s after appearance of a fine precipitate on the slide. Colonial morphology and broth characteristics were described after 3 days at 22 C on NA or in NB (30). Biochemical tests. All tubes were inoculated with 0.1 ml of a 24-h NB culture, plates and slopes being streaked from a similar source. The temperature of incubation for all tests was 30 C, the period being varied according to the test. Details of the tests employed are shown in Table 2. Carbohydrate reactions. The ability of strains to produce acid and/or gas from glucose, maltose, sucrose, and lactose was examined in Andrade peptone water. The carbohydrate solutions were sterilized by filtration and added to the basal medium to give a final concentration of 1.0% (wt/vol). Sole sources of nitrogen. Sulfur-containing nitrogenous compounds known to occur in meats (methionine, cystine, glutathione, taurine) were tested as sole sources of nitrogen. Filter-sterilized solutions of each N source (0.01 M) were added to a basal medium chosen to simulate the carbohydrate and salts composition of meat (16 water, and 40 ml of solution C and 10 ml of solution B were added. Computation of results. The results which were useful for differentiation were resolved into 58 features and presented as a table of strains versus features. The data were analysed by the program CLASP by which pairs of strains in all possible combinations were compared in turn. Similarity was defined as nJ/(n, + nd) (n, = ++, nd = + -). Negative matches were not counted (29). Strain numbers (123) were included in the computation; the remaining 36 isolates had identical responses with other isolates. Excision of sterile muscle sections. Sections of sterile muscle were excised from chicken breast (supra coracoid, pectoral proper) using a modification of the technique described by Sharp (24) and Gardner and Carson (10). Breast skin was carefully removed using sterile instruments and the underlying tissue was painted with aged, saturated solutions of crystal violet and brilliant green which were allowed to dry for 2 h. The muscle was not flamed as this procedure was found to cook into the depth of the tissue. The painted portion was sliced away using sterile instruments and large sections of the underlying muscle were excised and placed in sterile petri dishes. The large sections were cut into smaller portions (2 g) using sterile scissors and were stored in sterile screw-capped bottles. Sections were kept at refrigeration temperatures for at least 14 days and were examined visually and olfactorily before use. Five-milliliter quantities of nutrient broth were added to 10% of the sections and incubated at 22 C as sterility controls. Ability of representative strains to produce offodors. The ability of 15 pure cultures representing the various groups of organisms ( RESULTS The results of the computer analysis (summarized in Table 3) show mean similarities within and between the groups formed. The organisms within each group were recognized as follows: group 1, Pseudomonas putrefaciens; group 2, Proteus sp. (Proteus mirabilis and Proteus vulgaris); group 3, Citrobacter freundii; group 4, coryneform types. Summary characterizations of the groups are given in Table 4. In this survey, 48 of the 159 cultures isolated were strains of P. putrefaciens. These formed a very distinct group (9 = 87.0). The distribution of the strains can be seen in Table 1. It is of interest to note that all 26 isolates from spoiling steak stored at 5 C were P. putrefaciens. Other sources included frozen and chilled eviscerated chickens, poultry plant personnel, and equipment. The P. putrefaciens strains all grew well at 5 C and much more quickly at this temperature than the other H2S-producing types isolated. All five representative strains caused a typically sulfide-like spoilage odor when grown on sterile chicken muscle. Off-odors were detectable organoleptically after 7 days storage at 5 C. The group 2 strains all produced the characteristic swarming colonies of Proteus species. The group (53 strains) was distinct with an intragroup mean similarity of 83.2. All but three of the strains were recognized as P. mirabilis. The remaining three correspond to the description of P. vulgaris (6) and were grouped together at one extreme of the Proteus group. Growth at 5 C was recorded for 29 of 53 Proteus strains and four representative isolates developed faster than the Citrobacter types at this temperature, although much slower than P. putrefaciens. Despite the relatively slow growth rate at low temperatures, all Proteus strains tested produced the same characteristic odor. This was not recognized as sulfide-like but was described as "caramel" or "burnt sugar." (These strains liberate H2S when cultured at 5C.) The strains contained in group 3 were recognized as C. freundii (36 organisms, 9 = 82.2). Two anaerogenic types were included. Again, in relation to P. putrefaciens, these strains grow slowly at 5 C and only slight sulfide-like odors were detectable with two of the four representative strains tested after storage of inoculated chicken muscle for 14 days at 5 C. The coryneform organisms formed the least I distinct group; 19 strains were included in a group with an intragroup mean similarity of 64.1% S. The strains grew only very slowly if at all at 5 C and representative types did not produce any detectable off-odor after incubation on sterile chicken muscle for 14 days at 5 C. Seven of the eight strains isolated from beefburgers were coryneform types. It was not, however, possible to test these for spoilage ability against beefburger as sterile sections of this product could not be obtained. In view of their slow growth rate at low temperatures, members of this group are unlikely to be important spoilage agents of refrigerated meat products. DISCUSSION The results obtained indicate that a restricted range of bacterial types capable of producing detectable amounts of hydrogen sulfide on PIA occurs in the environs of meat and poultry plants. Of these, P. putrefaciens is recognized as potentially the most important spoiler. Both C. freundii and coryneform types are discounted as troublesome at 5 C. At higher temperatures, however, it is likely that these might develop much more rapidly with the subsequent onset of sulfide-like odors. Proteus strains provide apparently anomalous results; although developing only slowly at refrigeration temperatures, they produce a characteristic spoilage of chicken muscle which was not recognized organoleptically as sulfide-like. The role of P. putrefaciens as a spoilage organism is well documented. A number of workers have noted the presence of this organism and its role in the spoilage of chicken carcases (4,5). Lea and co-workers (17), however, concluded that P. putrefaciens and a pigmented pseudomonad produced changes little greater than those observed during sterile autolysis. All P. putrefaciens strains examined in this study produced potent off-odors from chicken muscle. P. putrefaciens has also been widely implicated in the spoilage of fish and fishery products (7,11,19). Other Pseudomonas strains have been reported in the literature as producers of sulfides and sulfide-like odors. Nichol et al. (21) recorded the formation of sulfmyglobin in prepacked beef by P. mephitica, but showed that sulfides were only produced under conditions of low oxygen tension. P. perolens was shown to produce a number of volatile sulfides when grown on sterile fish muscle (20) whereas P. putida, Pseudomonas group 1 (26), P. fragi, Pseudomonas group II, and Pseudomonas group IIJAV types possibly similar to P. putrefaciens also caused sulfide-like odors in fish muscle (11). In this study, the only sulfide-producing pseudomonad recovered from sources within meat and poultry plants was P. putrefaciens. Members of the Enterobacteriaceae have been shown to constitute a considerable portion of the flora of poultry carcases when the temperature is allowed to rise to 10 to 15 C (5). This study confirms that normal refrigeration temperatures permit at most only slow growth of the Citrobacter and Proteus types isolated. However, the latter are able to cause a characteristic off-odor on chicken muscle at 5 C in pure culture. The inherently slow growth rate, however, probably limits development of Proteus species in competition with more psychrotolerant organisms. Studies are being carried out to determine the number of cells of Proteus species required to cause off-odors in pure culture. Their development and the nature of spoilage in association with the faster growing P. putrefaciens is also of interest. It is possible that the odiferous compounds produced by Proteus strains are detectable organoleptically at extremely low levels and that relatively few cells are required to attain these levels.

v3-fos

2020-12-10T09:04:20.882Z

1975-07-01T00:00:00.000Z

237231680

s2

Deterioration of High-Moisture Corn Two small, leaky silos were filled with normal high-moisture corn (HMC), and two with HMC severely infested by Helminthosporium maydis. Counts of mesophilic bacteria, lactobacilli, coliforms, yeasts, and molds were made on corn samples as received and periodically thereafter during 220 days of storage. Temperature and gas levels also were monitored. Sequential changes in the populations of lactobacilli, yeasts, and molds were determined during spoilage of HMC. These population changes were compared on the basis of the variables encountered in the present study as well as with the results of previous studies conducted on normal HMC stored under adequate conditions. Heavy infestation by H. maydis had no appreciable effect on HMC preservation. Numerous studies are available on the roles that fungi play in the deterioration of stored corn (2,6,19,29), the prevalence of aflatoxins (21,27,30,34), and other toxic fungal products (7, 8, 20; 32, 36) in infested corn, and changes occurring in fungal populations during quality loss in corn (4,9,31). Few data are available, however, on alterations of the bacterial and yeast flora during storage of shelled high-moisture corn (HMC), and only a few workers (4,5,11) have examined the complex interactions of molds, yeasts, and bacteria that occur during HMC storage. For some of these studies (4,5), special attention was given to restricting gas interchange between the interior and exterior of the silos so that normal storage conditions, simulating those on the farm, could be duplicated. In one study (11), we attempted to induce spoilage of HMC by regulated periods of aeration, but the degree of aeration was insufficient to cause spoilage. Recently, we succeeded in obtaining deterioration of HMC at rates that approached those sometimes observed on the farm in defective storage units; these investigations are reported herein. In addition, comparisons were made of the genera and species recovered in the present study with those recovered in previous studies (4,5,11); the effects of heavy infestation by Helminthosporium maydis also were examined. IJournal MATERIALS AND METHODS Four metal storage structures were used (4); each was 1.8 m diameter by 3.4 m high. The structures had been damaged by high winds and contained numerous small air leaks. Two silos were filled (about 225 bushel/silo) on 6 October with 21.7% moisture, 9.9% damaged, grade 4 corn; the corn had been infested by the fungus causing Southern corn leaf blight (H. maydis). Two other silos were filled with 24.3% moisture corn of high quality. Sampling protocols and temperature and gas measurements were performed as described previously (4). Viable cell counts were made on a plate count agar (Difco) for aerobic bacteria; desoxycholate agar (Difco) for coliforms; tomato juice agar special (Difco), supplemented with 0.1% sorbic acid, and LBS agar (BBL) for lactobacilli; and Littmans Oxgall agar (Difco), supplemented with 30 ,g of streptomycin per ml, for yeasts and molds. Colonies were counted after incubation at 30 C for periods of 2 days for aerobic mesophilic bacteria and coliforms, 3 days for lactobacilli (incubated in CO2 GasPaks, BBL), and 4 days for yeasts and molds. Colonies for identification were randomly selected from plates representing alternate samples from the lower levels of each structure. Samples from the lower levels were used because they were more representative than samples from the upper levels of the microbial changes that would occur during storage of HMC in conventional silos. After purification on the isolation medium, lactobacilli were maintained in Micro Assay culture agar (Difco) deeps, yeasts on malt extract agar (Difco) slants containing 0.5% added agar, and molds on Czapek dox agar (Difco) slants. Cultures were incubated at 30 C for 48 h (5 days for the molds) and stored at 5 C. Coliforms were transferred to Trypticase soy agar (BBL) slants; fewer than half the cultures remained viable after storage 103 for about 6 months, however, and identification of the coliforms to species was not attempted. Extensive tests were conducted on 1,008 isolates: 308 lactobacilli, 357 yeasts, and 343 molds. For identification, lactobacilli were transferred to fresh Micro Assay culture agar deeps and then to 10 ml of brain heart infusion (Difco) broth in 15-ml dropping bottles, which were incubated for 48 h at 30 C and used as inocula. One drop of inoculum was used for each tube of medium; solid media in plates were inoculated (16 cultures/plate) with a capillary replicator (10). Data from 82 morphological, cultural, and physiological tests were scored according to the methods described by Lessel and Holt (18). The tests were largely routine (McMahon, M.S. thesis, Iowa State Univ., Ames, 1972), with the exception of the pH determinations in carbohydrate fermentation tests. Final pH determinations were made by using a semimicro-combination electrode; pH values of 5.0 or less, between 5.0 and 6.0, between 6.0 and 7.0, and above 7.0 were scored A, B, C, and D, respectively. Yeast identification (55 different tests) was performed according to the classification of Lodder (23) and culture techniques of Wickerham (35), using the dropping-bottle and capillary-replication techniques where possible (McMahon, M.S. thesis, 1972). Carbon-assimilation tests were recorded as: (i) negative, if, after 2 weeks at 30 C, the broth was clear with no evident sediment; (ii) weak, if there was a slight turbidity or sediment; and (iii) positive, if definite turbidity or sediment was present. Molds were identified to genus by their morphological characteristics. RESULTS AND DISCUSSION Bacterial populations. Counts of aerobic, mesophilic bacteria increased from about 10f/g at the time of ensiling to about 105/g by day 71 (Fig. 1). These increases in numbers were more gradual than those observed previously (4,11). There were few differences between counts of aerobic bacteria made on samples of normal and blighted HMC taken from either level of the four silos, although counts made on samples from one of the silos of blighted corn generally were lower than counts made on the other corn samples. We will return to this point later. In adequately preserved corn (4), numbers of coliforms increased 10to 100-fold during a few days of storage and then declined to fewer than 10/g during the next week or two. In the present study, however, coliform numbers remained high in the upper levels ( Fig. 2) or decreased slowly in the lower levels ( Fig. 3) of the silos. Thus, the presence of appreciable numbers (more than 102/g) of coliforms after 2 weeks of HMC storage at ambient temperature is suggestive of abnormal storage conditions; also, decreases in coliform numbers during storage cannot be used to ascertain adequate storage conditions. Reliable estimates of lactic acid bacteria were not obtained until day 21 after ensiling because of mold overgrowth on tomato juice agar plates; molds subsequently were inhibited upon the addition of 0.1% sorbic acid. LBS agar also was effective in this regard, but it was not used until day 56 of ensiling. Because counts and sequences of lactobacilli were similar for both media, the data were combined for this presentation. Numbers of lactic acid bacteria fluctuated considerably during storage in both levels of the four silos ( Fig. 4; only the lower levels are shown), in contrast to the results of Hesser et al. (11) who reported that lactobacilli maintained fairly constant levels of about 108/g during adequate sealed storage of HMC. The properties of lactic acid bacteria isolated from the corn samples were compared with descriptions in Bergey's Manual of Determinative Bacteriology (3) and data obtained on strains of Lactobacillus spp. isolated from silages (1,11,12,14,15) and other materials (13,26). Identification was aided by preliminary computer grouping (18,22); each strain, however, also was examined feature by feature. Some computer groups were split into subgroups, and many individual strains that did not appear in the computer groupings at high similarity values were later assigned to the groups on the basis of key properties. Detailed analyses of the data are available elsewhere (McMahon, M.S. thesis, 1972). Table 1 shows the numbers of lactic acid bacteria isolated after various times of storage, together with data reported by Hesser et al. (11). The types of lactobacilli isolated within treatment (normal versus blighted HMC) were similar, and the data from each type of treatment were pooled in the table. Lactobacillus plantarum was the most frequent isolate, although it was not as predominant in the normal corn as it was in a previous study (11). About 80% of the strains fermented arabinose strongly and may be designated as var. arabinosus; one culture (var. pentosus) fermented arabinose and xylose. Some variant strains did not coagulate milk, and raffinose was only occasionally fermented. Other strains varied in one or more specific carbohydrate fermentations. L. brevis was the second most prevalent species (Table 1); similar proportions of this species were isolated previously from normal corn in sealed storage (11). In the present study, L. brevis was first isolated on day 35 and remained throughout the storage period with few observable differences in dominance among silos. The delayed appearance of L. brevis is in agreement with the report of Hesser et al. (11) for good quality corn ensiled under adequate conditions. About half the L. brevis isolates were typical, with the exception of failure to hydrolyze arginine. The remaining half were negative in one or more of the following characteristics: arabinose fermentation, growth at 25 and 37 C, and resistance to 60 C for 45 min. In addition to L. brevis, another group of lactobacilli was not encountered until day 35 of storage. This group, intermediate between L. plantarum and L. casei, conformed to Keddie's group 2 (12), except that the cells were longer (up to 4.2 Aim long) and strongly fermented arabinose. Because of the prevalence of such strains in some natural habitats, perhaps another species should be promulgated. L. buchneri was recovered at all sampling dates. For some reason that we cannot explain, very substantial numbers were isolated from three of the four silos on day 93 of storage. L. casei-like cultures (15), isolated mostly during the latter half of the storage period (Table 1), resembled L. plantarum in some respects, but arabinose and melibiose were not fermented. Furthermore, these strains did not ferment lactose, coagulate milk, or reduce litmus; they are reportedly characteristic of poorquality silages (16,17). L. curvatus strains, only a few of which weakly fermented maltose, and L. coryniformis both appeared late in the storage period. The L. coryniformis isolates presented carbohydrate fermentation patterns that did not resemble any Lactobacillus spp. described in Bergey's Manual of Determinative Bacteriology (3); the cells, however, were kidney II VOL. 30,1975 shaped and occurred in pairs, similar to cells described by Abo-Elnaga and Kandler (1). Another group of isolates that did not match some definitive descriptions of lactobacilli (1,3,11,12,14,15), resembled L. sanfrancisco (13,26). Maltose, and sometimes xylose, were the only carbohydrates fermented. Other exacting nutritional requirements observed by Kline and Sugihara (13) were absent, and our isolates occasionally were 30 to 40 Aim long and did not produce C02. L. sanfrancisco was isolated only during the early storage period (Table 1), as were a few strains of Pediococcus cerevisiae that resembled the group 4 of Langston and Bouma (14). When the data obtained in the present study are compared with those of Hesser et al. (11), it is apparent that a more diverse range of species may be characteristic of ensiled HMC in the process of deterioration, relative to less diversity in good-quality ensiled HMC. In both types of HMC, strains of L. plantarum and L. brevis become predominant with time of storage. In deteriorating HMC, L. casei-like strains also seemed to compete favorably; these and several other Lactobacillus spp. were not observed by Hesser et al. (11) and seemed to have been replaced in Hesser's study by L. fermenti (Table 1). Yeast populations. Yeasts play a significant role in the preservation of ensiled HMC. Huxsoll (M.S. thesis, Purdue Univ., Lafayette, Ind., 1961) reported that marked increases of yeasts occurred in deteriorating HMC at moisture contents of 18 to 23%; less marked increases were observed in corn of 23 to 28% moisture. The yeasts grew well at 02 concentrations as low as 0.5%, and at 02 concentrations of about 8%, molds competed with the yeasts. Our data (Fig. 5) showed that, initially, there were about 105 viable yeast cells/g of corn in the lower levels of the storage units. Numbers of yeasts increased during the days 14 to 50 of storage to levels of about 10" to 108/g. The blighted corn contained as few as 0.01 of the numbers of yeasts contained in the undamaged corn, yet the 02 levels in the silos containing the blighted corn were much less than those in the silos containing the normal corn. Thus, contrary to the findings of Huxsoll (M.S. thesis, 1961), the yeasts competed favorably even in structures containing 10 to 15% 02; 02 and moisture levels were not the primary determinants for yeast growth in HMC. The yeast microflora of stored HMC may be divided into two groups: field species and storage species. Of the five species that predominated in our HMC samples (Table 1), Candida DETERIORATION OF HMC 107 parapsilosis and Torulopsis candida were isolated early in the study and were largely replaced after the first week of storage by Hansenula anomala-Candida pelliculosa and C. guilliermondii. H. anomala-C. pelliculosa accounted for 49% of the isolates. C. pelliculosa is essentially a biotype of H. anomala, differing by the production of matted colonies and failure to produce ascospores. C. guilliermondii constituted a substantial proportion of the yeasts present in the damaged corn at the time of ensiling; this yeast became predominant later (during about 1 to 12 weeks of storage) in the normal corn. In both instances C. guilliermondii was gradually replaced by H. anomala-C. pelliculosa; this occurred more rapidly in the damaged corn where the 02 levels were lower than in the normal corn. C. parapsilosis comprised about the same percentage of the yeast flora as was observed previously (11). In contrast to Lodder's description of this species (23), our isolates, except for two strains, all assimilated cellobiose and salicin. Only 4% of the yeasts were identified as Torulopsis candida; none assimilated raffinose or cellobiose. A single strain of C. albidus was identified. Four yeast species identified in a previous study (4) were not encountered in the present study; these seem to have been replaced by three other species (Table 1). Mold populations. Initial mold counts (probably consisting mostly of mold spores) were about 105/g of corn (Fig. 6). Since initial numbers of molds were approximately equal in both normal and H. maydis-infested corn, H. maydis and secondary fungal invaders did not appreciably alter the total mold (spore) population at the time of ensiling; this is contrary to the findings of some other workers (33). In the present study, mold counts reached between 107 and 108/g of corn in three silos during storage. Counts from one silo of H. maydis-infested corn fluctuated widely and were low during winter storage. The differences in total mold counts between the two silos containing blighted corn (Fig. 5) could not be explained, but a similar occurrence for yeast numbers in these two silos (Fig. 4) suggests that some factor(s) restricted growth of both molds and yeasts in the lower level of the same silo. These results are in contrast to those obtained on adequately ensiled HMC, in which mold counts increased from 0 to 2 weeks and then rapidly declined to less than 102/g within 30 days of storage (4). Mold microflora sequences followed similar patterns in both normal and H. maydis-infected corn (Table 1) and were similar to those observed by other investigators (see 6,29). Initially, the field (6) fungi (Fusarium, Mucor, Cladosporium, Alternaria, and Trichoderma) predominated. At this time, storage (6) fungi comprised no more than 4% of the isolates. H. maydis was not isolated from the blighted corn, probably because it was greatly outnumbered by Fusarium spp. (28); in addition, sporulation of H. maydis often is poor, and its isolation is difficult unless infected kernels are surfacesterilized before plating (33). No field fungi were recovered on day 7 after ensiling, excluding Fusarium spp. (consistently isolated through day 56 in two silos) and an occasional Cladosporium isolate. Storage species (Aspergillus and Penicillium spp.) were isolated on a regular basis by day 7 of ensiling. In three of the silos, Penicillium spp. generally appeared in large numbers only during the earlier and later stages of ensiling, while Aspergillus spp. predominated during winter storage. In the fourth silo, both species were present in approximately equal ratios during winter storage; in this silo, which also had lower mold counts and internal temperatures of 4 to 10 C lower than the other three silos, Aspergillus spp. did not multiply rapidly during winter storage. Three morphologically distinct groups of aspergilli and three groups of penicillia were observed, but these were not characterized further. The three groups of penicillia may represent the three species described by Mislivec and Tuite (24,25) in stored corn. Numbers of lactic acid bacteria and yeasts, and the sequences of species, in deteriorating HMC were similar to those reported for goodquality ensiled HMC (4,11). Numbers of molds, especially aspergilli and penicillia, were significantly greater, however, in corn undergoing spoilage. Therefore, as we suspected, molds are the primary factors in deteriorating HMC, even under conditions of low oxygen-high CO2 tension. Bacteria and yeasts play a relatively minor role in deterioration (except as scavengers of 02 in sealed structures) because bacterial and yeast populations were similar for both deteriorating and adequately stored HMC. Damage of corn by H. maydis does not substantially alter the fermentation pattern or susceptibility of the corn to spoilage during sealed storage.

v3-fos

2020-12-10T09:04:12.607Z

1975-10-01T00:00:00.000Z

237229726

s2

Optimum Membrane Structures for Growth of Coliform and Fecal Coliform Organisms The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 μm and a retention pore size of 0.7 μm. Since its introduction as a tentative method for coliform enumeration in the 10th edition of Standard Methods in 1955 (1), the membrane filter has gained wide usage not only for total coliform, but also for fecal coliform, total bacteria, and a variety of other bacterial tests. The unique advantage of the membrane over other test methods is its ability to concentrate and localize bacteria from large sample volumes. Hence, the membrane increases the sensitivity of quantitative bacteriology into the range well below one organism/ml.Once the bacteria are localized, the membrane provides a structure for counterdiffusion of nutrients and metabolic products as well as a "hospitable" growth environment. In these functions, the membrane differs little from the earlier pour-and streakplate methods. The earliest techniques for bacteriological analysis with membrane filters involved direct microscopic examination of bacteria trapped on the membrane surface. Here, the optimum structure required pores smaller than the organisms being trapped for examination, so that they would lie in a single microscopic plane. This surface planar retention facilitated finding the organisms under high-power microscopy. The above requirements evolved naturally to the practice of retaining organisms on the membrane surface for various culture techniques. At that time, not much thought was given to developing an optimal membrane structure for colony growth. The ideal characteristics of a membrane for quantitative bacteriology would appear to be pores small enough to retain bacteria but open enough to provide optimal diffusion of media and a hospitable surface for growth. However, upon examining the variety of bacterial methods utilizing membranes, one finds a considerable range of bacteria sizes, types, and metabolic requirements. These considerations led us to wonder if it was possible to develop membranes which would be especially favorable for the growth ofparticular types of organisms, such as coliform group. The critical step in development of a colony from a single bacterium is the onset of cellular division, and it is not unreasonable to postulate that this process could be affected by the extent and nature of the contact of the organism with the solid portion of the membrane filter and the extent and thickness of the nutrient film surrounding the organism. Further, nutrient supply by diffusion of medium and removal of subsequent metabolic waste products must be a function of membrane structure and pore morphology. With these factors in mind, we began this study with the objective of defining the optimum membrane structure for growth of coliform bacteria. (This paper was presented in part at the joint EPA/ASTM Symposium on Recovery of Indicator Organisms Employing Membrane Filters, Fort Lauderdale, Fla., 1975.) MATERIALS AND METHODS Several types of membranes were used in this study. There were type MF mixed esters of cellulose (Millipore Corp.), type GA cellulose acetate (Gelman Instrument Co.), and a noncellulosic polyaryl 686 SLADEK ET AL. ester membrane which is not available commercially. The surface structures of these were characterized using a Coates & Welter CWICSCAN 100-4 scanning electron microscope. Before observation, the membranes were coated with 1-to 2-nm layer of gold. Fecal coliform and total coliform determinations were performed in accordance with Standard Methods (2), sections 408 A and B, with the following modifications. To achieve the closest possible similarity between membrane tests and streak plate controls, the membranes were plated on a 0.34-cm thickness of agar medium in 47-mm petri dishes; each streak plate was prepared by spreading a 0.1ml aliquot of sample onto a 0.34-cm thickness of agar in a 90-mm dish. The reason for using a controlled thickness of agar is that we had found, in earlier experiments, that fecal coliform recovery is a function of agar thickness. M-FC broth and M-Coliform broth were obtained from the BioQuest Division of Becton, Dickinson, and Co. Agar (15 g/liter) was added when the media were prepared. Agar plates were stored at 5 C and were used within 48 h of preparation. Fecal coliform plates were incubated at 44.5 + 0.2 C in Blue M water baths equipped with calibrated recording thermistors. Total coliform plates were incubated at 35 ± 0.5 C in circulating air incubators. Most of the water samples were untreated sewage, obtained from the masher section of the Billerica, Mass., Sewage Treatment Plant. River samples were also used. Samples were stored at 5 C and were used within 30 h of collection. Some refinements of technique were needed to make it possible to run experiments involving large numbers of samples. Initially, it was found that noticeable die-off occurred in 15 min when the source water was diluted with phosphate buffer. The use of 0.1% buffered peptone, however, stabilized the count for a period of 1 h. It was also found to be important to restrict the time between plating and incubation to 15 min or less. The complete procedure was then as follows. A preliminary count was obtained when the sample was taken. The following day, a dilution was prepared to give a count of 200 to 1,000 bacteria/ml, using buffered peptone diluent. The diluted sample was mixed for 30 min on a mechanical shaker. Then groups of about 18 membranes and nine streak plates were prepared from 0.1-ml aliquots, plated, and incubated. This was repeated throughout the experiment. Using this method, up to 100 membranes plus associated streak plate controls could be run within the 1-h limit. To confirm fecal coliforms, typical blue colonies were transferred into lauryl tryptose broth and then into EC broth. Surface pore morphology. Membrane filter structure can be characterized by several parameters. The retention pore size is a measure of the smallest particle which is retained by the structure and is best measured by direct determination of passage ofparticles (or microbes) of known size. This technique is described by Rogers and Rossmoore (6). In the present investigation, we were interested not only in bacterial retention but also in how the bacteria are situated on the membrane. It is reasonable to expect that the environment of retained bacteria depends on the retention pore size as well as the structure of the surface layer in which they are retained. Figure 1 gives scanning electron photomicrographs of a series of eight membranes made from mixed esters of cellulose. The photomicrographs show similar structures which differ only in the size of the openings. In each photomicrograph relatively large surface openings can be seen overlying a system of finer pores. The large surface openings were characterized by the surface opening diameters reported on the figure. These were determined by direct measurements on each photomicrograph or, in the case of the smaller size openings, by measuring enlargements of the photomicrographs. The retention characteristics of these membranes for coliform organisms were determined by passage tests, as described in the following section. In summary, the way in which bacteria are situated on a membrane is determined by a new parameter, the surface opening diameter, which is observable from scanning electron photomicrographs. The retention of bacteria is determined by the more familiar retention pore size, which is found from passage tests. Figure 2 shows fecal coliform counts on the series of membranes described above. There is a remarkable increase in counts at surface opening diameters between 1.0 and 2.0 ,um. The decrease in counts at the largest opening size is evidently due to passage of organisms through this very coarse structure. The dotted line labeled "passage" was obtained by refiltering the effluent through a 0.45-,um retention pore size membrane and plating this membrane on M-FC agar in the usual way. On the basis of both growth and passage tests, the optimum membrane structure was determined to have a 2.4-,um surface opening diameter with smaller (fecal coliform retentive) voids of approximately 0.7 ,um internally. Results of this plus three other fecal coliform runs are given Fig. 3. In all four runs, the abrupt increase in recovery at a surface opening diameter of 1.0 to 2.0 ,um is evident, with the optimum structure, i.e., zero passage and optimum growth, occurring with 2.4-,um surface openings. membrane was compared with that of a 3.8-pum face opening sizes. Evidently, whereas the fecal coliform test requires a surface opening diameter of 2.4 pgm of optimum growth, the total coliform test is less demanding and works well on membranes of surface opening diameter in the range of 1 to 3 pm. RESULTS AND DISCUSSION At this point, it appeared that surface opening diameter was definitely a primary determinant of fecal coliform recovery. However, other factors, such as chemical composition and methods of sterilization, remained to be investigated. Effect of chemical composition. In the foregoing set of tests, membranes employed were composed of mixed esters of cellulose. A second series of experiments was designed employing cellulose acetate membranes. Cellulose acetate has a much smaller affinity for proteins, and TOTAL COLIFORM 30 COUNT ing diameter. eries may be affected by the method of sterilization. They did not, however, present data derived from comparing identical membranes, where the only variable was the method of sterilization. To test for possible sterilization effects, membranes were selected from the group exhibiting optimum growth characteristics (2.4-,um surface openings). These membjranes were then divided into four groups using random sampling techniques. One group was left unsterilized, one was autoclaved at 121 C for 15 min, one was exposed to ethylene oxide using a standard sterilization cycle (the cycle used a 2-h exposure to 12% ethylene oxide at 130 F [54.5 C] and 60% relative humidity [4]) and was aerated 3 days, and the fourth group was sterilized by irradiation at a dose of 1.0 megarads using gamma rays from a cobalt 60 source. Mean counts and 95% confidence limits on the means are given in Table 1. There are no significant differences between counts on the unsterilized membranes and counts on the membranes sterilized by the three methods used. The data collected to this point strongly suggest that neither chemical composition nor method of sterilization have any significant effect, but that the primary determinant of fecal coliform growth on a membrane filter is that of the surface pore morphology (specifically with respect to the size of upper surface openings). We speculated that since surface effects are strongest at surface void sizes which are close to coliform dimensions, some sort of fit of the organism into the pore might be required for optimum growth. To visualize the fit of the organism into the surface structure, electron photomicrographs of an Escherichia coli isolated from sewage on the 2.4-tum surface opening cellulose ester membrane were obtained. Figure 6 shows a field in which it appears that an organism has penetrated into a large surface void. Effect of nutrient supply. Concerning fecal coliform recovery, the mechanism of the effect could be that organisms which are deposited on very fine surface structures are incompletely surrounded by nutrient, whereas ones that fit into surface openings can be cradled below the level of nutrient that is drawn up by capillary forces. Because of evaporation, an incompletely surrounded bacterium might be subjected to a locally hypertonic solution, with resulting plasmolysis and death. This effect would be particularly evident at the elevated temperature (44.5 C) of the fecal coliform test. To test this hypothesis, three methods of supplying nutrient were compared. The 0.7-pum and the optimum 2.4-pim surface opening cellu- Ethylene oxide 44 ± 6 103 ± 9 sterilized Autoclaved 38 ± 6 108 ± 9 Irradiated 40 ± 6 94 ± 8 Streak plate 50 ± 6 82 ± 6 a Two different sewage samples were used. Each mean is an average of five replicates. lose ester membranes were used. One set was plated in the standard manner, one set was plated face down on the M-FC agar, and the third set was plated right side up with 2.0 ml of M-FC agar overlayed onto each membrane. Results are summarized in Table 2. Due to the confluence of colonies, accurate counts could not be obtained from the membranes plated face down. However, it was clear that the number of colonies on membranes having the smaller surface openings (0.7 ,um) was substantially increased by plating face down. Overlaying these membranes gave a dramatic increase in counts. The increase in growth thus seen from inverting the filter, plus the close agreement in counts of the two membrane groups when the lower yield filters were overlayed with nutrient, give strong evidence that complete nutrient coverage of the organisms is required and that this is achieved only with larger surface opening sizes. During the comparison testing of the membranes for the 0.7and 2.4-,um surface opening groups, some additional benefits were noted relative to the latter. These predictable, but nonetheless important, phenomena were an increase in the flow rate through the membrane, an increased diffusion rate ofmedia to the membrane surface, and, significantly, an increase capacity to filter larger volumes ofwater, particularly those where algae or other colloidal turbidity would otherwise limit the sample size. In summary, the factors expected to have an effect on fecal coliform recovery were investigated. The only one showing a significant effect was that of surface pore morphology. The evidence suggests that fecal coliforms must be cradled slightly below the membrane surface for optimum recovery at 44.5 C. This suggests an optimum membrane structure, with surface pores slightly larger than the fecal coliform organisms but with internal bacterial retentive pores. Until now, membranes recommended for bacterial testing have been specified by a retention pore size of 0.45 um. Typical 0.45-,um retention membranes have surface opening diameters of 1 to 2 ,um. A slight shift of position on the curve in the range of 1-to 2-,um surface openings can have a large and significant effect on recovery ( Fig. 2 and 3). Since membranes of different manufacture, all having 0.45-.um retention size, may exhibit differences in surface morphology (i.e., in relative surface opening diameters), they may also exhibit considerable differences in fecal coliform recovery. A change to the optimum 2.4-,um surface APPL. MICROBIOL. opening size will not only provide higher fecal coliform counts, but will also lead to a smaller sensitivity to small differences in surface morphology. For the total coliform test (Fig. 4), however, membrane performance is not sensitive to surface morphology (except in the range below 1-,um surface opening size). The new 2.4-,um surface opening/0.7-Im retention pore size membrane developed in this work should be regarded as an improvement for fecal coliform tests and may also be used for total coliform, with results equivalent to 0.45-Am retention membranes. ACKNOWLEDGMENT We are grateful to C. B. Neville for experimental assistance. LITERATURE CITED 1. American Public Health Association. 1955. Standard Methods for the examination of water and wastewater, 10th ed. American Public Health Association, Inc., New York.

v3-fos

2018-04-03T05:24:42.788Z

1975-11-01T00:00:00.000Z

42822736

s2

Purification and Properties of Intracellular Proteinase from Streptococcus cremoris. Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl(2) in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca and inhibited by Zn, Cu, Hg, Fe, ethylenediaminetetraacetate, and sodium lauryl sulfate. The K(m) value of the enzyme towards each casein fraction was almost the same, and the V(max) of the enzyme towards alpha(s)-casein was smaller than those towards the other casein fractions. At the early stage of milk fermentation, as well as at the ripening stage, proteolytic action of lactic acid bacteria on casein has been recognized to be very important for the development of the flavor and taste of fermented milk products. Grutter and Zimmerman (7), Williamson et al. (28), and Sasaki and Nakae (21) have detected extracellular proteases from lactic acid bacteria cultures. Baribo and Foster (3), Van der Zant and Nelson (24), and Cowman et al. (5), in addition to ourselves (22), revealed the existence of the proteolytic activity in the extract of the bacterial cells. The importance of the intracellular proteinase was demonstrated in successive studies (17,18), which indicate a lytic phenomenon of the cells when a marked increase in the proteolytic activity was observed in aseptic rennet curd containing cell pellets of Streptococcus cremoris or Lactobacillus helveticus. The enzymatic properties of the intracellular proteinase from S. cremoris, S. lactis, L. helveticus, and L. bulgaricus in the crude state were presented elsewhere (15,16). The present paper describes an attempt to purify the proteinase from the extract ofS. cremoris and reveal some properties of the purified intracellular proteinase. MATERIALS AND METHODS Materials. Whole casein was prepared from fresh skim milk by precipitation at the isoelectric point, pH 4.7. a,-, f-, and K-caseins were fractionated from the whole casein by the methods of Hipp et al. (9) and Tsugo and Yamauchi (23).All casein fractions were stored at low temperature in the lyophilized state until used. All the reagents used in this experiment were of guaranteed grade. Organism. S. cremoris (H-61) was a kind gift from the National Institute of Animal Industry, Chiba, Japan. Growth conditions of S. cremoris. The organism was grown in the media, the components of-which are listed in Table 1. The media were sterilized at 120 C for 15 min. The organism was activated by repeated subculture in the same medium used for the large-scale cultivation. Enzyme assay. (i) Determination of proteolytic activity. Routine assay was performed with casein (Hammersten casein) as the substrate at 30 C for 3 h. The activity was estimated by the increase in trichloroacetic acid-soluble materials resulting from the action of the proteinase on whole casein and fractionated caseins. One milliliter of the 0.5% solution of these casein fractions in tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer at pH 7.0 was incubated with 1 ml of enzyme solution in the same buffer. After the 3-h incubation, the reaction mixture was combined with an equal volume of 10% trichloroacetic acid solution to remove unhydrolyzed casein molecules. The amount of tyrosine liberated from casein in the deproteinized filtrate was determined from color intensity at 660 nm according to the Folin method (8). The enzymatic activity was described as micromoles of tyrosine released from casein during a 1-min reaction. (ii) Determination of dipeptidase activity. Hydrolysis of dipeptidase substrates by the enzyme was assayed by the estimation of liberated amino acid using modified Matheson and Tattrie ninhydrin reagent (13). 738 a The pH of each medium was adjusted to 7.0 with NaOH (salt medium) or NH4OH (tomato medium) before sterilization at 120 C for 15 min. b , Absence of the component in the media. c Results for tomato juice are given in milliliters per liter. Protein determination. The concentration of protein was estimated by the method of Lowry et al. (12) with bovine serum albumin as a standard. Molecular weight determination. Molecular weight was determined by the method of Andrews (1) on Sephadex G-200 in 0.1 M Tris-hydrochloride buffer, pH 7.0. The same column, sample application, and flow rate were used as described in Results. The void volume was known to be 65 ml from a calibration run with blue dextran (molecular weight, 2,000,000). Horse ferritin (450,000), human gamma globulin (160,000), bovine serum albumin (67,000), and cytochrome c (12,400), provided from Mann Research Laboratories, were chosen as standards. The molecular weight of the proteinase was determined by interpolation. RESULTS Production and preparation of the proteinase. The organisms harvested from each medium shown in Table 1 were washed well with physiological saline (0.9% NaCl solution). For purification of the intracellular proteinase, harvested cells of S. cremoris were frozen rapidly in a Hughes press in the freezer at -80 C and then disrupted with high pressure. After observing the almost perfect disruption of the cells by microscope, the disrupted cells were suspended in the Tris-hydrochloride buffer (0.01 M) at pH 7.0 and stirred for 30 min to extract intracellular substances. Cell debris was removed by centrifugation at 10,000 rpm for 15 min. The clear supernatant fractions containing proteinase activity were dialyzed overnight against the same buffer and are referred to as the crude extract. The amounts of the protein (about 1.5 g from 10 liters of culture) in the crude extracts from seven kinds of culture closely resembled each other. The proteolytic activities of the cell extracts, however, differed. (One unit [U] is 1 4mol of tyrosine released from casein during a 1-min reaction.) The activity in the extract from salt medium I (21 x 104 U/mg of protein) was remarkably greater than that from medium II (104 U/mg of protein), and the value from the tomato medium A (10 x 104 U/mg of protein) was also greater than those from media B and C (104 U/mg of protein) but smaller than those from media D and E (16 x 104 and 22 x 104 U/mg of protein, respectively). The extract from medium E contained the same or greater activity than the extract from salt medium I. The pH of the culture of S. cremoris (tomato medium E) was readjusted aseptically to pH 7 to 7.5 with NH40H after 24 h of cultivation, since the pH had dropped to about 4.6 or below as a result of lactic acid fermentation. Thereafter the cultivation was continued for an additional 24 h. At the end of the second cultivation, the pH of the culture had again been lowered to 4.6 to 4.9. Because of this pH readjustment, the amount of protein extracted from the bacterial cells increased about twofold, but the total activity was nearly the same as that of the cultivation for 48 h without the readjustment of pH. Therefore, the enzyme activity was decreased by a factor of 2. In the remainder of this paper tomato medium E without pH readjustment was usually employed for the preparation of crude extract. Purification procedure of intracellular proteinase. (i) Diethylaminoethyl-cellulose chromatography. This step and all subsequent steps for purification of intracellular proteinase were carried out at 0 to 5 C. The crude extract VOL. 30,1975 on May 5, 2020 by guest http://aem.asm.org/ Downloaded from containing about 500 mg of protein was applied to a diethylaminoethyl-cellulose column (3 by 50 cm) equilibrated with 0.01 M Tris-hydrochloride buffer (pH 7.0). The adsorbed protein was eluted with 2 liters of the same buffer, with increasing ionic strength of NaCl (0 to 1 M) linearly ( Fig. 1, broken line). The flow rate was 22 ml/min and 15-ml fractions were collected. The elution pattern is shown in Fig. 1 (solid line). Only one peak of proteinase activity ( Fig. 1, dotted line) was detected. It appeared at the middle of the large second peak of protein, which was eluted at about 0.25 M NaCl. The fractions having the highest proteinase activity were combined. The specific activity of the proteinase in the combined fraction was 0.033 U. (ii) Membrane filtration. The smaller-molecular-weight substances were removed from the combined fraction by membrane filtration (Diaflo, PM 10, Amicon Co.) under nitrogen pressure of 4 atm. Before removing all of the low-molecular-weight materials and buffer solution from the enzyme solution, about 50 ml of Tris-hydrochloride buffer (0.1 M, pH 7.0) was added to the residual solution with enzymatic activity in the Diaflo cell, and sieving was continuously carried out at low temperature until the solution was concentrated to about 3 ml. This sieving procedure (instead of the dialyzing procedure) was repeated three times for the purpose ofreplacement of the buffer and concentration of the enzyme solution, in addition to the removal of low-molecular-weight materials (less than 10,000 molecular weight). Depending on this procedure the total amount of protein contained in enzyme solution decreased to half and total activity decreased to two-thirds. (iii) Sephadex gel chromatography. The enzyme solution (containing about 20 mg of protein) concentrated from the preceding procedure was applied to a Sephadex G-200 column (2.6 by 40 cm) equilibrated with 0.1 M Trishydrochloride buffer (pH 7.0). The column was maintained at 5 C and eluted with an upward flow of 25 ml/h, and 6.5-ml fractions were collected. The activity towards casein was located at the second peak of protein as monitored by the absorbance at 280 nm (Fig. 2). The fractions in the major active peak were combined and reconcentrated to about 3 ml within the Diaflo cell under the same conditions as described above. Thereafter it was rechromatographed on the same gel column. The chromatographic patterns are presented in Fig. 3. Two peaks of protein appeared in fractions number 10 (void volume) and 14 (1.42 of Ve/Vo value). Only the second peak retained the activity. The specific activity of the enzyme (0.333 U/mg) was the same as that obtained by the first gel filtration Table 2). The first peak in Fig. 3 might be an inactivated enzyme protein polymerized during the reconcentration procedure. (iv) Polyacrylamide gel electrophoresis. Disc electrophoresis of the enzyme fraction obtained from the procedure of rechromatography on Sephadex G-200 was run by the method of Davis (6) on polyacrylamide gel at pH 8.9. The constant current was 3 mA/gel for 50 to 60 min at room temperature. Six milligrams of protein was applied and was detected by staining in amido black lOB. A single protein component appeared (Fig. 4), which had a mobility of onehalf that of bromophenol blue. This purified a Enzymatic activity was determined as micromoles of tyrosine released during a 1-min reaction. b Cell extract was prepared from 20 g of wet cells. Table 2. The overall yield was q 30 7%, and the specific activity of the purified°\ enzyme against casein was 0.333 U/mg. In this x 20 B way the enzyme was purified 150-fold. Properties of the purified intracellular I E proteinase. (i) Absorption spectrum. The ab-. sorption spectrum of the purified enzyme at pH . 10 7.0 revealed a maximum peak at 278 nm ( Tables 3 and 4. Zn2+, Cu2+, Hg2+, and Fe2+ caused 60 to 80% inhibition at a concentration of 1 mM, and Co2+ and Mg2+ did not affect on the activity. Ca2+ caused 37% activation ( Table 3). The activity was, however, inhibited by ethylenediaminetetraacetate as a result of the chelation of Ca2+. There is no effect of monoiodoacetic acid, Nethylmaleimide, p-chloromercuribenzoate, or cysteine on the enzymatic activity within the range of experimental error. Thus a thioor disulfide group does not seem to be essential for enzymatic activity. This characteristic is in conformity with that of an intracellular proteinase from a mutant ofS. lactis revealed by Westhoff et al. (27). (vi) Substrate specificity. Proteolytic activity of the enzyme was determined at a given substrate concentration, and their reciprocal numbers were plotted by drawing Lineweaver-Burk plots (Fig. 9) casein fractions and whole casein were found to be almost the same, 0.1%. This value was 1/20 that of trypsin (2) (vii). Dipeptidase activity. Dipeptidase activity of this purified intracellular proteinase was examined at 30 C for 3 h. The activities towards alanyl-glycine, alanyl-alanine, and glycyl-leucine were almost negligible. DISCUSSION Effect of medium components on proteolytic activity of S. cremoris. Seven kinds of media were used for cell production ( Table 1). Enhancement of proteolytic activity in the extract from the cells of S. cremoris was observed in tomato media; i.e., fairly high activity towards casein appeared when succinate (medium C) and citrate (medium B) were replaced by lactose (medium A) and by glucose (medium D) (see Results). The activity increased further by adding CaCl2 (medium E). These results suggest that intracellular proteinase is increased by lactose and glucose and also by casein and CaCl2. On the other hand, enhancement of activity was inhibited by the presence of citrate and succinate in the tomato media. This might depend on the property of succinate and citrate, which chelate Ca2+ contained in meat or yeast extract. In the salt media, however, chelation by succinate seemed to be released by casein, which could adsorb Ca2+ selectively. Since these media components, lactose, glucose, casein, and calcium ion, are the constituents of milk, milk is able to provide the best conditions for the production of intracellular proteinase of lactic acid bacteria during the fermentation of milk. This indicates that the starter lactic acid bacteria seem to have higher proteolytic activity during growth in milk than in any other synthetic media. The decrease in specific activity of the enzyme by the readjustment of the culture pH seems to assert that the proteinase could be enhanced in the acidic condition of a suitable culture containing salt medium I or tomato medium E. This phenomenon is consistent with the results reported by Sato and Nakajima (22), in which the activities of intracellular proteinases of lactic acid bacteria were the highest after cultivation for 48 h. For enhancement of the proteinase activity, there might be a possibility of proteinase induction by casein. If induction by casein is essential for the appearance of the enzymatic activity of the lactic acid bacteria, casein particles have to penetrate or to be carried across the cell membrane into the cells, or the proteinase must be located at the cell membrane for the formation of the complex between the enzyme and casein particle. Reasonable results about this problem will be presented in further studies. The proteinase activity usually appeared as a single peak (Fig. 1-3). According to this method of enzyme extraction and the purification technique, only one proteinase could be detected from S. cremoris. Hydrolytic properties of the proteinase towards casein fractions. Optimum pH of this intracellular proteinase from S. cremoris was 6.5 to 7.0. This value was also presented by Sato and Nakajima (22) and by Krishna and Dutta (11). Similar results are reported for the enzymes from S. lactis (3,10,11,22,25,26), L. casei (5,22), and L. bulgaricus (22). This optimum pH range for intracellular proteinase was slightly on the acidic side from those of extracellular proteinases from S. lactis (21), S. zymogenes (28), and S. faecalis var. liquefaciens (7). However, the value by Westhoff et al. (27) was slightly on the alkaline side from those of many other intracellular proteinase. Relative activity of this enzyme on casein at pH values encountered in cheese (pH 5.3 to 5.8; mean value, pH 5.5) was about 70% (Fig. 7). The Vmax ofthe purified intracellular proteinase towards a,-casein was smaller than those towards the other casein fractions and whole casein (Fig. 9). The hydrolytic reactions against these casein fractions were also studied by using crude extracts of S. cremoris (H-61) harvested from salt medium I and tomato medium E. Proteolytic properties of the crude extracts against these casein fractions were almost the same as that of purified intracellular proteinase from tomato medium E in this paper, as described above. Those results are, however, different from those presented in the other papers (15,16), where the same organism was grown in the same salt medium I. The conditions for the enzymatic reaction were also the same as those employed in this paper. In those papers, a,-casein was hydrolyzed by the crude extract more than the other casein fractions were. One of the reasons for these differences was caused by the changing properties of this organism by unknown factors during its storage for a long time at low temperature. More details, however, about these differences have to be studied further. The same Km values were evaluated from Fig. 9, in which reciprocal values of the concentration (grams per 100 ml) of each casein fraction were plotted on the abscissa. The evaluation of these values depends on the limited hydrolysis of casein by the enzyme, since one casein molecule contains many peptide bonds that can be hydrolyzed by the proteinase. When the molecular weights of each casein fraction determined by Mercier et al. (14) and Ribadeau-Dumas et al. (19,20) are taken into considera-APPL. MICROBIOL. on May 5, 2020 by guest http://aem.asm.org/ Downloaded from tion for the evaluation of these Km values, the value against K-casein would be larger than those against as-and ,-casein. In this paper, we tried to carry out the enzymatic reactions without any infectious factors and established the experimental conditions as described above. The reaction time, 3 h, is rather long compared with the usual enzymatic reactions; however, it could be curtailed to onefourth that employed by Westohoff et al. (27) and Krishna and Dutta (11).

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Nephrotoxicity of Dietary Ochratoxin A in Broiler Chikens Graded doses of pure ochratoxin A (0, 0.5, 1.0, 2.0, 4.0, and 8.0 μg of toxin per g of feed) were incorporated into a commercial diet which was fed to chicks from 1 day to 3 weeks of age, at which time the experiments were terminated. Growth was inhibited at 2.0, 4.0, and 8.0 μg/g, whereas the kidneys were enlarged at doses of 1.0 μg/g and above. Renal function as measured by clearance of phenol red was decreased 15 and 31% by doses of 4.0 and 8.0 μg/g, respectively. Uric acid was increased 38 and 48% over the control values by doses of 4.0 and 8.0 μg/g, respectively. The plasma electrolytes Na, Cl, Ca, and K were measured; however, only K was significantly (P < 0.05) altered, showing a decrease at doses of 4.0 and 8.0 μg/g. The percentage dry weight of the kidneys decreased significantly at dose levels of 4.0 and 8.0 μg/g, indicative of edema. Histological examination of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens. kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens. Ochratoxins include a group of structurally related, secondary metabolites produced by seven species of Aspergillus and six species of Penicillium (5). Aspergillus ochraceus, from which the toxins acquired their name, appears to be the predominant ochratoxin producer (5). Ochratoxin A, which is the most toxic and most prevalent form, is 7-carboxyl-5-chloro-8hydroxyl -3,4 -dihydro -3 -R-methylisocoumarin linked through an amide bond to L-fl-phenylalanine (25). The widespread occurrence of ochratoxin-producing fungi, their ability to grow on a variety of economically important feed and foodstuffs (5), and the natural occurrence of ochratoxin (8,17,18; P. Krogh, 2nd Int. Cong. Plant Pathol., Abstr. 0360, 1973) constitute a threat to both animal and public health. The dimensions of this threat are undefined as yet, but excellent reviews of the literature exist (5,20). Only a limited amount of research has been done on ochratoxicosis in poultry. Huff et al. (10) provided a general description of the disease in young broiler chickens and concluded that on the basis of acute mean lethal dose and minimal growth inhibitory concentration ochratoxin A was the most potent mycotoxin yet studied in chickens. They also reported that the most sensitive indicator of ochratoxicosis in young broiler chickens was enlarged kidneys. A IPaper number 4592 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. 48 microscopic evaluation of acute ochratoxicosis in young White Leghorn cockerels (6,12) revealed acute nephrosis, hepatic degeneration, and suppression of hematopoiesis in descending order of frequency. On the other hand, a histopathological study of ochratoxicosis in ducklings (23) revealed primarily hepatic degeneration characterized by an increase in fatty vacuolation, changes in the matrix of the mitochondria, and disorganization of the endoplasmic reticulum of hepatocytes. In laying hens ochratoxin at low concentrations delays sexual maturity and reduces egg production and hatchability (4). Because of the high toxicity of ochratoxin to chickens and because ochratoxin can be produced in a variety of feedstuffs used in the poultry industry, it was deemed desirable to study in more detail ochratoxicosis in the economically important broiler chicken. In particular, the nephrotoxicity of ochratoxin was investigated. (A preliminary report of part of this paper was presented at the 74th Annual Meeting of the American Society for Microbiology, Chicago, Ill., 1974.) MATERIALS AND METHODS Animal husbandry. Day-old male broiler chicks obtained commercially were used in these studies. The birds were housed in electrically heated batteries under continuous lighting. Feed and water were available ad libitum. The feed was a commercial VOL. 30,1975 broiler-starter from which all medications were omitted. Ochratoxicosis was induced by incorporating into small portions of the diet weighed amounts of crystalline ochratoxin A dissolved in 95% ethanol. The portions of feed containing the ochratoxin A were dried at 100 C to evaporate the ethanol before being mixed into the remainder of the feed. The experimental diets were fed from hatching until the experiments were terminated at 4 weeks. Ochratoxin A. A. ochraceus NRRL 3174 was grown on wheat by the method of Trenk et al. (24). Ochratoxin A was extracted from the wheat by the method of Steyn and van der Merwe (21) and purified by thick-layer preparatory chromatography on Silica Gel G using benzene-acetic acid (9:1, vol/vol) as the solvent (7). Ochratoxin A was eluted from the silica gel by making a slurry of the silica gel in hot benzene-acetic acid (9:1, vol/vol) solution. Approximately 100 ml of solution per g of Silica Gel G was used. The slurry was then filtered through paper. This procedure was repeated twice to insure that all of the ochratoxin A was removed from the silica gel. The combined filtrates were evaporated to 1% of the total extract volume. Cold benzene was then added to precipitate the ochratoxin A. The ochratoxin A crystals were then recrystallized twice from benzene and washed repeatedly with cold benzene before allowing to dry in the air. Assays. The growth rate of the chickens was determined by weighing the chickens weekly. A renal function test was conducted by the method of Pitts (14). Plasma uric acid levels were measured by the method of Caraway (3). Serum potassium, sodium, calcium, and chloride ion concentrations were measured by an independent laboratory (Rex Hospital, Raleigh, N.C.) using Technicon S.M.A. methodology. Tissue taken from the right kidney was fixed in neutral buffered formalin and prepared for histopathological examination by standard procedures. The relative kidney weight and percentage dry weight of the kidney were also measured. Experimental design. There were four replicates of 10 birds at each dose level. The dose levels were 0, 0.5, 1, 2, 4, and 8 ug of ochratoxin A per g of diet. The treatments and birds were completely randomized. The replicate means were evaluated statistically by analysis of variance in which an F ratio was calculated. If the F ratio were significant (P < 0.05), the treatment means were compared by the method of least significant differences (2). RESULTS The effect of graded doses of ochratoxin A on the parameters measured in this study are given in Table 1. The growth rate of broiler chickens was inhibited significantly (P < 0.05) at doses of 2.0 ttg/g and above, and the degree of inhibition was dose related. The weight of the left kidney relative to the total body weight was increased by doses of 1 ,gg/g and above, which agrees with the previous report (10). At the highest dose the relative kidney weight was more than doubled. The NEPHROTOXICITY OF OCHRATOXIN 49 enlargement of the kidney could be the result of edema, a general increase of protoplasm, or an increase of a specific constituent(s). The possible occurrence of edema in the kidney was tested by determining the dry weight ratios at the different dose levels. This ratio was decreased significantly at doses of 4 and 8 gg/g. Thus, the enlargement of the kidney during ochratoxicosis results in part from edema. The effect of dietary ochratoxin on the excretory function of the kidney was determined by measuring the rate of clearance of phenol red from the blood by the kidney. The rate of clearance was decreased significantly (P < 0.05) at 4 and 8 Ag/g. This impairment of renal excretory function should be reflected by alteration in the levels of blood constitutents which are excreted by the kidney. The primary product of nitrogen catabolism in the chicken is uric acid (22). The plasma uric acid levels were increased significantly (P < 0.05) at dose levels of 4 and 8 Ag/g, with an approximate 50% increase at the highest dose. During kidney impairment an electrolyte imbalance is often seen. Serum potassium levels were decreased, and this hypokalemia also was significant (P < 0.05) at 4 and 8 Mg/g. Sodium, calcium, and chloride concentrations in the serum were measured, but they were not altered from control values. The most striking histopathological changes seen in the kidney during ochratoxicosis were swelling of the tubular epithelial cells, tubular dilation, and proteinaceous material in the lumen. The severity of these changes appeared to parallel the dose of ochratoxin A administered. There was also a differential effect on the convoluted tubules in that the proximal portion was more severely affected than the distal portion to the extent that some generalized necrosis of the epithelial cells of the proximal tubules was observed. DISCUSSION The primary effect of ochratoxin A in chickens appears to be on the kidneys. The most sensitive visible indicator of ochratoxicosis is kidney enlargement (10). This enlargement, which occurred at doses as low as 1 jug/g, was not accompanied by changes indicative of altered excretory function, such as increased serum uric acid, hypokalemia, decreased dry weight, and decreased clearance of injected phenol red, until doses of 4 and 8 ,ug/g were reached. However, minor histopathological changes could be seen at doses of 1 and 2 Ag/g, which did cause enlargement. Presumably, the enlargement without an accompanying impair- ment of function represents a compensation by the chicken to the nephrotoxicity of ochratoxin. Once the compensating ability of the kidney is overmatched, alteration in function would be expressed. The suggestion that ochratoxin is primarily a nephrotoxin in broiler chickens agrees with prior observations on acute ochratoxicosis in White Leghorns (6,12), where the kidney was the organ most frequently affected. It differs from the observations with ducklings, where the basic lesion of ochratoxicosis is fatty infiltration of the liver, and from the rat, where both the liver and kidney appear equally affected (13) and the liver exhibits a hyaline degeneration and focal necrosis (23). Indeed, the nephrotoxicity of ochratoxin in broilers offers an easy differential diagnosis of ochratoxicosis from aflatoxicosis, which is primarily a hepatotoxin in broilers (19). The exact mechanism whereby ochratoxin exerts its nephrotoxicity cannot be stated on the basis of the present study, but some comments can be made. Extensive kidney damage is indicated by the renal function test, by the accumulation of uric acid in the blood, and by the severe histopathological changes observed. However, the differential effect on serum potassium and serum sodium suggests a differential effect on the kidney tubules. Sodium and chloride presumably are reabsorbed uniformly along the proximal and distal tubules (1). Potassium, on the other hand, is reabsorbed in the proximal and secreted in the distal tubules (11,15). Ochratoxin in rats differentially damages the proximal tubules (16) as it does in chickens. The lowered blood potassium can then be explained on the basis that its reabsorption is impaired during ochratoxicosis. It should not be assumed that ochratoxin is only a nephrotoxin in chickens. For example, growth is inhibited at 2 Mg/g, whereas a dose of 4 gg/g is required to depress the kidney excretory function, and at 8 ,ug/g the renal clearance of phenol red is decreased only 31%. A loss of 75% of the kidney causes only a slight decrease in an otherwise healthy chicken (9). This suggests a more direct effect on the growth process. It is interesting that ochratoxin A can cause an inhibition of carbohydrate and protein metabolism and oxidative phosphorylation in rats (5). In addition, ochratoxin appears to have an affinity for the gastrointestinal tract. In broilers treated the same as in the present study, an enlargement of the crop, proventriculus, and gizzard was noted (10). In acute lethal ochratoxicosis, chickens exhibit a severe enteritis (10,12). The neural system is also affected during acute lethal ochratoxicosis, although no neural disturbances were noted in the present study. It seems obvious that more detailed studies of ochratoxicosis are needed before this disease can be understood.

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1975-06-01T00:00:00.000Z

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Incidence of Molds on Pecan Nuts at Different Points During Harvesting Pecan nuts were selected at various points during routine harvesting, and nutmeats were analyzed for gross and internal fungal contamination and for the presence of Aspergillus flavus and A. parasiticus. Fungi were isolated from a large percentage of the nutmeats at all points of examination. No correlations could be made between increased incidence of fungi and particular harvesting procedures. (Carya illioensis [Wangenh. ] K. Koch) production (J. Taylor and R. E. Worley, Proc. Southeastern Pecan Growers Assoc., p. 29, 1972), as pecans enter the shelling plant (4), and during storage (3; E. K. Heaton, Proc. Southeastern Pecan Growers Assoc., p. 135,1972), no information is available describing the incidence of molds in general and potential aflatoxin-producing molds in particular on nutmeats as pecan nuts are sequentially subjected to various mechanical harvesting procedures. Substantial physical damage and exposure to field dirt may occur when nuts are mechanically shaken from the tree, swept in windrows, transported to shelling plants, and cleaned in preparation for cold storage. It has been suggested that increased levels of molds on nutmeats as they enter the processing plant may be a result of a specific mishandling procedure at some point during the harvesting scheme. Knowledge of increased contamination by A. flavus or A. parasiticus at a point(s) during traditional harvesting procedures might enable modifications to be made which would ultimately reduce the potential for aflatoxin contamination. With this in mind, 'Stuart,' a thick-shelled pecan cultivar, and 'Schley,' a thin-shelled cultivar, were mechanically harvested or hand selected on 23 to 26 October 1974 from groves near Albany, Ga. Points of examination are listed in Table 1. Samples were subdivided in the laboratory, and individual pecans were aseptically cracked. One-half (or a piece) of the kernel from each nut was plated on malt extract agar. The remaining half (piece) was surface sterilized (1, 4) by dipping for 2 min into a solution containing 20% commercial bleach (6% sodium hypochlorite as the active ingredient), 20% ethanol, and 60% water before plating on malt extract agar. All plates were incubated at room temperature (22 to 24 C) for up to 3 weeks, after which the number of pecan halves (pieces) showing fungal growth was recorded. Any colony suspected of belonging to the A. flavusoryzae group was subcultured on a second malt extract agar plate. Morphological characteristics were observed during growth and identification of A. flavus and A. parasiticus was made according to the classification given by Raper and Fennell (9). Data were analyzed by using the chi-square criterion. Results of visual inspection of pecan nutmeats at the time they were taken from the shell and the incidence of total molds as well as A. flavus and A. parasiticus on the nutmeats are summarized in Table 1. Any portion of the nutmeat of a single nut judged as inedible constituted a positive rejection. Criteria for inedibility included visual mold or insect damage, discoloration, and marked shriveling. The percentage of nutmeats judged as inedible increased in samples at collection points in the sequence after pecans were mechanically swept in windrows. Surprisingly low levels of inedibles were noted in uncracked "blow-outs," a term applied to low-specific-weight nuts which are separated from sound nuts by a high-velocity air stream prior to storage. Nutmeats are generally shrunken or unfavorably developed. though nutmeats of blow-outs were somewhat dehydrated, most were still judged as edible. Data show that pecan kernels are highly contaminated with molds while on the tree, the initial point of examination in this study. This observation was also reported by Hanlin (5), who noted that no fungi were present in pecan embryos but, as the seeds ripened, the level of fungi approached 100% at maturity. Substantial levels of internal fungal contamination of nutmeats were found in the present study, regardless of the sampling point in the harvesting scheme. Although significantly higher levels of gross and internal mold contamination were noted at points in the harvesting scheme, these levels were not noted exclusively or predominantly after a particular handling procedure. Therefore, neither gross nor internal build-up of mold levels can be correlated with a particular procedure used during pecan harvesting and handling. Furthermore, contamination does not appear to be associated with subjective judgments regarding inedibility. Table 1 also lists information on the incidence of A. flavus plus A. parasiticus on and in pecan kernels. Although these values are somewhat less than those reported for stored pecan halves (1) and bakery pecans (8), they are in line with recent data reported by Escher et al. (4) on sound and blow-out nuts as delivered to the shelling plant. On the other hand, Chipley and Heaton (2) found no A. flavus or A. parasiticus on small samples of aseptically shelled pecan meats. Differences in the populations of aspergilli and other fungi on pecans apparently are due to relative levels of particular genera at particular geographical locations as well as to climatic and storage conditions to which the pecans are exposed prior to examination. Reports have shown considerable variation in the distribution of fungal genera throughout the Southeast (6, 7). As in the case of total mold incidence, levels of A. flavus and A. parasiticus do not appear to be associated with a particular harvesting procedure or with a particular cultivar. Substantially higher levels were not noted to consistently occur in blow-outs. Data presented here are preliminary in nature and should be substantiated by repetitive examination of pecan nuts from several groves over a period of years. Nevertheless, observations from this study tend to disprove the theory that a particular mechanical harvesting practice might cause increased levels of mold contamination on pecan nutmeats. Alternative approaches may be necessary to control the incidence of potential toxin-producing molds on pecan nuts.

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Rumen Bacterial Degradation of Forage Cell Walls Investigated by Electron Microscopy The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues. The microorganisms comprising the rumen bacterial population vary in morphology and metabolic activity (7,19). Symbiotic relationships for the degradation and utilization of plant cell wall constituents have been shown by using known cultures of rumen bacteria (10,(13)(14)(15)(16). Investigations of the metabolic activities of these bacteria have helped clarify the complex nutritional system in the rumen. However, the initial steps in tissue digestion involving the mode of association with and degradation of intact plant cell walls by rumen bacteria have not been investigated extensively. Electron microscopic investigations have revealed certain interesting ultrastructural features of rumen bacteria (3,11,23,28). Leatherwood (23), using the scanning electron microscope (SEM), observed "tube-like appendages" on the cellulolytic coccus Ruminococcus albus only when grown on cellulose-containing media. Costerton et al. (11), using the transmission electron microscope (TEM), reported coats external to the outer membrane of three gram-negative rumen bacteria, Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii. Earlier TEM observations from our laboratory of the tropical forage Coastal Bermuda grass degraded by rumen bacteria in vitro revealed attachment, appar-ently by an extracellular matrix, of large cocci to the thick forage cell walls (3). In addition, observations of intact leaf sections by using the SEM have shown differences in the ease and extent of forage tissue digestion by rumen microorganisms (1,2). Investigations using the SEM at the level of microbial attachment should be useful in adding a new perspective to the complex problems of bacterial attachment and degradation of forage tissues by rumen bacteria. The objective of the work reported here was to examine the mode of bacterial attachment to and degradation of intact cell walls in tissues of the temperate forage grass Kentucky (Ky)-31 tall fescue (Festuca arundinacea Schreb.) by TEM and SEM. MATERIALS AND METHODS Substrate. Leaf blades of Ky-31 tall fescue were harvested after 4 weeks of summer regrowth, frozen immediately, and stored at -30 C until used. Sections 2 to 5 mm in length were cut from the midportion of the frozen blades and used as substrate for the bacteria. Microbial inoculum. To obtain a preparation relatively free from particulate debris which would obstruct TEM observation, whole rumen contents were squeezed through four layers of cheesecloth, centrifuged at 250 x g for 1 min, and prepared as previously described (1). Leaf sections were placed in 692 VOL. 29,1975 RUMEN BACTERIAL DEGRADATION OF FORAGE CELL WALLS flasks with 250 ml of the bacterial buffer suspension and continuously bubbled with CO2 at 39 C for a maximum of 72 h. Control leaf sections were incubated in buffer (9) with constant CO2 bubbling at 39 C for 72 h. For observation of degraded forage tissue by the SEM, rumen microorganisms were prepared by two methods. (i) Whole rumen fluid was strained through four layers of cheesecloth and diluted with an equal volume of McDougall's carbonate-phosphate buffer (24). (ii) Strained, whole rumen fluid was mixed with an equal volume of phosphate buffer (20) and incubated in a separatory funnel for 1 h at 39 C to permit sedimentation of heavy feed particles and large protozoa. Leaf sections were placed in 250 ml of each of these inocula and continuously bubbled with CO2 at 39 C for 4 or 6 h. TEM. Leaf sections incubated with the bacterial inoculum and with control buffer (without microorganisms) were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.0 for 2 to 3 days and postfixed in 1.5% buffered osmuim tetroxide for 4 h. Leaf sections were prepared for the TEM as previously described (3). SEM. Leaf sections incubated in the microbial inoculum for 4 to 6 h were fixed in 4% buffered glutaraldehyde for 30 h and postfixed in 1.5% buffered osmium tetroxide for 4 h. The degraded leaf sections were blotted free of excess moisture and affixed to SEM stubs so that the degraded edge of the cross-section was prominently displayed in a vertical position. The stubs were quick-frozen in dry ice-isopropanol or liquid nitrogen and allowed to reach room temperature as the specimens were dried in a vacuum evaporator before coating with gold-palladium (60:40) alloy wire. The specimens were then observed in a field emission SEM at about 15 kV. RESULTS Control leaf sections observed with the SEM revealed that the buffer did not solubilize the tissues after 72 h of incubation (Fig. 1). As observed with the TEM, even the easily digested phloem cell walls were intact after 72 h of incubation in buffer (Fig. 2). However, an intact leaf section incubated with rumen microorganisms and observed with the SEM revealed differential tissue destruction after 4 h (Fig. 3). Mesophyll (M) and phloem (P) cell walls were removed to a point beyond the depth of focus of the SEM (at x416); remnants of the outer bundle sheath (B) remained although this tissue had lost structural integrity. The epidermis (E) appeared to be partially degraded (arrow), whereas the sclerenchyma (S) and rigid vascular tissue (V) resisted microbial digestion. The bacterium-plant cell wall association investigated at higher magnifications revealed differences dependent on the tissue type. Non- descript areas of degradation were evident in the mesophyll cell walls near the bacteria but not necessarily in the location of bacterial association with the cell wall (Fig. 4, arrows). Similar phenomena were revealed by the TEM in that bacteria were seen near but not necessarily attached to the degraded areas in both mesophyll (Fig. 5, C) and phloem cells (Fig. 6, arrow). However, bacteria appeared at times to be attached by a dense extracellular substance to the mesophyll cell wall (Fig. 5, arrows). The tissues digested after longer incubation times (i.e., the outer bundle sheath and epidermal cells) appeared to require bacterial attachment prior to tissue degradation. Rod-shaped bacteria, lying on the epidermal cell wall, were surrounded at times by an apparent zone of degradation (Fig. 7, arrows). The TEM revealed similar conditions with bacterial degradation preceded by attachment of bacterial cells apparently by an extracellular substance to the epidermal wall (Fig. 8). The zones of degradation were sharply defined in contrast to the diffuse zones in the mesophyll and phloem cells ( Fig. 5 and 6). In addition, bacteria had tightly adhered to the outer bundle sheath cells (Fig. 9, B), and zones of hydrolysis surrounded rodshaped bacteria in the inner bundle sheath cell ( Fig. 9, arrow). Attachment to the outer bundle sheath by a large coccus as shown by the TEM appeared to be mediated by an extracellular substance (Fig. 10, arrow); similar electrondense substances were not apparent with the other three attached bacteria. Observations by SEM revealed a large coccus that appeared to be attached to the epidermal cell wall by rod-like appendages (Fig. 11, arrow). DISCUSSION That rumen microorganisms degrade the mesophyll and phloem cell walls more rapidly than other tissues has been shown for various forages (2). The current observations by the TEM and SEM, as well as previous ones by the TEM (3) on a grass of lower digestibility (i.e., Coastal Bermuda grass) (1), indicated that rumen bacteria at times degrade these tissues without prior attachment. The degradation of mesophyll and phloem cell walls by enzymes free from the surfaces of a specific microbe or group of microbes cannot be ruled out at this time. However, bacteria near degraded areas were diverse in morphology, indicating that no one species alone was involved. In addition, the bacterial attachment prior to degradation of bundle sheath and epidermal cells appeared to Outer bundle sheath cell wall observed by the TEM after 12 h of incubation with rumen microorganisms. Four bacteria appear to be attached to the plant wall. Attachment of the large coccus to the plant wall is mediated by an electron-dense, extracellular substance (arrow), whereas no such structure is apparent with the rods. However, the attachment appears to be so close that the bacterial shape of the attaching side is modified. x 16,000. occur with bacteria of diverse morphologies. Reports have shown that higher temperatures increase the cell wall constituents of tall fescue with a resultant decrease in in vitro digestibility (4,17). Research should be undertaken to examine the bacterial attachment phenomenon relative to the cell walls of plants that have undergone environmental changes with resultant changes in the rate of digestibility. The secretion of degradative enzymes has been reported for the rumen bacterium R. albus (23,26). Smith et al. (26) reported that extracellular enzymes from R. albus digested up to 65% of a small quantity of ground or blended cellulose. The cell walls of the mesophyll and phloem may be so structurally different from bundle sheath and epidermal cell walls that cell-free enzymes, or fractions of the degradative enzyme complexes (22), can degrade the former tissues. In addition, Leatherwood (23) proposed that in R. albus an "affinity factor" may be necessary to hold the "hydrolytic factor" of cellulase in position to the insoluble cellulose for multiple attacks to occur. Such a phenomenon may indeed be required for hydrolysis of the cell walls more resistant to bacterial degradation where attachment precedes degradation. Previously we reported that large rumen cocci possessed an electron-dense extracellular substance that appeared to adjoin the bacterial and plant cell walls (3). Leatherwood (23), using the SEM, reported tube-like appendages associated with R. albus cells only when grown on cellulose-containing media. We found similar structures in a large coccus that apparently was attached to the epidermal cell wall as shown by the SEM {Fig. 11). The rod-like appearance and amorphous, capsular-like appearance of this extracellular feature by the SEM and TEM, respectively, of the large cocci may be the same structure manifested in various ways by different drying techniques. Springer and Roth (27), using the TEM, had reported that the length and width of fibrils of capsules seen in Klebsiella pneumoniae varied with the dehydrating procedures. Perhaps electron microscopy of critical-point-dried (5) samples would elucidate these extracellular features in rumen bacteria as has been shown recently for other bacterial species (8). Jones et al. (21) and Fletcher and Floodgate (18) have reported the attachment of bacteria-to substrates by extracellular substances. In addition, Shilo (25) showed that close contact be-699 700 AKIN AND AMOS tween myxobacteria and the blue-green algae was required for algal lysis, although no capsule-like material was reported. Berg et al. (6) reported that Cellvibrio fulvus and Sporocytophaga myxococcoides cells grown on different types of cellulose media adjoined the cellulose fibers with distinct depressions made in the substrate; S. myxococcoides produced extracellular substances interpreted as bacterial envelopes when grown on cellulose. Costerton et al. (11) reported that three gram-negative rumen bacteria, B. ruminicola, B. succinogenes, and M. elsdenii, possessed extracellular coats outside the cell envelope. Although these authors (11) concluded that the extracellular coats provided protection in a highly competitive environment, our current and previous (3) observations indicated that an extracellular substance may also mediate the attachment of rumen bacteria to particular plant cell walls so that degradation may occur. Although attachment of large cocci to substrate by capsule-like material was noted often, we have not observed extracellular substances in all attached bacteria. It is possible that the coats are thin in some cases (11) and not seen without specific staining (i.e., with ruthenium red). Costerton et al. (12), reviewing the cell envelope of gram-negative bacteria, reported that the degradative enzymes associated with the cell wall provide a "facility unique among unicellular organisms in that complex food molecules are broken down into their component monomers in a zone immediately surrounding the cell." Our observations suggest that attachment to intact plant cell walls, mediated by extracellular substances in at least some rumen bacteria, is required before the hydrolytic fraction of the enzymes can degrade the complex organization of certain forage cell walls. Such a phenomenon would help explain why certain forages, whose microanatomy consists of a high ratio of bundle sheath and epidermal to mesophyll and phloem cells, are less rapidly digested than forages with a lower ratio of these tissues.

v3-fos

2020-12-10T09:04:20.514Z

1975-01-01T00:00:00.000Z

237230157

s2

Medium-Scale Production of Citrinin by Penicillium citrinum in a Semisynthetic Medium A convenient method is described for the production of up to 1.75 g of citrinin per liter by Penicillium citrinum growing in stationary culture in a 5-gallon (18.925 liters) carboy containing 4 liters of 4% sucrose and 2% yeast extract medium. In 1951 P. citrinum was isolated from yellowed rice imported from Southeast Asia to Japan. Saito et al. (12) reported that in 3-week experiments with rats fed P. citrinum-contaminated rice Tsunoda (16) found enlarged kidneys to be the characteristic lesions caused by this fungus, which has been frequently isolated from peanuts (3), corn (9), rice (12), and other raw and processed agricultural commodities. Citrinin was first isolated by Hetherington and Raistrick (5) in 1931 from a culture filtrate of Penicillium citrinum Thom. It has since been produced by 10 or more species of Penicillium and Aspergillus, and is regarded as a potentially important mycotoxin that may be ingested by man and animals as a contaminant of wheat, barley, rye, and oats (4, 7, 13). Hetherington and Raistrick (5) obtained yields of 525 to 700 mg of citrinin per 350 ml of medium with P. citrinum Ad. 23 in 12 to 16 days at 28 to 32 C. Timonin and Rouatt (15) obtained 500 to 700 mg of citrinin per 200 ml of mineral salts medium with Aspergillus candidus incubated for 20 days at 26 C. Highest yields of citrinin were produced by A. candidus on media containing honey (15) or maple syrup (17) rather than glucose as the carbon source. In 1966 Rodig (11) produced the equivalent of 2.6 g of citrinin per liter in 14 to 16 days at 40 C with Aspergillus niveus and 1.2 g of citrinin in 35 to 40 days at room temperature with P. citrinum using Timonin's medium containing twice as much glucose. This paper reports production of citrinin in 5-gallon (18.925 liter) carboys containing 4 liters of 4% sucrose and 2% yeast extract nutrient solution. This medium was previously described for ochratoxin A production by A. ochraceus (1, 2). A strain of P. citrinum, isolated from corn associated with toxicity in horses in Alabama, proved to be an efficient producer of citrinin. Subsequently, we found that Timonin's isolate of A. candidus deposited in a culture collection had lost its ability to produce citrinin and he no longer had a viable culture (personal communication with M. I. Timonin). Increased demand for citrinin for mycotoxicological investigations made it desirable to investigate the conditions for production of the toxin by this high-yielding strain of P. citrinum. P. citrinum AUA 532, used throughout this investigation, was identified by D. I. Fennell of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., and accessioned there as NRRL 5927. After sporulation, cultures were stored at 4 to 5 C on slants of agar containing 2% dextrose, 0.7% yeast extract (Difco), 0.5% KH2PO4, and 2% agar, and also as lyophilized cultures stored at -30 C. Wide-mouthed, 5-gallon Pyrex carboys containing 4 liters of 4% sucrose and 2% yeast extract nutrient solution were stoppered with cotton plugs and autoclaved for 30 min at 121 C (2). These were inoculated with a suspension of spores from 10to 14-day-old cultures of P. citrinum and incubated at room temperature (30 4 3 C) as stationary cultures. Carboys were placed on their sides to give maximal surfacearea-to-volume ratio. Two experiments with seven carboys per experiment were conducted with yields being measured as crude citrinin produced after 8, 10, 12, 14, 16, 18, and 21 days of incubation. A third experiment involved 10 carboys with data being taken on the above days and also after 25, 27, and 31 days of incubation. Streptomycin sulfate (0.2 g/liter) was added to the medium in the latter experiment to protect against bacterial contamination. The procedure for extraction and isolation of citrinin was essentially as described by Hetherington and Raistrick (5). The mycelium and culture solution were filtered through Whatman no. 1 filter paper and the filtrate was acidified to pH 1.5 by stirring in approximately 50 ml of concentrated HCl per 4 liters of medium to precipitate the crude citrinin. After precipitation was complete, the bright yellow mass of c rude citrinin was filtered onto Whatman no. 42 filter paper, dried in vacuo at 50 C, weighed, and reported as total crude citrinin per liter of medium. The crude citrinin was dissolved in a minimal amount (150 ml) of chloroform and filtered to remove nonchloroform soluble impurities. The chloroform solution was evaporated to dryness, and the residue was taken up in hot ethanol, filtered, and recrystalized three times by the hot alcohol method of Hetherington and Raistrick (5). Comparison of the chemical and physical characteristics of the purified material with the ultraviolet, infrared, mass, and nuclear magnetic resonance spectra, melting point, and optical rotary power of citrinin as reported in the literature (6, 8, 10, 12) confirmed identification of the compound. Production of crude citrinin by P. citrinum in sucrose-yeast extract medium for varying periods of incubation is illustrated by the regression curve in Fig. 1 (data for three experiments were averaged and the curve plotted by quadratic regression). Citrinin was detected on day 8 with the highest yield recorded on day 21. The addition of streptomycin sulfate to the medium in the third trial had no apparent effect on citrinin production. As much as 7.3 g of crude citrinin was produced per carboy (4 liters of medium). Yields seemed to plateau and did not decrease thereafter. Apparently citrinin is not reabsorbed and further metabolized. Thus, production runs may be terminated at the convenience of the researcher rather than according to a predetermined or rigid time schedule. Surprisingly, yields were comparable to those scaled up (by calculation) from smaller systems (5, 11,15,17) rather than diminished as often occurs when small systems are scaled up. About 45 to 50% of the crude citrinin was recoverable in pure form by the method used (5). However, Timonin and Rouatt (15) reported that they recovered 70 to 80% of the crude product as pure crystalline citrinin using the dioxane method of Tauber et al. (14). Timonin and Rouatt (15) conducted a very thorough study on factors influencing citrinin production by A. candidus. Results of their investigation on A. candidus seem directly applicable to citrinin production by our P. citrinum isolate AUA-532. This isolate is maintained by lyophil in our cultun collection and is available for distribution. Small amounts of citrinin are also available as qualitative standards, or in larger amounts where circumstances warrant.

v3-fos

2018-04-03T03:55:43.131Z

1975-01-01T00:00:00.000Z

24630903

s2

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery ofbacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed. Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery ofbacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed. Bacteria associated with the gut mucosa have been described in a number of mammals (2,5,6) but the phenomenon has received only brief mention in ruminants (7). In a study of the rumen epithelium of sheep with the scanning electron microscope, objects resembling bacteria were observed on the epithelial surfaces. The persistence of these bacteria through extensive and thorough washing of the tissue suggested that they might be firmly attached to the epithelium. The present study was undertaken to investigate the extent and nature of the epithelial bacterial population. MATERIALS AND METHODS Romney wether sheep, aged from 1 to 4 yr and fed on mixed white clover (Trifolium repens L.)-perennial ryegrass (Lolium perenne L.; "Grasslands Ruanui") pasture, were slaughtered by severing the jugular vein. The rumen and reticulum were rapidly exteriorized and pieces of wall about 2 cm square were removed from specific sites. Each tissue sample was washed by shaking vigorously in 500 ml of 0.85% (wt/vol) sodium chloride at room temperature, and when free of adhering rumen contents (approximately 10 a) was immediately fixed in 10% (wt/vol) formalin. To minimize postmortem changes in the tissue, the manipulations were performed as rapidly as possible and all samples were fixed within 3 min of sacrificing the animal. The pieces of fixed tissue were washed further with jets of distilled water from a squeeze-bottle, attention being paid to the areas between the papillae, and were freeze-dried at -30 C. Dried tissue pieces were mounted, suitably orientated, on aluminum stubs with rubber cement. The preparations were coated with 20 nm of carbon, followed by 20 nm of gold-palladium, while being rotated in a vacuum chamber. The coated preparations were examined and photographed with a Cambridge Stereoscan scanning electron microscope, Mk 2a. Each epithelial sample was assessed for degree of bacterial cover by examination of a number of sites on two or more papillae. The degree of cover was scored on a scale from 1 (a few scattered bacteria) to 4 (dense mat of bacteria). The types of bacteria present were also recorded. Magnifications were mainly in the range x 2,000 to 5,000. RESULTS The distribution and degree of bacterial cover on the epithelial surfaces were investigated by examining pieces of tissue from 14 sampling sites in the ruminoreticulum of a single sheep. The sites examined are shown in Fig. 1. With each sample a search was made of surfaces of suitably orientated papillae. An example of the papillae in a typical preparation from the roof of the dorsal rumen is shown in Fig. 2. Bacteria were found on all of the surfaces examined but the numbers varied greatly in different areas of the ruminoreticulum. Most of the bacteria seen were on the dorsal, caudal, and lateral surfaces of the rumen, with the most dense populations on the roof of the dorsal rumen and on the floor of the caudodorsal blind sac (positions 4 and 5, Fig. 1). The rest of the rumen surfaces had only light or moderately heavy cover of bacteria. With the exception of one sample (see Fig. 5b) only scattered bacteria were seen on the reticulum samples. Areas on some of the papillae were covered with populations composed almost entirely of a single morphological type. To substantiate the observations that bacterial populations varied in both density and type on surfaces from various sampling sites, samples were obtained from nine other sheep. In each of these sheep pieces of tissue were taken from four areas in the rumen (positions 3, 4, 5, and 7 as described in Fig. 1). These areas had been found to vary in density and type of bacterial cover in the first sheep examined. A summary of the results for all 10 sheep is shown in Table 1. The differences found in the original animal were confirmed; similar bacterial cover and types were found in the same epithelial sampling sites in all animals. Examples of the degrees of cover observed are illustrated in Fig. 3a and b. In Fig. 3a, only a few bacteria are present on the epithelial surface and the granular projections covering the surface of the epithelial cell can be clearly seen. In Fig. 3b, the surface of the sample from the floor of the caudodorsal blind sac is densely covered with a mixed population. Minute local areas with a "Pure" population were frequently found and Fig. 4a shows such an area on a papilla from the roof of the dorsal rumen. A coccus can be seen colonizing the raised portion of the epithelial surface while the lower surfaces are covered by a population composed almost entirely of a long rod. Figure 4b illustrates the discrete spatial separation of the two forms. Details of structure of the two morphological types are shown in Fig. 4c (14) 3. peared to have a rough, pimpled surface. The rods were 0.3 to 0.5 gum by 3 to 7 ,um, usually curved and thickened in the polar region. Figure 5a represents a degree of cover intermediate between those in Fig. 3a and b, and shows a relatively pure population of a curved rod on a sample from the floor of the caudoventral blind sac. Figure 5b shows a localized high concentration of a short rod on a sample from the mid-reticulum. This was the only dense population of bacteria seen in any reticulum sample. In a number ofsites throughout the ruminoreticulum, pockets of a spiral organism were found. Figure 5c is typical of the heaviest population seen. Figure 5d gives more detail of its morphology. Although this spiral is associated with mixed populations on the epithelial surfaces, it does not correspond with any of the spiral organisms previously reported from rumen contents (1). It has been isolated and cultured and is being characterized. It differs in its small size (0.3 by 4 pum) and in the extremely tight coiling of the spirals, the coils of which are just visible under critical phasecontrast illumination. At high magnifications, thin thread-like structures were frequently seen between bacterial cells (Fig. 6a and b). DISCUSSION The bacteria found on the epithelial surfaces of the ruminoreticulum are occupying a specific ecological niche or are merely contaminants from the bacteria-rich rumen contents. A number of observations indicate that the bacteria may be true inhabitants of the epithelial surface. First, there was variation in bacterial cover in different areas of the ruminoreticulum. Second, when four areas in each of the nine animals were examined in detail, two areas consistently had heavy cover and two were consistently lightly covered. Third, epithelial areas were found with a single morphological type predominating, and these areas were consistent in location in all sheep. Fourth, at least one of the bacteria, the spiral ( Fig. 5c and d), has not been reported previously from rumen contents. However, failure to demonstrate its presence might be explained by its small size. The main types of bacteria found on the epithelium are rod shaped, as is the case in normal rumen contents. It is not possible to identify them on the basis of morphology so comparison with known rumen bacteria cannot be made. Isolation and characterization of some of these bacteria are in progress. Tamate et al. (7), in their study of bovine rumen epithelium, described colonies of a coccus with thread-like structures between the cells. The similar structures observed by us were associated with both coccal and rodshaped bacteria (Fig. 6a and b). The threads appear similar to mucus. However, mucus is APPL. MICROBIOL. on February 7, 2021 by guest http://aem.asm.org/ Downloaded from not secreted by the epithelium of the ruminoreticulum and it seems unlikely that the material could be derived from salivary mucus. The close association of the threads with individual bacterial cells suggests that they may be an extracellular product of bacterial metabolism. Nothing is known of the functional significance of these bacteria on the rumen epithelium. Indigenous populations of bacteria have been observed, by conventional histological methods and by transmission electron microscopy, on the mucosal epithelia of various regions of the gut of many animals. These animals include man, monkeys, swine, hamsters, rats, mice, and birds (2,5,6). The bacteria involved are believed to interact physiologically with the epithelium (5, 6) but the mechanisms are not fully understood. Some of these indigenous populations are firmly attached to the epithelium and some are merely embedded in mucin; most are in a position to receive nutrients from the adjacent epithelial cells. Interference with alkaline phosphatase has been postulated for bacteria in the duodenal epithelium of mice (8). The epithelium of the ruminoreticulum is an important absorptive surface and passage of some metabolites into the rumen also occurs (3,4). Attached bacteria would thus be in a favorable position to utilize nutrients passing through the wall. Other benefits of attachment to the epithelium can be postulated. An advantage would be conferred on organisms whose growth rate was insufficient to avoid being washed out of the gut. Also, in the rumen, ingestion by ciliate protozoa might be avoided. Further studies of these bacteria, including their isolation and growth in vitro, and determination of their substrate affinities may provide evidence for their role in this niche. ACKNOWLEDGMENTS The microscopy was carried out at the Physics and Engineering Laboratory, Department of Scientific and Industrial Research, Lower Hutt, and we are indebted to the staff of the electron microscopy section for their assistance and guidance. We thank C. S. W. Reid and D. Dellow for slaughtering and sampling the animals.

v3-fos

2020-12-10T09:04:22.639Z

1975-06-01T00:00:00.000Z

237230766

s2

Ultrastructure of Cell Envelopes of Bacteria of the Bovine Rumen Most of the bacteria found in rumen fluid samples taken from cows fed hay, or a concentrate diet, had cell walls of the gram-negative type. Most were intact, with only a small proportion of lysed cells, and many of the cells contained electron-translucent cytoplasmic deposits similar to the carbohydrate reserve material described in pure cultures of rumen organisms. All of the bacteria observed in these samples had an external “coat” layer outside the outer membrane when fixed in glutaraldehyde and osmium, stained with uranyl acetate and lead citrate, and examined as sectioned material. These coat layers varied from thin (ca. 8 nm) structures to very extensive fibrous systems, sometimes including concentric arrangements and radial fibers extending up to 1,200 nm from the cell. The thin-coat layers sometimes exhibited a rough periodicity. In all, 10 different types of coat layers were distinguished on a morphological basis. It is proposed that these external coat layers have protective and adherence functions for the rumen bacteria in the environment. Mixed microbial populations of certain specific environments have been described recently by direct observation with electron microscopy (2)(3)(4)15). Fletcher and Floodgate (15) examined marine bacteria adherent to surfaces, and Casida's group (3,4) described bacteria immediately after their elution from soil. A common finding in these and other studies is that each of the gram-negative bacterial cells that make up most of these populations is enclosed by an extensive and complex capsular structure external to the outer membrane. The marine organisms adhere to their substrate by a fibrous polysaccharide (15), and the capsule surrounding the soil bacteria is often fibrous in nature (2,3). These findings suggest that bacteria living in natural, challenging environments may depend for their survival on the production of external structures on the cell wall that dictate their adhesion pattern and provide a measure of protection for the cells (12). Salmonella typhimurium shows little external polysaccharide in shaken laboratory culture but produces a very extensive (150 nm) lipopolysaccharide microcapsule in infected tissue (26), indicating that cells in laboratory cultures may differ from cells in their natural environment. In contrast, some bovine rumen bacteria, grown in the rumen (6) or in pure cultures that have been repeatedly transferred in the laboratory (11,23), showed the presence of layers of fibrous polysaccharide outside the cell wall or of external patterns of globular units resembling the protein coats of Spirillum (5) and many marine bacteria (29). In one case (23), the fibrous polysaccharide coat of the cells of a rumen bacterium has been shown to mediate their attachment to cellulose fibers in pure culture. In this study, we used direct transmission electron microscopy to determine the extent to which complex cell coats are formed by bacteria within the rumen. MATERIALS AND METHODS Rumen contents were collected from eight fistulated cows fed a daily ration of 5.4 kg of a pelleted all-concentrate diet, or 8.2 kg of alfalfa hay, in two equal feedings. Samples were collected before the morning feeding and 4 and 8 h after this feeding on days 21, 27, and 35 after initiation of each of these diets. Rumen contents were filtered through four layers of cheesecloth and centrifuged at 48,000 x g at 4 C for 20 min. The pellet from this centrifugation contained most of the bacteria in the sample, and this pellet was prefixed for 1 h by the addition of 0.5% glutaraldehyde in 0.067 M cacodylate buffer at pH 6.8. Fixation was carried out by resuspending the material in 5% glutaraldehyde in 0.067 M cacodylate buffer at pH 6.8 for 2 h at room temperature. The material was enrobed in agar by resuspension in 4% agar at about 40 C and expressed by Pasteur pipettes 841 as a cylindrical core. The cores were washed five times in the cacodylate buffer, postfixed in 2% osmium in the buffer, washed five times in the buffer, and dehydrated through a graded acetone series before embedding in Vestopal (23). Thin sections were stained with uranyl acetate (2% aqueous) and then lead citrate (25) and were carbon coated before examination, using an A.E.I. 801 electron microscope. Glutaraldehyde and osmium were obtained as concentrated solutions, under argon, and the embedding materials were kept under freon to minimize oxidation and standardize block hardness. RESULTS Most of the bacteria seen in about 100 rumen samples showed the gram-negative pattern of cell wall structure, and very few were seen to have a thick peptidoglycan layer similar to that seen in pure cultures of Megasphaera elsdenii (11) and Ruminococcus albus (23). All of the cells examined had outer membranes of the usual dimensions (about 8.5 nm), and all had some structure external to this double-track layer ( Fig. 1-5). Ten different morphological variants of this external structure were discerned. One of the simplest of the extracellular coats was a single, thin, electron-dense layer, separated from the outer membrane by a regular space ( Fig. 1 and 2a, F). An irregular periodicity ( Fig. 1 and 2a, arrows) similar to that seen in cells of pure cultures of Bacteroides ruminicola (11,12) and short, irregularly spaced connectives between the coat layer and the outer membrane ( Fig. lb, C) were apparent in highmagnification electron micrographs of this structure. The absence of a fibrous coat on these cells is an important observation because it suggests that the fibrous coats seen on other cells are not the result of the nonspecific adhesion of fibers produced elsewhere in the rumen. A relatively simple coat structure was seen in the diffuse coat of particles and fibers that appear to be anchored directly to the outer membrane of some cells (Fig. lb, 2, 3, 4a, G). The particles were intensely electron dense and the fibers moderately electron dense, and the possibility must be considered that the particles are cross-sections of the fibers. These thin fibers extended up to 1.2 gm from the cell surface and, where similar cells were clustered, produced small areas of continuous fibrous material ( Fig. 2a, P). In a few cells, the extracellular coat consisted of a thin deposit of electron-dense material in the outer aspect of the outer membrane ( Fig. 2b, H). More often, cells showed a thicker (50 nm) layer of electron-dense material (Fig. 4a, I). In both cases, this intensely stained material appeared to be composed of fine granules that were aggregated into clusters in the thicker structure. One of the most common forms of the extracellular coat in these rumen organisms was a discrete mat of fibrous material (80 to 200 nm thick) with a distinct outer boundary ( Fig. 1 and 4c, J). This fibrous coat often served to connect the cell to a piece of detritus, to a different cell (Fig. 1), to a similar cell or, rarely, to a series of similar cells (Fig. 4c). Other types of extracellular coats, which were only rarely seen, were a highly convoluted, double-track structure outside the outer membrane with adherent bleblike structures (Fig. 2b, K), a homogeneous electron-dense mass maintained at a constant distance from the outer membrane by radial connective structures (Fig. 4b, L), and a thick electron-dense layer with thick and irregular radiating fibers (Fig. 5, M). Small numbers of cells in these rumen samples were enclosed by a single, thin, electron-dense layer, with apparent periodicity in tangential section. This layer was maintained FIG. 1-5. Electron micrographs of sections of bacteria in embedded samples of rumen fluid from cows with normal digestive processes. The cows whose rumen bacteria are illustrated in Fig. 1, 3, 4b, 4c, and 5 were fed on alfalfa hay, and those whose bacteria are shown in Fig. 2 and 4a were fed on an all-concentrate diet. All of the cells were labeled according to the type of external coat layer they possess, as follows. (F) These cells have a thin electron-dense coat that shows a rough periodicity where the sectioning angle is favorable (arrows). In some areas (C), fine electron-dense connectives can be seen between this layer and the outer membrane. at about 75 nm from the outer membrane by radial fibers that extended to it and beyond it into the menstruum (Fig. 3, N). A few cells were enclosed by two concentric electron-dense layers maintained at considerable distance from the outer membrane, and from each other, by radial fibers (Fig. 4a and 5, 0). Sectioned material is not ideal for the study of adhesion, but the fibrous extracellular coats of bacteria often appeared to mediate an adhesion of these rumen bacteria to food particles. In the bacterial rumen populations, we always found some cells that contained electrontransparent masses (Fig. 2b and 4b) within their cytoplasm. Each of these masses, like the a-1,4 glucan deposits (9) seen in cells of a pure culture of a rumen organism (M. elsdenii), were delimited by a single electron-dense layer. Because cells of all physiological ages were present in these samples, the appearance of their cytoplasm and the degree of condensation of their nucleoids was highly variable, but very few lysed cells were seen. DISCUSSION The relationship between a microbial population and its environment is mediated by the cell envelope of the bacterial cells. The cell envelopes of bacteria growing in the normal bovine rumen are predominantly of the gram-negative type, and all have additional cell coats outside the outer membrane. The bacteria of freshwater environments are also predominantly gram negative (M. Franklin, "Hotpack" lecture of Canadian Society of Microbiologists, Montreal, 1974), as are those of marine environments (17), and many of these bacteria have been shown to possess extracellular coats of fibrous carbohydrate (15,18) or of globular protein (5,29). Many enteric pathogens have been shown to produce externally located carbohydrate materials (16), and lipopolysaccharide, which is a component of the outer membrane, is actively shed into the medium (19,30) in shaken batch culture or accumulated around the bacterial cells in a capsular form in infected tissue (26). Similarly, bacteria eluted directly from the soil are often surrounded by a mat of fibrous material (2-4) that forms an enclosing capsule, and gliding bacteria exude a slime (22) that is important in their motility (13). Thus, it is clear that many bacteria can produce and assemble complex and often extensive coat layers on the outer surface of their already complex gram-negative cell wall (12). These gram-negative cell walls by themselves confer protection from antibodies (24), antibiot-ics (20), and other hazards of microbial life (12) and also maintain a molecular environment so that cell wall-associated enzymes are conditioned (28) and protected (8). Part of this protection is provided by the limited penetrability of the outer membrane, but the Donnan effect exerted by ions bound within the structural molecules that constitute the cell wall is also important in conditioning the molecular environment within the cell wall and in limiting the access of extraneous molecules and ions to the cytoplasmic membrane (12). Coat layers have been observed to confer protection from attack by predatory bacteria (Bdellovibrio) (F. L. A. Buckmire, Bacteriol. Proc., p. 43, 1971) and to inhibit phagocytosis (14). Whether coat layers are composed of carbohydrate or of protein, they must be expected to contain bound ions that would act in the manner of a complex ion exchange resin to further condition the molecular environment of the cell envelope and to limit its penetrability (12). Cell coats are also sometimes important in the adhesion of bacteria to surfaces in their environment (10,18). At least one species of rumen bacteria adheres to cellulose fibers by means of its polysaccharide coat layer (1,23), and the secondary and irreversible attachment of aquatic bacteria to surfaces is a function of their production of a carbohydrate material (10,15,21). That this attachment may be of physiological and ecological significance is indicated by the finding that Myxobacteria must adhere to the surface of blue-green algae for the enzymes associated with their cell wall to digest the cell walls of the algae (27). The predominance of gram-negative bacteria with extracellular coat layers in these environments may also result, in part, from their content of wall-associated enzymes. These enzymes have been shown to be located in the periplasmic space and at the cell surface of gram-negative cells (12), and some rough strains of S. typhimurium release an alkaline phosphatase-lipopolysaccharide complex into their environment (19). Studies of pure cultures of rumen organisms have shown that one "marker" enzyme (alkaline phosphatase) for the wall-associated group of enzymes is tenaciously bound to structural elements in the periplasmic space (7). The retention of degradative enzymes within the gram-negative cell wall and at its surface allows the enzymes access to external "food" molecules, even if these are insoluble polymers, and prevents the loss of these enzymes into the menstruum. The activity of these enzymes provides products that are spatially very close to the permeases that will 848 APPL . M ICROBIOL . BACTERIA OF THE BOVINE RUMEN transport them into the cell and that are vital to cellular growth. Thus we find that the predominant bacteria of the bovine rumen have a gram-negative cell wall with an additional external cell coat. This cell coat, which may be composed of protein or of carbohydrate, may function in adhesion of the cells to surfaces, and the whole cell envelope probably functions in the protection of the cell and the retention of cell wall-associated enzymes. This external coat layer takes 10 morphological forms in the material we examined and, although there is a possibility that capsules may change as the cells age (9), further studies indicate that there is an even greater variety of distinct capsular types among rumen bacteria.

v3-fos

2018-04-03T02:02:19.643Z

1975-06-01T00:00:00.000Z

32005846

s2

Laboratory Method for Fermentation of Meat and Poultry Sausages in Fibrous Casings' The construction and operation of a relatively inexpensive cabinet for sausage fermentation studies is described. Temperature can be controlled to 1 C with a relative humidity of approximately 95%. Laboratory-scale fermented meat studies generally employed rather conventional casings to hold the meat is the disadvantages that (i) the ratio of air to meat surface is considerably different from Laboratory-scale fermented meat studies have generally employed beakers rather than conventional sausage casings to hold the meat (3), is economical but has the disadvantages that (i) the ratio of air to meat surface is considerably different and restricted from that ) controlled via warm water bath heated by found with casings, and (ii) the fermented material cannot readily be smoked, cooked out, or dried for possible additional study. Small commercial scale air-conditioned smokehouses, providing temperature, humid-ity, and air-flow control, are quite expensive and not always available. Therefore, we designed a simple, relatively inexpensive cabinetheater combination that will permit fermentation studies in typical fibrous or natural casings. The cabinet (Fig. 1) was a conventional vat with lid, both made of a reinforced plastic material and of the type previously used for icing down poultry (Model GE795, Goodyear Aerospace Corp., Jackson, Ohio). The vat is available through most meat supply houses. Holes were drilled as shown to accommodate eight stainless-steel rods from which sausage chubs or links were suspended. Temperature control within the cabinet was achieved with a 16-liter external water bath equipped with a 1,000 W heater-circulator pump. This pump was used to circulate heated water from the external bath to the cabinet via tygon tubing, 9.5-mm inside diameter by 1.6-mm wall (0 by %A6 inch). A coil of copper tubing, 6.1 m by 9.5-mm inside diameter (20 feet by % inch), in the bottom of the cabinet was connected to the tygon tubing and served as a heat exchanger by transferring heat from the external water bath to approximately 25 liters of water covering the coil in the bottom of the cabinet. Cabinet air temperatures (dry bulb) above the coil-heated water of 30 and 38 C were achieved with external water bath settings of approximately 38 and 52 C, respectively. Sausage material with an initial internal temperature after stuffing of 10 to 12 C rose to 30 C in about 2.5 to 3 h and to 38 C in a total of about 4.5 h. The preceding rates were found for sausage stuffed in 52 mm diameter dry sausage fibrous casings (Union Carbide). Use of smaller or larger diameter casing would change the heat penetration rate. Humidity within the chamber averages approximately 95%, based on wet-bulb and drybulb readings. In industrial practice, a humidity control between 90 to 98% is obtained by introducing steam as the only heat source. It may be possible to achieve a lower humidity, near 90%, through use of various salt solutions although none have been tested in our laboratory. The rates of pH reduction for replicate sausage fermentations using the above cabinet and an air-conditioned industrial scale smokehouse have shown no significant differences. The rates of pH reduction in these units for a summer sausage mix inoculated with a frozen concentrate of Pediococcus cerevisiae (Lactacel, Merck and Co.) and held at 38 C are shown in Fig. 2. Lactic acid bacteria, as enumerated on the V-8 medium of Fabian et al. (4), were initially (postinoculation) at 8.6 x 106 cells/g and increased to 6.3 x 108 cells/g within 24 h. Studies of microbiological, meat chemistry, physical, and processing parameters involving fermentation with the above system have been reported elsewhere (5-7).

v3-fos

2020-12-10T09:04:22.678Z

1975-02-01T00:00:00.000Z

237235158

s2

Ecology of Soil Arthrobacters in Clarion-Webster Toposequences of Iowa Toposequence variations in soil properties were characterized and related to variations in populations of total isolatable bacteria and arthrobacters. Increases in soil NO3-N, available phosphorous, NO3-N-producing power, Arthrobacter counts, and the percentage of the total counts represented by arthrobacters were correlated with decreases in soil acidity. The total bacterial counts were not correlated with soil acidity but were associated with percentage of soil organic matter and percentage of clay. The percentage of the total counts represented by arthrobacters was lowest at the summit position and increased downslope to the highest value in the toeslope position. Factor analysis of the data revealed that 67 to 81% of the total variance exhibited by all variables per site-sampling period could be accounted for by soil acidity, soil structure, soil fertility, soil moisture, and bacterial factors. A selective medium was developed for soil arthrobacters and tested on a wide variety of central Iowa soils to determine its potential as a medium for enumeration as well as isolation. The medium developed in this study was found to be superior to the other available direct-isolation media for soil arthrobacters. Various studies have shown that members of the genus Arthrobacter are often among the more numerically predominant bacteria routinely isolated from soils (15,25). These soil arthrobacters are nutritionally very diverse (20,27), and many isolates can be found that exhibit the ability to degrade various pesticides (9,14,24). Very little work has been done, however, to determine possible correlations between variations in soil properties and variations in any particular group of soil bacteria. Soil pseudomonads have been found associated with slightly acid rhizosphere soil samples, whereas arthrobacters have been associated with slightly alkaline non-rhizosphere soil samples (22). Increased numbers of arthrobacters have been associated with soil samples adjusted to higher moisture contents, whereas pseudomonads have been predominant in soil samples adjusted to lower moisture contents (21). Topography is a very complex soil formation factor that could affect the bacterial populations by influencing certain soil properties through climate or drainage-related functions (11). Proceeding downslope from the shoulder position to the toeslope in Clarion-Webster toposequences of Iowa, the percentage of soil 'Present address: Department of Microbiology, Oregon State University, Corvallis, Ore. 97331. organic matter increases to a maximum while the mean particle size decreases to a minimum. The thickness of the A-horizon and the depth to carbonates or mottles decreases as the slope gradient becomes steeper (29). The toposequence soils in Clarion-Webster toposequences represent a gradation in textural classes; several studies have associated nematode populations with texture variations (18,19), but no attempts have been made to do this with bacterial populations. Soil organic matter levels are interrelated with other soil properties, and little is known about the effects of this relatively stable soil property of bacterial populations. Arthrobacters have normally been isolated from soils by using either enrichment techniques or by randomly picking and identifying isolates from media used to determine total counts (15). Mulder and Antheunisse (16) developed a selective procedure for arthrobacters involving two separate media where the identification was based on observation of a morphological cycle possessed by members of this genus. Their method was not intended to serve as a means of enumerating Arthrobacter populations and, because of the lack of a suitable enumeration procedure, one was developed in our study. 211 This study investigated variations in total isolatable bacteria and Arthrobacter populations in two toposequences in the Clarion-Webster soil association area in Iowa. Toposequence variations in soil properties were characterized in relation to their effects on the total bacterial and Arthrobacter populations. MATERIALS AND METHODS Site location and description. The two toposequences were located in the Clarion-Webster soil association area in north-central Iowa and are described in Table 1. Site I was in a corn-soybean rotation from 1963 to 1968 and in continuous corn from 1968 to 1973, whereas site II was in a cornsoybean rotation from 1963 to 1973. During this interval site I received no lime treatments, whereas site II received the appropriate amount of lime to maintain the soil pH at 6.9. Sample collection and sampling periods. Four adjacent rows of corn that extended parallel to the toposequence transect were chosen at each sampling site, and nine core samples were removed, three each from the middle of the furrow between adjacent rows of corn. Three sampling sites were chosen in each of the four soil types comprising the toposequences. The core samples were obtained and processed individually on 30 August at site I (soil temperature, 33 C) and 25 October at sites I and II (soil temperature, 26 C). All core samples were taken from the Ap-horizon at a depth of 10 cm at both sampling sites. Total bacteria analyses. All core samples were placed in plastic bags and transported to the laboratory, and platings were performed on the same day that each core sample was obtained. A 5.0-g sample was aseptically removed from the previously unexposed center of each core sample and suspended in 495 ml of sterile 0.5% peptone broth. Each sample was then agitated in a Waring blender for 3 min at low speed, serial dilutions in 0.5% peptone broth were Harps, 0%, Harps, 0%, none none made, and 0.1-ml portions of appropriate dilutions were spread over the surface of sterile media in petri plates. Total counts were made from a medium containing 0.1% peptonized milk (Difco), 0.1% yeast extract (Difco), 0.01% Acti-Dione (Upjohn Co.), and 1.5% agar. The pH was adjusted to the pH of the particular soil being plated and plates were incubated at 25 C for 10 days, after which colonies were counted. All platings were done in triplicate. Arthrobacter selective medium and analyses. Seventeen Arthrobacter named strains from the American Type Culture Collection (ATCC, Rockville, Md.) and 20 Arthrobacter, 6 Bacillus, 6 Micrococcus, 4 Nocardia, 4 Streptomyces, 4 Flavobacterium, and 6 Pseudomonas soil isolates were screened on 31 dyes, 13 antibiotics, and 11 assorted compounds to determine possible selective properties for the arthrobacters. The soil isolates were taken from the medium used to determine total counts and were identified to the genus level according to procedures outlined by Buchanan and Gibbons (5). All 67 cultures were tested on a wide range of concentrations of each of the 55 potential selective agents to detect any differential as well as selective properties. The screening was performed by incorporating the various concentrations of the potential selective agents in either Trypticase soy agar (BBL), peptonized milk agar, or nutrient agar (Difco). The three basal media were tested at a variety of concentrations with additions of various amounts of yeast extract as well as with the potential selective agents. Those agents that were heat sensitive were filter-sterilized and added aseptically to the cooled, autoclaved media. The media were adjusted to a variety of pH values ranging from 5.0 to 8.5. The cultures were transferred to the surface of the media with a multipoint inoculation device (7). All plates were incubated at 30 C for 72 h, after which plates were examined. Those compounds that exhibited either selective or differential properties for the arthrobacters were retested on various concentrations of the three basal media at varying pH values. A total of 720 different variations were examined to determine the best possible combination of a basal medium plus yeast extract plus various concentrations of different selective ingredients. The best selective medium had the following composition: 0.4% trypticase soy agar, 0.2% yeast extract, 2.0% NaCl, 0.01% Acti-Dione, 150 pig of methyl red (Harleco) per ml, and 1.5% agar. The methyl red was filter-sterilized and added aseptically to the autoclaved, cooled medium (see Results and Discussion). Soil samples were diluted and plated, and plates containing the selective medium were incubated at the temperature used for the total count medium. The pH was adjusted to the pH of the particular soil being plated. The selective medium was tested, on a variety of soils, to determine what percentage of the isolates were arthrobacters by subculturing and microscopic examination of all the colonies on various randomly selected plates for each soil type. The colonies were transferred to trypticase soy agar plus 0.2% yeast extract and examined microscopically for possession of a rod-to-coccus morphological cycle, snapping divi-sion, pleomorphism, and V-forms (5). The same procedure was also performed on plates containing the total count medium and the media developed by Mulder and Antheunisse (16). For enumeration of the arthrobacters in the four soils examined in the ecological survey ( Table 1), 78% of the counts on the selective medium was taken as the Arthrobacter counts. Soil analyses. After the bacterial analyses were performed, eight portions were taken from each core sample, two each for the following determinations: soil NO,-N (4), soil NH4-N (2), NO.N-producing power (26), and soil moisture (28). One particle size analysis was performed on each core sample by using a modified pipette method (28). The remainder of each core sample was air-dried and screened through a 4.0-mm sieve, and two replicate determinations for all procedures were made on each core sample. Soil pH, total exchangeable bases, exchangeable hydrogen (10), soil organic matter (6), available phosphorous (17), and soluble salts and saturation percentage (3) determinations were then performed. Statistical analyses. Simple correlation matrices were computed, and a preliminary set of factor-loading values for the factor analysis was computed from these matrices by using the principal components method (8). These factor-loading values were subjected to a varimax rotation (12) to maximize the factor loadings without changing the specific variance of each variable. The linear factor analysis model (23) used for each of the 16 variables was z l = a F, + aF. + aF3 +cE,. This model equation expresses each variable, z, in terms of three factors, F1 to F,, and an error factor E. The factor loadings, a and c, indicate the extent to which each factor participates in the test. The specific variance of the error factor for each variable indicates how much of the variation exhibited by the variables is not explained by the three factors. This particular factor analysis model was used because the results of a test of significance for the total number of factors indicated that there were not more than three factors involved at any one site-sampling period (13). In using this model we assumed that the sample size of 108 toposequence samples per site-sampling period was large enough to avoid sampling error. To insure this, only factor-loading values larger than 0.50 or smaller than -0.50 were considered significant correlational values. RESULTS AND DISCUSSION Of all the compounds tested as possible selective agents, only a few exhibited any selective properties for the arthrobacters. The combination of Acti-Dione at 0.01% and NaCl at 2.0% effectively inhibited all fungi and most streptomycetes, nocardia, and gram-negative bacteria. The methyl red at 150 ,g/ml inhibited other gram-positive bacteria (bacilli and micrococci) but did not affect the arthrobacters. The pH of the medium, between 5.0 and 8.5, did not affect its selectivity, and the combination of trypticase soy agar at 0.4% and yeast extract at 0.2% gave the highest yield of arthrobacters with the addition of the selective ingredients over the other basal media (data not shown). In testing the selective medium (Table 2), the percentage of the colonies identified as arthrobacters was much higher (74%) than that of either the total count medium (14%) or the nutritionally poor medium (24%). From the soils tested, approximately 25% of the colonies on the selective medium were not arthrobacters, and microscopic examination was necessary to distinguish them. In examining the selective medium for enumeration potential ( Table 3) the percentages of arthrobacters from the selective medium were close to or slightly higher than the percentages from the total count medium. Because of this close agreement, it was decided to use the selective medium for enumeration purposes by taking a percentage of the colonies growing on the plates as being the arthrobacter counts and comparing these with a Percentage of arthrobacters for the NPM and SM were obtained by using the numbers of arthrobacters from both of these media as determined from the data in Table 2. These figures were then compared with the total counts for each soil type to obtain the percentage of the total counts represented by arthrobacters for each of the media. AND HOLT APPL. MICROBIOL. the counts from the total count medium to arrive at the percentage of arthrobacters contained in any one sample. The nutritionally poor medium (Table 3) was not suitable for enumeration purposes. Further tests on the four soils used in the ecological survey (data not shown) indicated that 78% of the counts on the selective medium was a suitable figure for determining the arthrobacter counts from the respective soils. The largest total bacterial and Arthrobacter populations occurred at the toeslope position of both toposequences during each sampling period. The smallest total bacterial and Arthrobacter populations occurred at the backslope position and increased down to the toeslope and up to the summit position ( Table 4). The percentage of the total counts represented by arthrobacters was lowest at the summit and increased downslope to the highest percentage in the toeslope position. Pronounced changes in soil variables accompanied these variations in bacterial populations at each toposequence (Table 4). However, due to the higher pH caused by the limed conditions in the soils at site II, the variation in most of the variables was not as great as for either sampling period at site I. Proceeding from the summit to the toeslope position, there were increases in soil pH, percentage of clay, percentage of silt plus percentage of clay, soluble salt levels, percentage of organic matter, soil NO,-N, NO-N-producing power, available phosphorous, total exchangeable bases, percentage of moisture relative to field capacity, and percentage of moisture relative to percentage saturation. There were decreases downslope in the exchangeable hydrogen and soil NH,-N (Table 4). Due to the interpretational method of factor analysis, each of the factors was arbitrarily named, depending upon which variables appeared to be consistently interrelated ( Table 5). The soil fertility factor name was chosen because NO-N is the end product of nitrification and therefore is a useful indicator of the ability of the soil to supply plant-available NOU-N. The other factors were named according to the obvious combinations of variables composing the various factors. More of the variation in the soil fertility, acidity, structure, and bacterial variables was accounted for in factor analysis than variation soil moisture variables, which was indicated by higher specific variance values of the soil moisture variables compared with the other variables (Tables 6-8). At site I during both sampling periods (Table 6, 7), the soil acidity factor was positively correlated with soil NO-N, NO-Nproducing power, available phosphorous, Arthrobacter counts, and the percentage of the total counts represented by arthrobacters, and negatively correlated with soil NH,-N. The soil structure factor was negatively correlated with percentage of moisture relative to field capacity, total bacterial counts, and Arthrobacter counts. The absence of a soil fertility factor at site I on 30 August (Table 6) was probably due to interference by the roots of the corn plants with the soil fertility variables (uptake of available P and NO-N). By 25 October (Table 7) the roots were dead, the interference was removed, and a soil fertility factor, which was positively correlated with percentage of moisture relative to percentage of saturation, was generated. At site II (Table 8) the soil acidity factor was positively correlated with the same variables as at site I, but the degrees of correlation were not as great. The soil structure factor was not correlated with any variables, whereas the soil fertility factor was negatively correlated with the percentage of moisture relative to field capacity and positively correlated with the percentage of moisture relative to percentage of saturation, total counts, and Arthrobacter counts. At site I, increased soil acidity resulted in decreased soil NOO-N and increased soil NH,-N (Table 5). This was a measure of the lessened activity, due to acid sensitivity, of the nitrifying bacteria Nitrosomonas and Nitrobacter (1). The increased soil acidity was responsible for the decreased Arthrobacter counts and percentages of the total counts represented by arthrobacters. The total bacterial counts were influenced strongly by the soil structure factor (percentages of clay and organic matter) and, to a lesser degree, by the soil acidity factor. The Arthrobacter counts were positively correlated with acidity, but the degree of correlation was not as great (Tables 6-8) as was that of the percentage of the total counts represented by arthrobacters. This was due to the effects of the soil organic matter and clay content on the Arthrobacter counts, especially at the shoulder position (Table 4), whereas these variables did not significantly affect the percentage of the total counts represented by arthrobacters. The same relationships were found at site II, but the significant correlations were not as great due to the decreased variation in many of the variables caused by the limed conditions. During the 30 August sampling period at site I, 76.80% of the total variance removed by all factors was accounted for, whereas 80.92% was accounted for in the 25 October sampling period site I (Table 9). At site II 67.84% of the total variance was accounted for. The increased contribution of the soil acidity factor at site I accounted for the greater total percentage of the variance removed as compared with site H. The 20 to 30% of the total variation unaccounted for represented other unmeasured and/or unknown environmental factors. Factor analysis of the data from the toposequence soils examined in this study indicated that the arthrobacters in these soils were acid sensitive and their numbers decreased in a cause-and-effect relationship with increasing acidity. At site I on 25 October (Table 4), the percentage of the total counts represented by arthrobacters was 4.68% at the summit (pH 5.79) and increased to 23.39% at the toeslope as the acidity decreased (pH 7.42). At site H on 25 October (data not shown), the variation was less due to the limed conditions since, at the summit, 14.81% of the total counts were arthrobacters (pH 6.91) and increased to 20.26% at the toeslope as the acidity decreased (pH 7.38). The total bacterial counts were not correlated with soil acidity during any of the site-sampling periods, which probably indicated that as the acidity increased and the Arthrobacter counts decreased, the numbers of some type of acidtolerant bacterium were increasing. This study demonstrated that the distribution and abundance of certain types of bacteria (in this case, arthrobacters) were, to a large extent, determined by certain ecological variables. If the assumption is valid that microbial populations are selected by their environments, then the methodology applied in this study might find additional uses in determining which environmental variables most strongly influence the distribution of any of a wide range of microorganisms.

v3-fos

2018-04-03T02:57:53.691Z

1975-07-01T00:00:00.000Z

35665684

s2

Nephrotoxicity of Dietary Ochratoxin A in Broiler Chickens' Graded doses of pure ochratoxin A (0, 0.5, 1.0, 2.0, 4.0, and 8.0 ,g of toxin per g of feed) were incorporated into a commercial diet which was fed to chicks from 1 day to 3 weeks of age, at which time the experiments were terminated. Growth was inhibited at 2.0, 4.0, and 8.0 ug/g, whereas the kidneys were enlarged at doses of 1.0 Mg/g and above. Renal function as measured by clearance of phenol red was decreased 15 and 31% by doses of 4.0 and 8.0 ig/g, respectively. Uric acid was increased 38 and 48% over the control values by doses of 4.0 and 8.0 jg/g, respectively. The plasma electrolytes Na, Cl, Ca, and K were measured; however, only K was significantly (P < 0.05) altered, showing a decrease at doses of 4.0 and 8.0 ,g/g. The percentage dry weight of the kidneys decreased significantly at dose levels of 4.0 and 8.0 ,g/g, indicative of edema. Histological examination of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens. Ochratoxins include a of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens. Ochratoxins include a group of structurally related, secondary metabolites produced by seven species of Aspergillus and six species of Penicillium (5). Aspergillus ochraceus, from which the toxins acquired their name, appears to be the predominant ochratoxin producer (5). Ochratoxin A, which is the most toxic and most prevalent form, is 7-carboxyl-5-chloro-8hydroxyl -3,4 -dihydro -3 -R-methylisocoumarin linked through an amide bond to L-fl-phenylalanine (25). The widespread occurrence of ochratoxin-producing fungi, their ability to grow on a variety of economically important feed and foodstuffs (5), and the natural occurrence of ochratoxin (8,17,18; P. Krogh, 2nd Int. Cong. Plant Pathol., Abstr. 0360, 1973) constitute a threat to both animal and public health. The dimensions of this threat are undefined as yet, but excellent reviews of the literature exist (5,20). Only a limited amount of research has been done on ochratoxicosis in poultry. Huff et al. (10) provided a general description of the disease in young broiler chickens and concluded that on the basis of acute mean lethal dose and minimal growth inhibitory concentration ochratoxin A was the most potent mycotoxin yet studied in chickens. They also reported that the most sensitive indicator of ochratoxicosis in young broiler chickens was enlarged kidneys. A IPaper number 4592 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. 48 microscopic evaluation of acute ochratoxicosis in young White Leghorn cockerels (6,12) revealed acute nephrosis, hepatic degeneration, and suppression of hematopoiesis in descending order of frequency. On the other hand, a histopathological study of ochratoxicosis in ducklings (23) revealed primarily hepatic degeneration characterized by an increase in fatty vacuolation, changes in the matrix of the mitochondria, and disorganization of the endoplasmic reticulum of hepatocytes. In laying hens ochratoxin at low concentrations delays sexual maturity and reduces egg production and hatchability (4). Because of the high toxicity of ochratoxin to chickens and because ochratoxin can be produced in a variety of feedstuffs used in the poultry industry, it was deemed desirable to study in more detail ochratoxicosis in the economically important broiler chicken. In particular, the nephrotoxicity of ochratoxin was investigated. (A preliminary report of part of this paper was presented at the 74th Annual Meeting of the American Society for Microbiology, Chicago, Ill., 1974.) MATERIALS AND METHODS Animal husbandry. Day-old male broiler chicks obtained commercially were used in these studies. The birds were housed in electrically heated batteries under continuous lighting. Feed and water were available ad libitum. The feed was a commercial on March 18, 2020 by guest http://aem.asm.org/ Downloaded from VOL. 30, 1975 broiler-starter from which all medications were omitted. Ochratoxicosis was induced by incorporating into small portions of the diet weighed amounts of crystalline ochratoxin A dissolved in 95% ethanol. The portions of feed containing the ochratoxin A were dried at 100 C to evaporate the ethanol before being mixed into the remainder of the feed. The experimental diets were fed from hatching until the experiments were terminated at 4 weeks. Ochratoxin A. A. ochraceus NRRL 3174 was grown on wheat by the method of Trenk et al. (24). Ochratoxin A was extracted from the wheat by the method of Steyn and van der Merwe (21) and purified by thick-layer preparatory chromatography on Silica Gel G using benzene-acetic acid (9:1, vol/vol) as the solvent (7). Ochratoxin A was eluted from the silica gel by making a slurry of the silica gel in hot benzene-acetic acid (9:1, vol/vol) solution. Approximately 100 ml of solution per g of Silica Gel G was used. The slurry was then filtered through paper. This procedure was repeated twice to insure that all of the ochratoxin A was removed from the silica gel. The combined filtrates were evaporated to 1% of the total extract volume. Cold benzene was then added to precipitate the ochratoxin A. The ochratoxin A crystals were then recrystallized twice from benzene and washed repeatedly with cold benzene before allowing to dry in the air. Assays. The growth rate of the chickens was determined by weighing the chickens weekly. A renal function test was conducted by the method of Pitts (14). Plasma uric acid levels were measured by the method of Caraway (3). Serum potassium, sodium, calcium, and chloride ion concentrations were measured by an independent laboratory (Rex Hospital, Raleigh, N.C.) using Technicon S.M.A. methodology. Tissue taken from the right kidney was fixed in neutral buffered formalin and prepared for histopathological examination by standard procedures. The relative kidney weight and percentage dry weight of the kidney were also measured. Experimental design. There were four replicates of 10 birds at each dose level. The dose levels were 0, 0.5, 1, 2, 4, and 8 ug of ochratoxin A per g of diet. The treatments and birds were completely randomized. The replicate means were evaluated statistically by analysis of variance in which an F ratio was calculated. If the F ratio were significant (P < 0.05), the treatment means were compared by the method of least significant differences (2). RESULTS The effect of graded doses of ochratoxin A on the parameters measured in this study are given in Table 1. The growth rate of broiler chickens was inhibited significantly (P < 0.05) at doses of 2.0 ttg/g and above, and the degree of inhibition was dose related. The weight of the left kidney relative to the total body weight was increased by doses of 1 ,gg/g and above, which agrees with the previous report (10). At the highest dose the relative kidney weight was more than doubled. The NEPHROTOXICITY OF OCHRATOXIN 49 enlargement of the kidney could be the result of edema, a general increase of protoplasm, or an increase of a specific constituent(s). The possible occurrence of edema in the kidney was tested by determining the dry weight ratios at the different dose levels. This ratio was decreased significantly at doses of 4 and 8 gg/g. Thus, the enlargement of the kidney during ochratoxicosis results in part from edema. The effect of dietary ochratoxin on the excretory function of the kidney was determined by measuring the rate of clearance of phenol red from the blood by the kidney. The rate of clearance was decreased significantly (P < 0.05) at 4 and 8 Ag/g. This impairment of renal excretory function should be reflected by alteration in the levels of blood constitutents which are excreted by the kidney. The primary product of nitrogen catabolism in the chicken is uric acid (22). The plasma uric acid levels were increased significantly (P < 0.05) at dose levels of 4 and 8 Ag/g, with an approximate 50% increase at the highest dose. During kidney impairment an electrolyte imbalance is often seen. Serum potassium levels were decreased, and this hypokalemia also was significant (P < 0.05) at 4 and 8 Mg/g. Sodium, calcium, and chloride concentrations in the serum were measured, but they were not altered from control values. The most striking histopathological changes seen in the kidney during ochratoxicosis were swelling of the tubular epithelial cells, tubular dilation, and proteinaceous material in the lumen. The severity of these changes appeared to parallel the dose of ochratoxin A administered. There was also a differential effect on the convoluted tubules in that the proximal portion was more severely affected than the distal portion to the extent that some generalized necrosis of the epithelial cells of the proximal tubules was observed. DISCUSSION The primary effect of ochratoxin A in chickens appears to be on the kidneys. The most sensitive visible indicator of ochratoxicosis is kidney enlargement (10). This enlargement, which occurred at doses as low as 1 jug/g, was ment of function represents a compensation by the chicken to the nephrotoxicity of ochratoxin. Once the compensating ability of the kidney is overmatched, alteration in function would be expressed. The suggestion that ochratoxin is primarily a nephrotoxin in broiler chickens agrees with prior observations on acute ochratoxicosis in White Leghorns (6,12), where the kidney was the organ most frequently affected. It differs from the observations with ducklings, where the basic lesion of ochratoxicosis is fatty infiltration of the liver, and from the rat, where both the liver and kidney appear equally affected (13) and the liver exhibits a hyaline degeneration and focal necrosis (23). Indeed, the nephrotoxicity of ochratoxin in broilers offers an easy differential diagnosis of ochratoxicosis from aflatoxicosis, which is primarily a hepatotoxin in broilers (19). The exact mechanism whereby ochratoxin exerts its nephrotoxicity cannot be stated on the basis of the present study, but some comments can be made. Extensive kidney damage is indicated by the renal function test, by the accumulation of uric acid in the blood, and by the severe histopathological changes observed. However, the differential effect on serum potassium and serum sodium suggests a differential effect on the kidney tubules. Sodium and chloride presumably are reabsorbed uniformly along the proximal and distal tubules (1). Potassium, on the other hand, is reabsorbed in the proximal and secreted in the distal tubules (11,15). Ochratoxin in rats differentially damages the proximal tubules (16) as it does in chickens. The lowered blood potassium can then be explained on the basis that its reabsorption is impaired during ochratoxicosis. It should not be assumed that ochratoxin is only a nephrotoxin in chickens. For example, growth is inhibited at 2 Mg/g, whereas a dose of 4 gg/g is required to depress the kidney excretory function, and at 8 ,ug/g the renal clearance of phenol red is decreased only 31%. A loss of 75% of the kidney causes only a slight decrease in an otherwise healthy chicken (9). This suggests a more direct effect on the growth process. It is interesting that ochratoxin A can cause an inhibition of carbohydrate and protein metabolism and oxidative phosphorylation in rats (5). In addition, ochratoxin appears to have an affinity for the gastrointestinal tract. In broilers treated the same as in the present study, an enlargement of the crop, proventriculus, and gizzard was noted (10). In acute lethal ochratoxicosis, chickens exhibit a severe enteritis (10,12). The neural system is also affected during acute lethal ochratoxicosis, although no neural disturbances were noted in the present study. It seems obvious that more detailed studies of ochratoxicosis are needed before this disease can be understood.

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Production of Aflatoxin on Soybeans Probable factors influencing resistance to aflatoxin synthesis in soybeans have been investigated by using cultures of Aspergillus parasiticus NRRL 3240. Soybeans contain a small amount of zinc (0.01 μg/g) bound to phytic acid. Autoclaving soybeans at 15 pounds (6803.88 g) for 15 min increases the aflatoxin production, probably by making zinc available. Addition of zinc to both autoclaved and nonautoclaved soybeans promotes aflatoxin production. However, addition of varying levels of phytic acid at a constant concentration of zinc depresses aflatoxin synthesis with an increase in the added phytic acid. In a synthetic medium known to give good yields of aflatoxin, the addition of phytic acid (10 mM) decreases aflatoxin synthesis. Aflatoxins are hepatotoxic metabolites produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. Production of aflatoxin by A. flavus strains on agricultural crops like groundnut, cottonseed, wheat, rice, barley, coconut, corn, dried peas, oat, sweet potato, millet, and cassava is well documented (4). In view of the wide acceptance of soybeans as an excellent source of nutrition to both man and poultry, due to its high protein content, the question of whether soybeans can become contaminated with aflatoxin assumes importance. There is little information on aflatoxin production in soybeans by A. parasiticus. Reports available are controversial. In a field study involving a survey of 866 commercial samples in the United States, Shotwell et al. observed that the incidence of aflatoxin contamination was only 0.8% although 50% of the samples were contaminated with A. flavus (16). Chang et al. (1) could not detect aflatoxin in moldy soybeans contaminated with toxigenic isolates of A. flavus though they were able to demonstrate aflatoxin production with A. parasiticus NRRL 2999. However, Davis and Diener (2) obtained considerable yields of toxin (48 to 138,ug/ml) on the Bragg variety of soybean after 21 days of incubation with a toxigenic strain of A. parasiticus. Nagarajan et al. (12) also found toxin production (0.12 to 31.25 ,g/ml), using different isolates of A. flavus and A. parasiticus. Therefore, the present study was undertaken to find out the probable factors controlling the production of aflatoxin on soybeans. MATERIALS AND METHODS The Lee variety of soybean and the A. parasiticus strain NRRL 3240 used in the present study were obtained from the Indian Agricultural Research Institute, New Delhi, India, and the Northern Regional Research Laboratory, Peoria, Ill., respectively. Phytic acid was obtained from B.D.H., England. Synthetic medium. Sucrose-low salt medium used in this study has the following composition: 85 g of sucrose, 10 g of asparagine, 3.5 g of (NH4)2S04, 10 g of KH2PO4, 2 g of MgSO4.6H,0, 75 mg of CaCl2 2H20, 10 mg of ZnSO4.7H20, 5 mg of MnCl2.-4H20, 2 mg of ammonium molybdate, 2 mg of Na2B4O7, and 2 mg of FeSO4.7H20 made up to 1,000 ml with double distilled water. The pH of the medium was adjusted to 4.5. Twenty grams of finely powdered soybean was placed in each 500-ml conical flask and enough water (about 20 ml) was added to just moisten the powder. The flasks were divided into two groups. Group I was autoclaved at 15-pounds pressure for 15 min, and group II was not autoclaved. A spore suspension, prepared in sterile double distilled water from a 5-to 6-day-old culture of A. parasiticus grown on glucosepeptone agar, was distributed equally among the flasks (the inoculum usually contained 3 x 106 to 3.5 x 106 spores). The flasks were incubated at 26 + 1 C for 8 days. At the end of the incubation period flasks were sprayed with 95% alcohol and dried overnight at 80 C as reported by Nagarajan et al. (12). The dried samples were first defatted with n-hexane and then extracted with chloroform. The chloroform extract was dried overnight over anhydrous sodium sulfate. The sodium sulfate was filtered off, and the extract was evaporated and made up to a known volume. Aflatoxins were separated by thin-layer chromatography using the solvent system toluene:iso-amyl alcohol:methanol (90:32:3, vol/vol/vol) (15). Individual aflatoxin bands were eluted with methanol and estimated by spectrophotometry using extinction coefficients reported by Nabney and Nesbitt (11). Experiments were carried out in triplicate and data presented are the average of values from three separate flasks. 834 Table 1 shows the effect of autoclaving on the production of aflatoxin in soybeans. Total aflatoxin production with nonautoclaved soybeans was only 0.34 mg per 100 g as compared to 6.85 mg per 100 g for autoclaved soybeans. This clearly indicates that exposure of soybeans to high temperature even for a short period promotes high production of aflatoxin. A similar observation has been made by Kratzer et al. (8) regarding the availability of dietary zinc for turkey poults. Many reasons have been advanced for the resistance to aflatoxin synthesis in soybeans by A. flavus. These are conditions of moisture unfavorable to the fungus at the time of soybean maturity, development of the seed in a closed pod, and the possibility of an inhibitor in soybeans preventing growth of the fungus (7). According to Nagarajan et al. (12), soybeans produce aflatoxin but the extent of production depends on the variety of soybean and the toxigenic potential of the isolate used. Soybeans are known to contain a high amount of phytic acid. In terms of phosphorous content, the phytic acid present constitutes 690 mg per 100 g of the edible portion as compared to 190, 390, 306, and 298 mg in rice, groundnut, wheat, and peas, respectively (5). Phytic acid is known to bind with zinc (13). It is likely that the negligible amount of aflatoxin synthesis observed with nonautoclaved soybeans was due to binding of zinc with phytic acid. Many workers have shown that swine develop parakeratosis, a zinc deficiency disease, when fed on soybean protein. O'Dell et al. (14) and Smith et al. (17) have reported that availability of zinc to animals fed plant protein was poor as compared to that in those fed animal protein. Zinc is known to have a pronounced stimulatory effect on aflatoxin production (3,9,10). Mateles and Adye (10) reported a reduction of 88% in toxin yields in the absence of zinc. The observed synthesis of high amounts of aflatoxin on autoclaved soybeans is probably due to the destruction of phytic acid by heat, resulting in the availability of zinc for aflatoxin synthesis. Table 2 shows the effect on aflatoxin production by addition of zinc sulfate to nonauto- claved soybean. There is a gradual increase in aflatoxin formation with an increase in zinc sulfate from 1.0 to 5.0 g per flask. Addition of 5.0 g of zinc sulfate is observed to give a total yield of 9.60 mg of aflatoxin per 100 g of soybean. In the case of autoclaved soybeans, it was expected that it would increase the production of aflatoxin (from Table 1) because on high temperature the phytic acid structure is broken down and zinc would be released. To find out whether addition of zinc has any effect on autoclaved soybeans, only one concentration, i.e., 5.0 g of zinc sulfate, which gave maximum yield in the case of nonautoclaved soybeans, was studied. It was found that aflatoxin synthesis increased from 6.55 mg per 100 g of soybean to 9.60 mg per 100 g of soybean. This shows that on autoclaving the soybean zinc is released but it is not enough to synthesize maximum aflatoxin (Table 3). Glick (6) has shown that the zinc content of soybeans is only 0.01 sg/g, whereas that of rice, barley, legumes, oats, and wheat is 18 to 19, 14 to 15, 22 to 23, 1 to 35, and 1 to 24 ,g/g, respectively (6). The observation in the present study of a synthesis of aflatoxin on autoclaving soybeans and also on addition of zinc sulfate to both autoclaved and nonautoclaved soybeans can be explained on the basis of availability for aflatoxin synthesis of zinc, which under usual conditions is low in amount and bound to phytic acid. This would lead to the conclusion that soybeans contain only a small amount of zinc which is present in a bound form with phytic acid. In view of the stimulation by zinc of aflatoxin production in soybeans, it was of interest to find out whether phytic acid blocks aflatoxin synthesis. The results are presented in with phytic acid and is essential for the formation of aflatoxin. On the basis of the observation that phytic acid inhibits aflatoxin formation by combining with zinc, a further study was carried out to find out the effect of phytic acid on sucrose-low salt synthetic medium which gave good yields of aflatoxin. The results are presented in Table 5. Sucrose-low salt synthetic medium gives good yields of aflatoxin, about 24.0 mg per 100 ml of medium, but addition of 10 mM phytic acid to the medium reduces aflatoxin production to 3.15 mg per 100 ml of medium, indicating that phytic acid binds with zinc which is an absolute requirement for aflatoxin synthesis. APPL. MICROBIOL.

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Use of pH Gradient Plates for Increasing the Acid Tolerance of Salmonellae Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9- to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains. The minimum pH for growth of salmonellae has been studied by a number of workers. Stokes and Bayne (8) did not observe colony formation in trypticase soy agar buffered with phosphate-phosphoric acid at pH 5.0 with any of several dozen strains and serotypes of salmonellae. Even at pH 6.0 Salmonella gallinarum and S. pullorum grew very poorly. The optimum for all strains was pH 7.0, with many strains growing equally well at pH 8.0. On the other hand, in liquid egg white, Banwart and Ayres (1) found S. pullorum and several other strains to grow at a more rapid rate at pH 6.0 than at 7.0 and faster at 7.0 than at 8.0. Reduction in numbers occurred at pH values of 9.0 or above. Prost and Reimann (6) indicated that pH values above 9.0 and below 4.5 had a killing effect on salmonellae with destruction in a matter of minutes in lemon or lime juice (pH 2.3 to 2.5), whereas tomato juice (pH 4.3 to 4.5) was less bactericidal, with survivors found after 10 to 30 days. Chung and Goepfert (2) showed that the type of acid was important in determining the minimum pH for growth. Hydrochloric or citric acids required a pH of 4.05 to inhibit growth of S. anatum, S. tennessee, and S. senftenberg; lactic acid inhibited at pH 4.40, whereas acetic and propionic acids inhibited at pH 5.40 and 5.50,respectively. Levine and Fellers (5) also showed that the type of acid was important for determining minimum pH for growth, acetic acid being more toxic than lactic or hydrochloric acids. The bactericidal action of volatile fatty acids on S. typhimurium was shown to increase when the pH was lowered from 6.0 to 5.0 (3); there also appeared to be a decrease in bactericidal activity with increasing chain length of the acid. Many foods are naturally or artificially protected against microbial contamination. Smith and Palumbo (7) recently reported on the microbiology of Lebanon bologna manufacture. They found a considerable population of lactic acid bacteria during the fermentation, with a final pH of from 4.6 to 5.6, due most likely to lactic acid. Lactic acid was demonstrated to be the chief acid produced in Swedish sausage (4). Salmonellae are found in meat products and could be present in the raw materials for sausage manufacture. The study reported here was undertaken to determine if salmonellae could survive the acidity of fermented sausage and become adapted to growing at increasing lactic acid concentrations. MATERIALS AND METHODS Gradient plates. The two-layer gradient agar technique of Szybalski and Bryson (9) was used with square plastic petri dishes having bottom dish dimensions of 90 mm with a depth of 14 mm. The bottom layer was 25 ml of tryptic soy agar (TSA; Difco) poured while the lugs of one end of the dish rested on plastic tubing with a diameter of 6 mm. After solidification, the plates were placed on a level surface and a second layer of 27 ml of TSA containing 0.34 or 0.68% lactic acid was poured. The plates were then placed in a refrigerator for 20 to 24 h. Cultures and media. Salmonella serotypes were received from B. Blackburn of the National Animal Disease Laboratories, U.S. Department of Agriculture, Ames, Iowa, and were maintained on TSA slants. Cultures were streaked on the plates by means of a needle with a semicircular curve of about 2 mm diameter, using several back-and-forth strokes. Incu-bation was at 35 C for 20 to 24 h. pH measurements. The pH of the surface of the gradient plates was determined by placing Whatman no. 1 filter paper strips (10 by 35 mm) on appropriate portions of the equilibrated plates for 20 min. The strips were then placed in a 10-ml beaker, and 1.5 ml of distilled water was added. Minimum pH for growth. The lowest pH for growth in tryptic soy broth was determined by adjusting the pH with 20% lactic acid. The pH values did not change appreciably after autoclaving. The inoculum was 1 drop per 4 ml of a small amount of growth from the gradient plates or slants emulsified in 4 ml of H20. RESULTS The growth of the salmonellae on pH gradient plates is shown in Table 1. The cultures used as inocula were from 12-day-old TSA slants kept at room temperature. The pH values of 10-mm increments, as measured by the filter paper method, were 4.60, 4.55, 4.65, 4.75, 5.05, 5.35, 5.90, 6.40, and 6.75. The most acid-resistant culture was S. grumpensis, growing at a distance of 30 mm from the most acid end. The next most resistant was S. dublin (34 mm) followed by S. saint-paul and S. tournai (39 mm). These were in the 10-mm area, showing a pH of 4.75. All the other strains grew in the area with a pH of 5.05. Eleven of the salmonellae were further studied. Fresh slants were prepared from the original cultures used for the experiment of Table 1 and these (wild) cultures were compared with inocula taken directly off the gradient plates at the point of growth nearest the acid end (acid conditioned) ( Table 2). There was an appreciable increase in acid resistance with all cultures after growing on the gradient plates. The greatest differences of growth termini, indicating the greatest degree of acid conditioning, were with S. madelia (32 mm), S. braenderup (31 mm), S. cerro (29 mm), S. meleagridis (28 mm), and S. havana (25 mm). The least acid conditioning noted was with S. montevideo (7 mm). The change in growth-limiting pH of S. madelia appeared to be from 5.20 (50 to 60 mm) to 4.60 (20 to 30 mm), using the filter paper strip method of measuring surface pH values. A more accurate determination of the minimum pH values permitting initiation of growth of several of these salmonellae and S. typhimurium (similarly acid conditioned) was made in tryptic soy broth adjusted with 20% lactic acid to pH values of 4.2 to 5.3, in increments of 0.1 pH units. The pH values remained constant when measured after autoclaving and cooling (Table 3). S. madelia, which showed the great-TABLE 1. Growth of salmonellae and other Enterobacteriaceae on pH gradient platesa est increase in acid resistance in agar, was also the most acid resistant in broth, growing at pH 4.2; however, 2 days were required for growth to be evident. S. montevideo, which apparently showed the least acid conditioning in agar, showed an appreciable increase in acid tolerance in broth, with growth at pH 4.4 for the conditioned strain and 5.3 for the wild strain. Most of the salmonellae tested showed increases in acid tolerance of about 0.8 pH units. S. dublin (wild type) failed to grow at pH 5.3, the highest tested, although in another test it grew at 5.2 (wild) and 4.6 (acid conditioned). The conditioned strains quickly lost their acid resistance. After transferring two times on TSA slants, the conditioned strains were no more resistant than the wild. Tests for survival of the original inoculum in a Surface pH values (by the filter paper strip method) were 4.55 (0 to 10 mm from most acid end), 4.55 (10 to 20 mm), 4.60 (20 to 30 mm), 4.65 (30 to 40 mm), 4.95 (40 to 50 mm), 5.20 (50 to 60 mm), 5.80 (60 to 70 mm), 6.25 (70 to 80 mm), and 6.70 (80 to 90 mm). the tryptic soy broth were performed after 1 day of incubation on the two tubes, with pH values immediately below those showing visible turbidity. For these tests, 0.25 ml of the tryptic soy broth, previously inoculated with either wild or acid-conditioned strains, was placed into 4.5 ml of new broth and the tubes were reincubated for 3 days. The pH values of the new tubes, without further adjustment, were 5.40 for the original pH 4.20, 5.65 for the 4.40, 5.85 for the 4.60, and 6.05 for the 4.80. These pH values were high enough to permit growth of any of the salmonellae. All tests were negative, indicating that the effect of these pH values was bactericidal rather than bacteriostatic. DISCUSSION The results reported here indicate that salmonellae can become adapted to growing under more acid conditions, although this does not appear to be a genetic change, the characteristic being lost very soon after transferring to a neutral agar medium. Fermented foods such as Lebanon bologna ordinarily require several days for the pH to reach low enough levels to inhibit salmonellae. During this time salmonellae, if present, could conceivably become adapted to the acidity and persist in the finished product. A more rapid increase in acidity, such as that produced by adding starter lactic cultures, could possibly prevent such adaptation, although Chung and Goepfert (2) could not demonstrate any degree of increased acid tolerance in liquid media. This may indicate that the acid conditioning, as reported here, takes place only during aerobic growth as on the surface of pH gradient plates. This may be a reflection of the difference in metabolism of these cultures under aerobic conditions (tricarboxylic acid cycle) and in liquid, nonaerated systems (Meyerhof-Embden-Parnas system). LITERATURE CITED

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Degradation of 3-Hydroxybenzoate by Bacteria of the Genus Bacillus The pathway whereby certain bacterial strains of the genus Bacillus degrade m-hydroxybenzoate is delineated. Of 12 strains examined, nine were tentatively classified as representatives of the species Bacillus brevis, two of Bacillus sphaericus and one of Bacillus megaterium. All strains degraded m-hydroxybenzoate via the same pathway. m-Hydroxybenzoate was hydroxylated to 2,5-dihydroxybenzoate (gentisate), which was oxidized by a gentisate 1,2-dioxygenase yielding maleylpyruvate. Maleylpyruvate was hydrolyzed without prior cis, cis to cis, trans isomerization yielding pyruvate and maleic acid. Numerous soils were examined by plate-count procedures and found to contain 104 to 106 aerobic sporeformers able to grow on m-hydroxybenzoate per g of dry soil. The pathway whereby certain bacterial strains of the genus Bacillus degrade m-hydroxybenzoate is delineated. Of 12 strains examined, nine were tentatively classified as representatives of the species Bacillus brevis, two of Bacillus sphaericus and one ofBacillus megaterium. All strains degraded m-hydroxybenzoate via the same pathway. m-Hydroxybenzoate was hydroxylated to 2,5dihydroxybenzoate (gentisate), which was oxidized by a gentisate 1,2-dioxygenase yielding maleylpyruvate. Maleylpyruvate was hydrolyzed without prior cis, cis to cis, trans isomerization yielding pyruvate and maleic acid. Numerous soils were examined by plate-count procedures and found to contain 10( to 106 aerobic sporeformers able to grow on m-hydroxybenzoate per g of dry soil. Bacteria of the genus Bacillus (bacilli) are an important component of the microflora of most soil and water environments and thus are potential agents ofbiological transformation and degradation of aromatic compounds that enter soil/water ecosystems. Unfortunately, little is known concerning the ability of Bacillus species to catabolize aromatic molecules. Certain Bacillus strains appear to degrade benzenoid compounds via reaction sequences similar to those described in other bacterial genera (1,2,9,17,(22)(23)(24)(25). However, recent investigations indicate that many bacilli may catabolize aromatic molecules via reaction sequences involving novel chemistry (4, 6, 10-12, 20, 21). The following describes my recent investigation into the catabolism of 3-hydroxybenzoate by various species of Bacillus. This investigation is part of a continuing project which has a goal of determining the mechanisms whereby bacteria ofthe genus Bacillus degrade aromatic molecules. MATERIALS AND METHODS Isolation, identification and growth of the microorganisms. Bacillus strains were isolated from pasteurized soil after selective enrichment on various aromatic compounds (Table 1). All strains were identified using the key and procedures of Gordon et al. (7). Stock cultures were maintained on brain heart infusion (Difco) slants that were stored at 4 C and subcultured biweekly. Microorganisms were grown in the minimal medium previously described (4), except that m-hydroxybenzoic acid replaced phydroxyphenylpropionic acid. One liter of medium contained in a 2-liter flask was inoculated with the growth of one stock slant and magnetically stirred at room temperature until cells reached early stationary phase. Cells were collected by centrifugation and washed by resuspension in 0.1 M potassiumsodium phosphate buffer, pH 7.2. This buffer was also used in all reaction mixtures. Preparation of cell extracts. Washed cell pastes were suspended in 2 to 3 volumes of buffer which contained 25% (by volume) glycerol. The resulting cell suspensions were passed through a French pressure cell (American Instrument Co., Silver Springs, Md.) at >10,000 lb/in2 applied with a hydraulic press (American Instrument Co.). Extracted cells were centrifuged at 26,000 x g for 20 min to give clear cell extracts containing 5 to 15 mg ofprotein/ml as determined by the method of Gornall et al. (8). All procedures were performed at 0 to 5 C. Enzyme assays. 2,5-Dihydroxybenzoate 1,2-dioxygenase (EC 1.13.11.4; gentisate 1,2-dioxygenase) was assayed by the procedure of Crawford et al. (5). Maleylpyruvate hydrolase was assayed by observing decrease in absorbance at 334 nm resulting from hydrolysis of maleylpyruvate to pyruvate and maleate (14). The hydrolase assay was performed after the gentisate 1,2-dioxygenase assay in the same reaction mixture while maleylpyruvate concentration was between 3 x 10-5 and 5 x 10-5 M. Emax of maleylpyruvate was assumed to be 10,800 (5) and 1 U of hydrolase activity is defined as the amount of protein required to hydrolyze 1 jLmol of maleylpyruvate per min. In no instance was there observed an increase in the rate of maleylpyruvate degradation by Bacillus extracts on addition of reduced glutathione (GSH). Fumarase (EC 4.2.1.2.) and maleate isomerase activities were assayed essentially as described by Scher and Jakoby (18). Fumarase activity was assayed by observing the decrease in absorb-439 Spectrophotometric determinations of pyruvate formed enzymatically from gentisate were performed using lactate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) as previously described (3,4). Values shown in Table 2 are averages of two or more determinations, each initiated with a different concentration of gentisate. The second product formed on hydrolysis of maleylpyruvate is maleate (14; see below). Cell extracts of m-hydroxybenzoategrown Bacillus strains did not attack protocatechuate or catechol. Counting of bacterial populations. Total viable counts of soil were performed by dilution/plating of soil suspensions onto plate-count agar (Difco). Counts of total aerobic sporeformers were performed on the same medium after pasteurization of soil suspensions at 80 C for 15 min. Counts of total mhydroxybenzoate utilizers and of m-hydroxybenzoate-utilizing aerobic sporeformers were performed on minimal medium containing 500 mg of m-hydroxybenzoate per liter and 15 g of purified agar (Difco) per liter before and after pasteurization, respectively. Plates were incubated at 25 C. Thin-layer chromatography (TLC) and gaschromatography/mass spectrometry. Eastman chromatogram sheets (13181 silica gel; Eastman Ko- dak Co., Rochester, N.Y.) were used for analytical chromatography of aromatic compounds. Developing solvents were (A) benzene-methanol-acetic acid (45:8:2 by volume) and (B) benzene-ethyl acetate-80% formic acid (9:1:1 by volume). Aromatic compounds on plates were viewed under light of wavelength 253.7 or 375.0 nm. Gentisic acid exhibited a characteristic fluorescence when viewed on chromatograms under the former, but not the latter, wavelength of light. Organic acids were chromatographed on sheets of cellulose (Eastman, 13254 cellulose) using butanol-acetic acid-water (12:3:5 by volume; solvent C) or ethanol-ammonium hydroxidewater (16:1:3 by volume; solvent D) and were located by dipping chromatograms through AgNO3 in acetone followed by alcoholic NaOH (19). In solvent C maleic acid showed a spot of Rf 0.45, whereas fumaric acid showed a spot of Rf 0.80. Corresponding Rfs in solvent D were maleic acid, 0.68, and fumaric acid, 0.25. Analyses using an LKB-9000A gas-chromatograph/mass spectrometer were performed as previously described (16). Identification of maleate as an enzymic hydrolysis product of maleylpyruvate. The unsaturated, dicarboxylic acid formed on hydrolysis of maleylpyruvate by cell-free extracts of m-hydroxybenzoategrown Bacillus sp. was identified as maleate by the following procedures. A solution (total volume, 10 ml) containing 20 nmol of gentisate and approximately 50 mg of cell extract protein (prepared from m-hydroxybenzoate-grown Bacillus strain A2a or Cla) was incubated at room temperature with stirring for 3 h. The pH of the solution was adjusted to 4.0 and sufficient absolute ethanol was added to give a final concentration of 95%. Precipitated material was removed by centrifugation and the clear solution was concentrated to 1 to 2 ml by evaporation. Examination of this concentrate by TLC in solvents C and D revealed the presence of pyruvic acid (R. 0.6 and 0.7, solvent C) (19) and maleic acid (R. 0.45, solvent C; 0.67, solvent D), but no fumaric acid. When such enzymic reaction solutions were acidified to pH 2.0 and incubated at room temperature overnight prior to work-up, TLC revealed the presence of fumaric acid (Rf 0.8, solvent C; 0.24, solvent D), but no maleic acid. Control experiments indicate that, under the latter procedure, maleate is nonenzymically isomerized to fumarate. Materials. Enzymes and co-factors were purchased from the Sigma Chemical Co. All compounds listed in Table 1 as well as gentisic acid, organic acids, protocatechuic acid, and catechol were purchased either from the Sigma Chemical Co. or the Aldrich Chemical Co. Commercial compounds were examined for purity by TLC and recrystallized prior to use where necessary. RESULTS Soils sampled during this investigation typically yielded 108 to 1010 bacteria per g of dry soil, as determined by counting on plate-count agar. Of these approximately 10% survived pasteurization and represent the aerobic, sporeproducing component of the soil microflora. Of the total countable population of any particular soil, about 1% were able to grow on minimal media containing m-hydroxybenzoate as the only source of carbon and energy. Of the aerobic sporeformers in these soils approximately 0.1% were able to germinate and grow on mhydroxybenzoate plates. This represents, as an average for all soils examined, about 0.01% of the total viable count. Though this percentage seems small, it represents 104 to 106 aerobic sporeformers that are able to utilize m-hydroxybenzoate as a carbon/energy source per g of dry soil. Soils examined during this investigation were collected at numerous locations around downtown Albany, N. Y., and served as the source of all microbial strains used in this study. The Bacillus strains used during this investigation are listed in Table 1, along with their genus/species identification and the aromatic compounds upon which they were isolated. Results of enzymic assays of each strain, after growth on m-hydroxybenzoate, are summarized in Table 2. Cell extacts prepared from m-hydroxybenzoate-grown bacilli did not attack catechol or protocatechuate and none contained maleylpyruvate isomerase, maleate isomerase, or fumarase activities. Such extracts did readily degrade fumarylpyruvate. Succinateor glucose-grown cells lacked detectable amounts of aromatic pathway enzymes. The ring-fission product produced by oxidation of gentisate by Bacillus extracts showed spectral characteristics expected of maleylpyruvate; a Xmax in neutral or basic solution of 334 nm which is abolished upon acidification (5,15). It was possible to demonstrate directly the conversion of m-hydroxybenzoate to gentisate by using the Fe2" chelator a,a'-dipyridyl as an inhibitor of gentisate oxidation. Whole cells of Bacillus strain B9a were harvested after growth on m-hydroxybenzoate and allowed to oxidize m-hydroxybenzoate in the presence of a,a'-dipyridyl, as described by Hopper and Chapman (13). After a 3-h incubation, cells were removed by centrifugation. The supernatant was acidified to pH 2.0 and an excess of FeCl2 was added to trap a,a'-dipyridyl as its water-soluble iron complex. The cherry-red solution was extracted three times with ethyl acetate. Ethyl acetate extracts were combined, washed once with 0.1 M FeCl2 and twice with water, dried over anhydrous Na2SO4, and evaporated to yield a semicrystalline solid. This organic residue was examined by TLC using solvents A and B and shown to contain mostly gentisic acid and some residual m-hydroxybenzoic acid. Gentisic acid was unequivocally identified by its retention time and molecular ion (trimethylsilyl derivative, m/e = 370) as revealed by gas-chromatography mass spectrometry. Figure 1 illustrates the spectral changes ob- VOL. 30, 1975 served at 340 nm during oxidation ofm-hydroxybenzoate by a supplemented extract of m-hydroxybenzoate-grown Bacillus strain C5f. Oxidation was dependent upon addition of reduced pyridine nucleotide to the reaction mixture. No changes in the ultraviolet spectrum of m-hydroxybenzoate were observed when NADH was omitted from the reaction mixture. The depicted spectral changes (Fig. 1) were also produced using other extracts prepared from strains chosen at random from the list in Table 1. The unsaturated, dicarboxylic acid formed on hydrolysis of maleylpyruvate by cell extracts of m-hydroxybenzoate-grown Bacillus sp. was identified as maleate (cf. Materials and Methods). DISCUSSION The data summarized above indicate that all Bacillus isolates examined degrade m-hydroxybenzoate by the reaction sequence shown in Fig. 2A. This gentisic acid pathway is a modified version of the sequence originally delineated by Lack (15; Fig. 2B) and has been observed previously only in two strains of Pseudomonas isolated by Hopper et al. (strains 2,5 and 3,5; references 13, 14 and personal communication). Spectral changes shown in Fig. 1 are consistent with requirements of the sequence of Fig. 2A. Thus on addition of m-hydroxybenzoate to a reaction mixture containing NADH and cell extract prepared from m-hydroxybenzoate-in-duced cells, one observes an initial decrease of A340 resulting from oxidation of NADH by an enzymatic hydroxylation of m-hydroxybenzoate forming gentisate. Gentisate formed is immediately oxidized by a gentisate 1,2-dioxygenase present in cell extracts, forming maleylpyruvate (Amax = 334 nm) which absorbs strongly at 340 nm. Its formation results in an increase of A 340. The final decrease in A340 reflects hydrolysis of maleylpyruvate by its hydrolase forming pyruvate and maleate. Hydrolysis of maleylpyruvate is not speeded on addition of GSH. When a,a'-dipyridyl is included in the reaction mixture, gentisate 1,2-dioxygenase is inhibited. Thus NADH oxidation is no longer masked by maleylpyruvate formation and A340 continues its initial decrease. It is conceivable that maleylpyruvate is isomerized to fumarylpyruvate by a GSH-independent isomerase prior to hydrolysis to pyruvate and a 4-carbon, dicarboxylic acid (particularly since cell extracts prepared from m-hydroxybenzoate-grown cells readily degrade fumarylpyruvate). This possibility is ruled out by our observation that maleic acid, rather than fumaric acid, accumulates from gentisate when the latter is oxidized by extracts prepared from mhydroxybenzoate-grown Bacillus A2a or Cla. Like the nonfluorescent pseudomonad of Hopper et al. (14), the bacterial strains examined here induce an apparently nonfunctional fumarylpyruvate hydrolase when grown on m-hydroxybenzoate. Hydrolysis of maleylpyruvate and fumarylpyruvate may be catalyzed by a single enzyme. The answer to this question awaits purification of the hydrolase activities. The enzymic specific activites and pyruvate yields shown in Table 2 are as expected for the pathway of Fig. 2A. Gentisate pathway enzymes, not present in glucoseor succinategrown cells, are induced to high levels during growth on m-hydroxybenzoate. Also, one molecule of gentisate yields one molecule of pyruvate (Table 2) in a GSH-independent, enzymic reaction. Actual isolation of gentisic acid as a metabolite of m-hydroxybenzoate after inhibition of cells with aa'-dipyridyl is direct evidence of gentisate participation in the catabolic pathway. Most of the Bacillus strains examined are tentatively classified as isolates of B. brevis (nine strains); however, strains of B. sphaericus (two strains) and B. megaterium (one strain) are also represented. Thus, it seems unlikely that degradation of m-hydroxybenzoate via the GSH-independent, gentisate pathway will be of taxonomic value in distinguishing species of Bacillus. It may, however, be possible to identify a small group of species with the taxonomically valuable characteristic of ability to grow on m-hydroxybenzoate. Our results indicate that the GSH-independent gentisate pathway may be of general occurrence in the genus Bacillus, rather than the GSH-dependent pathway. Investigation of many additional strains of Bacillus species that utilize gentisate as a catabolic intermediate will be necessary to determine whether or not this is a valid generalization. As far as I know this is the first demonstration of the presence of a gentisate pathway among bacteria of the genus Bacillus (other than a preliminary report by Crawford and Chapman [R. L. Crawford and P. J. Chapman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, 03, p. 1921) as well as the first report ofdegradation of m-hydroxybenzoate by microorganisms of this classification.

v3-fos

2020-12-10T09:04:20.793Z

1975-06-01T00:00:00.000Z

237232998

s2

Salmonella Survival on Pecans as Influenced by Processing and Storage Conditions Survival of Salmonella senftenberg 775W, S. anatum, and S. typhimurium during exposure to currently practiced, as well as abusive, pecan processing and storage conditions was studied. Thermal treatments normally carried out during the processing of pecans are inadequate to consistently destory salmonellae in highly contaminated inshell nuts. Pecan nut packing tissue was toxic to salmonellae, thus affording some protection against high initial contamination and subsequent survival of the organisms. Examinations of inoculated inshell pecans stored at -18, -7, 5, and 21 C for up to 32 weeks revealed that the extent of survival was inversely correlated to the storage temperature. S. senftenberg 775W and S. anatum were not detectable on inshell nuts after 16 weeks of storage at 21 C. Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18, -7, and 5 C for 32 weeks. Due to organoleptic quality deterioration in pecan nutmeats at elevated temperatures, sterilization methods other than thermal treatment appear to be required for the elimination of viable salmonellae from pecan nuts. The production and utilization of pecans has increased significantly in recent years, mainly due to the advent of mechanical processing equipment and improved storage practices. With this increase, pecan processors and food manufacturers using pecans as product ingredients have become increasingly concerned with the presence of potentially pathogenic microorganisms. Concern is especially intense among manufacturers of certain confectionery, dairy, and snack products wherein pecans are incorporated without processing treatments which would be lethal to microorganisms. Research attention has been given to the incidence of these microoganisms on pecans and other nuts (3,7,8,12) and to their destruction during processing (2,11) and storage (1). The present study was initiated to determine the behavior of salmonellae as they are exposed to commercial pecan processing and storage conditions. Pilot plant experiments were designed to simulate currently practice processing and storage procedures as well as conditions which represent process abuse. MATERIALS AND METHODS Pecans. Standard grade Stuart and Schley cultivar pecans used in this study were a gift from Nut Tree Pecan Co., Albany, Ga. Nuts in 28-pound (ca. 13 kg) sealed boxes had received a double propylene oxide treatment to reduce natural microflora counts and simplify salmonellae enumeration in planned inoculation studies. Test organisms. Salmonella senftenberg 775W, S. anatum, and S. typhimurium were maintained on nutrient agar slants, at room temperature. Working cultures were transferred daily in tryptic soy broth containing 0.5% yeast extract (TSY) and grown at 30 C on a gyratory shaker (150 rpm). Inoculation of pecans. Four separate tests were carried out to determine the behavior of salmonellae during processing and storage of pecans. These tests are illustrated in Fig. 1, together with a schematic diagram of the procedures generally followed in processing and handling pecans after the nuts are delivered from the grove. Test 1 was made to determine the survival of salmonellae on inshell pecans during prolonged storage. Test 2 was run to determine the lethality of the hot water treatment to salmonellae. Test 3 was included to determine if a water-floatation vacuum treatment resulted in a decrease in recoverable organisms. Finally test 4, yielding information concerning potential public health problems which might be associated with pecans contaminated with salmonellae, was carried out to assess the survival of the organisms on nutmeats which would be distributed and possibly consumed without further processing. Both Stuart and Schley cultivars were examined in tests 2 and 3, whereas Stuart alone was used for test 1 species grown in TSY at 30 C on a gyratory shaker were collected by centrifuging for 15 min at 5,000 x g. The cells were resuspended in sterile tap water and applied to inshell pecans (test 1) and halves (test 4) using an aerosol spray. Nuts were dried at 40 C in a forced-air oven, and 50-g quantities were deposited in 28-ounce (ca. 1.8 kg) glass jars. Sealed jars were stored at -18, -7, 5, and 21 C for 2, 4, 8, 16, 24, and 32 weeks. For test 2, S. senftenberg 775W was studied. Inshell pecans were submerged for 5 min in a tap water suspension containing 4 x 107 viable salmonellae per ml. Nuts were drained briefly and sealed in a plastic bag at 22 C overnight, thus simulating the conditioning treatment a sheller would carry out prior to cracking. Conditioned nuts were submerged in tap water at 60, 71, 82, 93, and 99 C (104, 160, 180, 200, and 210 F, respectively) for 1, 2, 3, 4.5 and 6 min, cooled, and examined for viable S. senftenberg 775W. Conditioned nuts inoculated with S. senftenberg 775W were also submerged in tap water at 71, 82, and 93 C for 2 min, cracked and shelled (Champion Pecan Machine Co., model C, San Antonio, Tex.), and examined for viable organisms. Test 3 consisted of subjecting pecan halves which had been freshly inoculated with a tap water suspen-E SALMONELLA SURVIVAL ON PECANS sion of S. senftenberg 775W to two alternate 3-min treatments of 13-inch (33 cm) vacuum and atmospheric pressure. Viable cells were then enumerated. Enumeration of salmonellae. Fifty grams of inshell pecans, pecan halves, or shells were combined in a 28-ounce glass jar with 50 ml of sterile 0.1% peptone and shaken on a mechanical shaker for 3 min. Serial dilutions of the peptone wash were plated in TSY containing 1.5% agar, and counts were made after 24 h incubation at 35 C. Supplementation of tryptic soy broth with yeast extract has been reported to enhance the recovery of cold-injured bacteria (9,10). The initial natural microflora population was less than 10 per 100 g. For this reason, traditional salmonellae enrichment and identification procedures were not required in this study. All samples were brought to room temperature before viable count procedures were initiated. Counts reported are averages from at least two samples run in duplicate for each test. Antimicrobial activity of pecan packing tissue. Packing tissue (see Fig. 2) was removed from noninoculated Stuart pecans, ground with a mortar and pestle, and passed through a 30-mesh screen. The material was added to 100-ml quantities of TSY (pH 7.0) at levels of 0.05, 0.1, 0.2 and 0.4% (wt/vol) and dispensed in 250-ml Erlenmeyer flasks. A 16-h culture of S. senftenberg 775W was harvested by centrifuging at 5,000 x g for 15 min. Cells were resuspended in 0.1 M phosphate buffer (pH 7.2), and 1 ml of the suspension was used to inoculate each of the test media. Cultures were incubated at 35 C on a gyratory shaker (150 rpm). Aliquots were withdrawn over a 7.5-h growth period, serially diluted, and plated on TSY containing 1.5% agar. Colony counts were made after 24-h incubation at 35 C. Heat penetration of inshell pecans. Holes, Y16 inch (ca. 0.159 cm) in diameter, were drilled at one of three positions (see Fig. 2) in inshell Stuart pecans. Thermocouples were inserted and sealed by applying an epoxy glue. Nuts were submerged in a water bath at 60, 71, 82, and 93 C for up to 5.2 min, and temperatures were recorded on a Honeywell Electronik 16 Multipoint recorder. Analytical. Procedures for moisture determination were reported earlier (1). Data presented throughout this paper are averages from at least two independent trials run in duplicate. RESULTS AND DISCUSSION A flow diagram of the processing and handling procedures which would generally be followed by a pecan processor is shown in Fig. 1. The information presented in the diagram reflects practices considered to be optimum for maintenance of high quality pecans. Substantial deviation from these procedures would result in decreased product quality. Realizing that process deviations do exist within the industry, specific tests were designed to include possible conditions of abuse. These conditions, in addition to those recognized as acceptable for the preservation of high quality pecans, were studied with regard to their effect on the survival of salmonellae. Figure 3 shows the survivor curves for three species of Salmonella on inshell Stuart variety pecans stored at -18, -7, 5, and 21 C for up to 32 weeks. Data were derived from test 1 (Fig. 1). Although initial counts for the three species were different, their behavior throughout the examination period was similar. No S. senftenberg 775W or S. anatum were detected after 16 weeks of storage at 21 C. S. typhimurium counts dropped significantly on nuts stored at 21 C, but detectable levels were still observed after storage for 32 weeks. Populations surviving the storage period were inversely related to the storage temperature. Only slight reductions in viable salmonellae were noted for inoculated inshell nuts stored at -18 C for 32 weeks. Data from test 2 (Fig. 1) on inshell Stuart and Schley pecans heated in tap water for periods of time ranging to 6.0 min. Control experiments showed that low numbers of microflora naturally present on the nuts did not proliferate during the overnight holding period and that less than 50 viable cells per 100 g of inshell pecans were contained on nuts run through the cracking and shelling operations. The initial salmonellae population on both varieties was 6 x 105/g. A trend toward reduction in viable population at the higher temperature, longer treatment times is weak at best. The inconsistent data reveal a lack of uniformity among the nuts within sample lots with respect to initial contamination levels and/or a lack of uniformity with respect to rates of heat penetration in the nut. To test these theories, thermocouples were used to measure the internal temperature of sound inshell pecans heated at 60, 71, 82, and 93 C. Temperatures were monitored in the nutmeat, middle septum and packing tissue as illustrated in Fig. 2. Surprisingly, only small differences were noted in temperatures at the three positions after exposure to the same time-temperature treatment. The nutmeat temperature was always about 2 F (ca. 1 C) lower than septum and tissue temperatures. The data were averaged and plotted as heat penetration curves (Fig. 4). Come-up times were not reached within sound pecans even after 5.2-min exposure to heat. The 2-min water dip treatment at 180 and 200 F (82 to 93 C) as carried out by processors would result in internal nut temperatures of approximately 160 and 185 F (71 and 85 C), respectively. Poor heat conductivity of the porous packing tissue accompanied by the high lipid content of kernels apparently retards heat transfer within the pecan shell. A portion of the salmonellae which were imbibed by nuts in various quantities during inoculation remained viable and thus resulted in part for the inconsistent data presented in Table 1. Dissolved solids within the nut may also have afforded some protection against thermal inactivation of S. senftenberg 775W. Salmonellae are reported to have increased heat resistance as the water activity of heated menstrua is decreased (5). In any case, when present in the internal portions of inshell pecans, S. senftenberg 775W survives the 2-min water dip treatment at 180 to 200 F. Test 2 was extended to determine the fate of surviving S. senftenberg 775W as the nuts were passed through a cracker-sheller (Fig. 1). Data are summarized in Table 2. Counts are again slightly higher for the Schley pecans, possibly indicating greater numbers of nuts with hairline cracks which may have initially facilitated the entrance of higher populations of salmonellae. Apparently, the viable population tends to equilibrate between shell and nutmeat samples, probably due to surface contact. Results from test 3 (Fig. 1) revealed that the alternating vacuum-atmospheric pressure treatment had no effect on the number of salmonellae recoverable from pecan halves. Figure 5 shows results from test 4 (Fig. 1). The three salmonellae test species were enumerated from Schley pecan halves stored at -18, -7, 5, and 21 C for times ranging to 32 weeks. With the exception of persistent survival of S. anatum at 21 C, the three species behaved similarly. Neither S. senftenberg 775W nor S. typhimurium were detectable after 24 weeks of storage at 21 C. Slight losses in viability were noted for the duration of the study for all species when storage was at -18, -7 or 5 C. Unfortunately these lower temperatures are required if high organoleptic qualities of pecans are to be maintained over prolonged storage. During initial experiments in this study, attempts were made to inoculate nutmeats of inshell pecans by submerging the nuts in heavy suspensions of salmonellae followed by drying. The inshell pecan will readily absorb liquid through fibrovascular bundles at its base and through suture separations at its apex as illustrated in Fig. 2, thus providing ports of entry for salmonellae. Repeated trials using this inoculation procedure, however, were unsuccessful. Aseptic separation and analysis of nutmeats from inoculated inshell pecans after drying yielded low numbers of viable salmonellae. These results prompted an investigation of the packing tissue for possible bacteriocidal activity (Fig. 6). Packing tissue, when present in TSY at a level as low as 0.05%, retards the growth of S. senftenberg 775W. At 0.2%, tissue exerts a bacteriocidal effect. The toxic effect is probably due to tannins and polyphenolic compounds which are present in high concentrations in the packing tissue. The moisture contents of nutmeats and shells have a pronounced effect on the ability of salmonellae to remain viable during prolonged storage. The moisture contents of nutmeats studied in tests 1 and 2 were 4.5% at the beginning of the storage period. Previous studies indicated that inshell pecans having a nutmeat moisture content of 4.5% have an outer- shell moisture content of 11.5% (6,13). This higher moisture level is probably accompanied by higher water activity which, according to a report on the survival of S. newport in "dry" foods over a range of water activity (4), is likely to have a lethal effect. This line of reasoning may explain in part the decline in viable salmonellae on inshell pecans at all storage temperatures in test 1 compared to relatively little decline in population on nutmeats stored at -18, -7, and 5 C in test 2. Data from this study point up the necessity for considering several factors when and if microbiological limits are established for pecans and possibly other tree nuts. Due to morphological differences among pecan cultivars, susceptability to cracking and, hence, the potential for water uptake with subsequent microbial contamination differs greatly. Realizing that inshell pecans are often marketed as mixed variety lots, it will be important to design sampling schemes which reflect the true incidence of contamination over large quantities of product. Results from test 2 of the present study indicate that this criterion was not achieved. Storage conditions after processing affect the viability of all microbial contaminants on pecans. Response to environmental conditions by particular groups of organisms may differ, however, thus rendering total aerobic counts of questionable value in setting up microbiological limits on pecans. It is not reasonable to extrapolate from survival data presented here for sal-monellae, or from survival studies involving other pathogenic organisms, a maximum total microbiological count which would insure pecans as being microbiologically safe from a public health standpoint. Additional research is required to determine the behavior of naturally occurring bacterial and fungal contaminants during pecan processing and storing. On a practical level, results from this study indicate that presently established heat treatment procedures used during pecan processing are not adequate for the elimination of salmonellae from internally contaminated inshell nuts. However, morphological features and composition of nut packing tissue and shell do afford protection against high initial contamination and subsequent survival of salmonellae on the nutmeat. Surface to surface contact between contaminated shells and "clean" nutmeats during cracking and shelling is likely to result in nutmeats haboring salmonellae which are extremely tolerant to commercial refrigerated storage conditions. Destruction of these salmonellae will need to be achieved by means other than thermal treatment, such as gassing with propylene oxide, due to organoleptic quality deterioration in pecans subjected to temperatures sufficient for pasteurization.

v3-fos

2020-12-10T09:04:20.587Z

1975-07-01T00:00:00.000Z

237230600

s2

Toxigenic Fungi in Food Forty-five fungal isolates from moldy supermarket foods were tested for toxicity to brine shrimp, and twenty-two of these isolates were subsequently tested for toxicity to chicken embryos. Highly toxigenic fungi were Cladosporium sphaerospermum from a bakery product, Fusarium oxysporum from carrots, F. solani from cabbage, Aspergillus niger and Penicillium corylophilum from bread, P. cyclopium and P. herquei from corn meal, P. lanosum from onions, P. steckii from chocolate syrup, Penicillium sp. from jelly, and Rhizopus nigricans isolates from sweet potato, applesauce, and strawberries. Approximately one-third of the fungal cultures were moderately to highly toxigenic to brine shrimp and chicken embryos, while several additional cultures were slightly toxigenic. Mycotoxicoses have been well documented in animals, but there is an increasing need for research in the area of human health (7). Some of the implications of mycotoxins to human health have been recently reviewed (1,4). The extent of mycotoxin hazard to man can be better assessed, if the identity of the fungi involved is known. This report presents results of a portion of a screening program in which fungi were routinely isolated from a variety of human foods, identified, and tested for toxicity to brine shrimp in primary bioassays and chicken embryos in secondary bioassays. Forty-five fungi were isolated from visibly moldy foods purchased or obtained with the cooperation of personnel in a local supermarket and obtained from home refrigerators during 1973-74. Isolation was by direct plating on YE agar (2% dextrose, 0.7% yeast extract [Difco1), 0.5% KHXPO,, and 2% agar), from which the fungi were monocultured at room temperature (25 to 30C) on either Czapek-Dox or potatodextrose agar for identification as described by Morgan-Jones and Booth (Toxin-Producing Fungi, in preparation). The fungi were then grown in 1-liter Erlenmeyer flasks on nutrientamended shredded wheat (3,6), which had been autoclaved 15 min at 121 C twice in 24 h. The cultures were incubated 14 to 21 days at 25 C. Moldy substrates were extracted with chloroform-ethanol (80:20), filtered, and evaporated under an airstream at room temperature. Extracts for the brine shrimp bioassay were taken up in 95% ethanol, while extracts for chicken embryo bioassays were suspended in corn oil by methods previously reported (3). Controls con-sisting of extracts of uninoculated nutrientamended shredded wheat were included in all bioassays. Initially screening was with brine shrimp, which are regarded as sensitive test organisms for mycotoxins (5,9). However, Curtis et al. (2) have shown that some fungi elaborate naturally occurring fatty acids that are toxic to brine shrimp. Thus, toxicity towards brine shrimp should be confirmed with at least one additional test organism. In this research, fungi that were toxic to brine shrimp were subsequently tested with chicken embryos (11 except in the case of M. globosus (#595), which died in culture between bioassays. However, certain isolates not toxic to brine shrimp were also tested with chicken embryos, namely, C. sphaerospermum and F. solani. R. nigricans (#620) was included in the second bioassay because previous tests had indicated that it was toxigenic. Extracts from approximately one-third of the fungi used in this study were moderately to highly toxic to brine shrimp and chicken em-bryos. Saito et al. (10) found that 22.5% of 247 fungal isolates from foods in Japan were moderately to highly toxic to HeLa cells and 30% to mice. Approximately 36% of 531 fungal strains from foods in the diet of rural Bantu in Eastern Transvaal and Swaziland (8) were moderately or very highly toxic to ducklings. Our isolates were from conspicuously molded foods from a local supermarket, and from the refrigerators and kitchens of consumers. One wonders what happens to such foodstuff in localities where extreme hunger exists and concern is on quantity rather than quality. A potential hazard could be compounded by the fact that the undernourished are generally more susceptible to toxicants than are individuals with more adequate diets. This investigation did not determine whether the foodstuffs actually contained known mycotoxins or were toxic per se. It was only determined that 42% of the moldy foodstuffs investigated were invaded by strains of fungi capable of elaborating toxic substances. Additional work appears warranted to determine the toxic compounds and whether the toxigenic isolates elaborate mycotoxins on the foods from which they were isolated. Several of the toxigenic isolates are not generally recognized mycotoxin producers. These are C. sphaerospermum, F. lateritium, P. corylo- Check 647 769 533 553 597 625 665 664 765 535 630 650 748 551 707 644 648 598 620 675 606 618 philum, P. herquei, P. lanosum, P. steckii, and R. nigricans. Fungi from meat were not included in this investigation, since moldy meat was not observed in the supermarket. Wu et al. (12) in their investigation of fungi on meat found that toxigenic species of Aspergillus were more prevalent than toxigenic species of Penicillium. Penicillium was the most frequently isolated group of fungi in our study, with 41% of 17 isolates being toxigenic to brine shrimp and chicken embryos. Thus, our data are similar to that of Saito et al. (10) in that the most prevalent toxigenic fungi in foods were Penicillium species. The apparent predominance of Penicillium species may reflect the influence of refrigeration; it has been our observation that Penicillium is very competitive at low temperature storage of high moisture foods.

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2020-12-10T09:04:12.726Z

1975-10-01T00:00:00.000Z

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Detection of Lactobacillus acidophilus in Feces of Humans, Pigs, and Chickens Lactobacilli in fecal material from humans, pigs, and chickens were enumerated on lactobacillus selective agar (LBS). In all samples, higher numbers of lactobacilli were detected when plates were incubated in a system flushed with CO2 rather than in air. Much higher numbers of bacteria from human feces were detected when the LBS agar plates were incubated anaerobically in a hydrogen-carbon dioxide atmosphere (GasPak) than when incubated in CO2. The bacteria from human feces isolated on LBS agar incubated anaerobically were predominately bifidobacteria. Cultures from all three sources isolated on LBS agar incubated under CO2 were lactobacilli, including Lactobacillus acidophilus. Differences were observed in biochemical characteristics of some of the L. acidophilus isolated from all three sources. Guanine plus cytosine base ratios of deoxyribonucleic acid isolated from L. acidophilus cultures from humans were lower, in most cases, than those from pigs and chickens. Many reports have been published regarding the importance of lactobacilli in maintaining balanced intestinal flora necessary for the health of humans (for a review, see reference 18). Therapy involving the consumption of viable Lactobacillus acidophilus has been beneficial in treating certain gastrointestinal disorders (18). There are indications that the biotypes of lactobacilli present in the intestinal tract vary among different host animals (11). A possible "host specificity" for intestinal microorganisms has been suggested (12). For these and other reasons there is a need for more selectivity in the quantitation of lactobacilli in the intestinal tract; also more detailed information is needed on lactobacilli found in the intestines of different hosts. In the present study, lactobacillus selection (LBS) agar incubated under different conditions was used in efforts to differentiate various groups of lactobacilli. Characteristics of cultures ofL. acidophilus isolated from the feces of humans, pigs, and chickens also were observed. MATERIALS Recherches Zootechnique (Jouy-en-Josas, France). Other lactobacilli were isolated from fecal material of either humans, pigs, or chickens and are so designated. All L. acidophilus strains were routinely propagated in sterile lactobacillus MRS broth (Difco Laboratories, Detroit, Mich.) by using a 1% inoculum and incubation at 37 C for 18 h. At least three successive transfers were made prior to characterization experiments. The cultures were stored at 5 C between transfers. The Bifidobacterium cultures were propagated in a similar manner except that the tubes were incubated and stored between transfers at 5 C in a GasPak system (BBL, Cockeysville, Md.). Enumeration procedures. The fecal samples were diluted with sterile 1% peptone, and duplicate plates were prepared for each dilution. The plates were poured with LBS agar (BBL) prepared from individual ingredients, and an overlay of the same medium was added. Some plates were placed in a plastic bag, flushed for 1 min with carbon dioxide, and sealed (LBS-CO2 counts). For incubation in an atmosphere of hydrogen and carbon dioxide, plates were placed in a GasPak system (LBS-GP counts). This atmosphere contained approximately 7% CO2 (BBL). In addition, some plates were incubated aerobically. All plates were incubated for 48 h at 37 C. Isolation procedures. Colonies were picked from countable plates of LBS-CO2 and LBS-GP and inoculated into tubes containing 10 ml of sterile MRS broth. The tubes containing isolates from the LBS-CO2 plates were incubated aerobically, and those from the LBS-GP plates were incubated anaerobically in a GasPak system. Cultures that grew within 24 to 48 h were diluted and plated by the pour plate method with MRS agar (MRS broth plus 1.5% agar). Isolated colonies from these plates were picked and maintained in MRS broth for characterization test. Classification of isolates. Only those isolates that were gram-positive rods and catalase negative were considered for further identification. Tests for catalase were made by adding 5 ml of 3% hydrogen peroxide to the cell pellet obtained by centrifuging (12,000 x g for 10 min) 10 ml of an MRS broth culture. Cultures were considered catalase negative if no visible gassing was observed. Cultures were examined for growth at 15 and 45 C in MRS broth and for gas production by the methods of Rogosa et al. (17); for the latter, 5 ml of 1.5% sterile agar was used as the overlay. MRS broth with glucose and beef extract omitted (19) was the basal medium used for determination of ammonia production from arginine, esculin hydrolysis, and carbohydrate fermentations. Esculin hydrolysis and ammonia production were determined as described by Davis (4). Ability of cultures to ferment various carbohydrates was evaluated as described by Rogosa and Sharpe (16), except that amygdalin, melibiose, and raffinose were not employed. Cultures of anaerobic organisms were incubated in a GasPak system. To check for branched cellular forms ofthe anaerobic isolates, the cultures were grown in the lowcalcium medium of Kojima et al. (7) in which yeast extract (0.5%) was substituted for the beef liver infusion. Cellular morphology was determined by examining, with a microscope, methylene blue stains of the cultures. Determination of DNA base composition. Deoxyribonucleic acid (DNA) was isolated from representative lactobacilli and bifidobacteria by the method of Marmur (9). The percentage of guanine plus cytosine (G+C) in the DNA samples was determined from the thermal melting point of the DNA by using procedures described by Marmur and Doty (10). The change in absorbance of the heated DNA was measured automatically by using a Beckman DU spectrophotometer equipped with a Gilford model 2000 automatic recording photometer (Gilford Laboratories, Inc., Oberlin, Ohio). The cuvette chamber was heated by a Haake model FE constanttemperature circulation bath (Gilford Laboratories, Inc., Oberlin, Ohio) filled with ethylene glycol. RESULTS Enumeration of lactobacilli. L. acidophilus NCFM did not form colonies on LBS agar incubated under aerobic conditions (Tgble 1). How- ever, the culture grew equally well on LBS-CO2 and LBS-GP. Colonies developed from chicken and pig feces plated on LBS agar and incubated aerobically. Few differences were observed between the colony counts on LBS-CO2 and those on LBS-GP. The LBS-CO2 and LBS-GP counts were approximately 2.5 and 5.5 times greater than the LBS-aerobic counts for chickens, and pigs, respectively. Marked differences in counts were obtained when human feces was plated on LBS-CO2 and LBS-GP (Table 2). In all subjects studied, much higher numbers were observed when the fecal material was plated on LBS-GP. Aerobic conditions were not evaluated because of the poor growth of L. acidophilus incubated aerobically on LBS agar. Furthermore, the lactobacilli from pig and chicken feces grew less well on LBS agar incubated aerobically than on LBS-CO2 and LBS-GP. Isolation of lactobacilli. Cultures isolated from the highest dilutions of human fecal material on LBS-GP were anaerobic and had characteristics of Bifidobacterium species ( Table 3). All of the isolates, with the exception of BA1, formed branched rods in the medium of Kojima et al. (7); this is typical for this species. Furthermore, all of the isolates from human feces plated on LBS-GP (with the exception of BS3) had biochemical characteristics of Bifidobacterium species (15). The G+C contents of five strains (including BS3) tested were within the range reported for Bifidobacterium (1). Cultures isolated from human, pig, and chicken fecal material plated on LBS-CO2 were all classified as lactobacilli. Of the 20 isolated from human feces, 6 possessed characteristics closely resembling L. acidophilus. Three additional isolates did not closely fit the fermentation pattern described by Rogosa and Sharpe (16) for L. acidophilus; however, their characteristics were closer to those of L. acidophilus than to any other organism of the thermobacterium group. Of 12 lactobacilli isolated from pigs, none was identical toL. acidophilus. However, eight of the isolates had characteristics more nearly similar to L. acidophilus than to any other lactobacilli. Of 12 isolates obtained from chickens, 3 had characteristics closely resembling L. acidophilus and 2 additional isolates more closely resembled L. acidophilus than any other lactobacilli in the thermobacterium group. Complete fermentation patterns for five representative isolates characterized as L. acidophilus from humans, pigs, and chickens are presented in Table 4. Four laboratory strains of L. acidophilus were included for comparison. The fermentation patterns for these strains conformed closely with the characteristics of L. acidophilus (15) with the exception of CNRZ 216. Isolates HA3 and HM2 (human origin) possessed the same characteristics as L. acidophilus. Isolate HA1 was similar except for an apparent variation in the ability to ferment maltose and galactose. Isolate HA2 differed from L. acidophilus in that it failed to ferment trehalose. Isolate HM6 was variable with respect to the fermentation of cellobiose and salicin, and failed to ferment trehalose or hydrolyze esculin. All of the isolates of pig origin differed from the fermentation pattern of L. acidophilus because of negative or variable actions on cellobiose, salicin, and trehalose. All except PA3 were also variable with respect to the hydrolysis of esculin. Of the five representative isolates from chickens, isolates C1, C2, and C3 possessed characteristics identical to those of L. acidophilus. Isolate C7, however, was variable on mannitol, and isolate Cl1 fermented mannitol but did not attack esculin, cellobiose, or salicin. G+C content of DNA. The G+C content for isolates identified as L. acidophilus ranged from 36.7 to 43.7 mol%, except for isolate C11, which was 29.8 mol% (Table 4), and therefore could not be considered as L. acidophilus. The G+C contents for two of the three isolates of human origin (HA3 and HM2) were closer to the percentages observed for the laboratory strains than were those for the isolates obtained from chicken and pig fecal material. Isolate PA 19 (of pig origin) also appeared to have a G+C content close to that observed for the laboratory strains. In general, G+C contents for the isolates (except PA19 and C11) obtained from the chicken and pig fecal material were higher than those observed for the laboratory strains. Isolate HA1 (of human origin) also possessed a higher G+C content than those observed for the laboratory strains. DISCUSSION In evaluating the effect(s) offeeding L. acidophilus on the microbial flora of the intestinal tract, the selection of the proper medium for detection of the organism being fed is of great importance. In addition, it appears that selection of an L. acidophilus strain compatible with the host is also important. LBS agar has highly selective qualities for lactobacilli. The conditions under which LBS agar is incubated have been shown in this study to yield different counts for the same sample of fecal material, indicating a possible means of more selectively enumerating the lactobacilli. Although colonies from chicken and pig feces were observed on LBS agar when incubated aerobically, L. acidophilus NCFM failed to show growth under these conditions. Rogosa and Sharpe (16) indicated that high concentrations of acetate caused by evaporation during aerobic incubation may be inhibitory to some lactobacilli. L. acidophilus NCFM and other strains may be sensitive to these higher levels of acetate caused by evaporation. Thus, this medium should not be used aerobically for the enumeration ofL. acidophilus. On the other hand, incubation under strictly anaerobic conditions (GasPak system) enables anaerobic Bifidobacterium species to grow and completely prevents the detection of L. acidophilus from some sources (i.e., human feces). Anaerobic incubation of LBS plates would provide a method of enumerating bifidobacteria. Not all organisms detected on LBS-CO2 were classified as typical L. acidophilus; therefore, this method of incubation cannot be used to enumerate L. acidophilus selectively. Such results also indicate that lactobacilli other than L. acidophilus are present in the intestinal flora of humans, pigs, and chickens. Comparison of the fermentation patterns of isolates identified as L. acidophilus indicate that different biotypes exist. Mitsuoka (11) reported that different biotypes of L. acidophilus existed which were host specific. Morishita et al. (12) described a similar phenomenon in that a strain of L. acidophilus of human origin would not become established in the intestines of chickens. The differences observed in the fermentation patterns and G+C content of the DNA among the isolates of the present study also lends support to the theory that relationships exist between the host and the strain or biotype ofL. acidophilus that can become established in the intestine. Thus, it appears that care must be taken in selecting strains of L. a#idophilus for use in dietary preparations intended as a source of lactobacilli for establishment in the intestinal tract.

v3-fos

2020-12-10T09:04:17.271Z

1975-11-01T00:00:00.000Z

237231907

s2

Fibrous Material in Feedlot Waste Fermented by Trichoderma viride Trichoderma viride QM9123 fermented fiber isolated from feedlot waste at concentrations up to 16.7% solids. The fermented fiber solids decreased by 32%, and carbohydrate decreased by 60%. Cellulolytic enzyme production was better with fiber substrates that had been alkali pretreated and had a lower hemicellulose-to-cellulose ratio. Trichoderma viride QM9123 fermented fiber isolated from feedlot waste at concentrations up to 16.7% solids. The fermented fiber solids decreased by 32%, and carbohydrate decreased by 60%. Cellulolytic enzyme production was better with fiber substrates that had been alkali pretreated and had a lower hemicellulose-to-cellulose ratio. Griffin et al. (2) demonstrated that whole feedlot waste at 2.5% concentration is a complete and convenient medium for the production of cellulolytic enzymes by Trichoderma viride (5). However, higher concentrations were inhibitory to the fungus. We have attempted to eliminate this inhibition and improve the overall amount of feedlot waste digestion through fermentation of fibers isolated from the whole waste. Three samples of fiber (Table 1) were isolated and compared with whole waste as a fermentation substrate. Isolated fiber differed from the whole waste in that no inhibition was detectable at substrate concentrations up to 16.7%, but nutrient deficiencies appeared. With the alkali-washed Ariz:OH fiber, all of the nutrient supplements described by Mandels and Weber (4) were necessary for good enzyme production. Enzyme production was also influenced by the amount and kind of nitrogen nutrients added to the fermentation flasks ( Fig. 1). At a semisolid substrate level of 16.7%, enzyme production reached a maximum at ammonium sulfate-urea and peptone concentrations of 200 and 15 mg per flask, respectively. In all three samples, approximately 60% of the carbohydrate disappeared ( Table 2) and only 68% of the solid was recoverable by centrifugation. Lignin content, which is generally 13 to 20%, increased to 20 to 28% in the fermented fibers, and there was a 0.5 to 1.8% net increase of insoluble nitrogens. Significant difference in the fermentation of cellulose appeared among the samples, however, which correlated with the hemicellulose content of fibers. Hemicellulose is defined here as those sugars not found in the cellulose fraction (Tables 1 and 2). When the ratio of hemicellulose to cellulose was rela-I Address reprint requests to: Dr. G. E. Inglett, Northern Research Regional Laboratory, Peoria, Ill. 61604. tively large (e.g., 1.33 for the Illinois fiber), the hemicellulose fraction appeared to be degraded preferentially. For example, the ratios of hemicellulose to cellulose were 1.33, 0.82, and 0.36 for the Illinois, Arizona, and alkali-treated Arizona fibers, respectively. These ratios became 0.87, 0.84, and 0.53 upon fermentation of the fibers. Also, cellulolytic enzyme production was inverse to the hemicellulose content of fibers. Cellulolytic activity described as milligrams of glucose released per flask per hour (2) was 120, 160, and 250 for the Illinois, Arizona, and alkali-treated fiber substrates, respectively. Presumably, a smaller quantity of cellulolytic enzyme is needed with hemicellulose-enriched substrates to give growth. The sequence of fermentation events starting from a spore inoculum is shown in Fig. 2. Between days 3 and 7, 60 to 70% of the digestible nutrients was consumed, whereas cellulolytic 1. Amounts ofammonium sulfate-urea nitrogen supplement (A) and peptone (B) required by Trichoderma viride cultures grown on 16.7% feedlot waste fiber to produce (0) cellulolytic enzyme and (X) total protein in the supernatant after centrifugation. Supernatant protein was calculated by a colorimetric method described by Layne (3), and cellulolytic enzyme was assayed by the procedure of Griffin et al. (2). Except for either the nitrogen supplement or peptone, each 500-ml cultural flask contained 10 g of fiber in 50 ml of nutrients (163 mg ofammonium sulfate, 35 mg of urea, 15 mg ofpeptone, and 25 mg of Tween 80 adjusted to a final pH 4S with H3P04), and the spore-inoculated culture was incubated aerobically at 28 C for 10 days. enzyme was detectable only during the later days. Other soluble proteins appeared after the release of cellulolytic enzyme and may have been released upon cellular death and lysis. T. viride seemed to be as effective as Thermoactinomyces (1) in reducing the volume of waste solids. Furthermore, the fiber substrates could be fermented at high concentrations, and cellulolytic enzyme was produced conveniently in the fermentation broth. cellulolytic enzyme (@), and total solids (A) recovered per flask. Total solids include insoluble residue recovered by centrifugation and supernatant solids after Iyophilization. VOL. 30, 1975

v3-fos

2018-12-01T06:08:18.292Z

1975-01-01T00:00:00.000Z

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s2

ACTIVITY PATTERNS OF GULL CHICKS IN RELATION TO FEEDING BY PARENTS - THEIR POTENTIAL SIGNIFICANCE FOR DENSITY-DEPENDENT MORTALITY THIS study compares the activity of Glaucous-winged Gull (Larus glaucescens) chicks after unsuccessful begging for food with their activity after having been fed. We wished to test the hypothesis that differences might exist in the behavior of hungry and well-fed chicks that would result in the hungry chicks wandering from their territories and thus being exposed to more frequent attacks by neighboring adult gulls. If hunger resulted in inappropriate behaviors by chicks, we could then link chick survival with the availability of food to foraging adults. METHODS We studied young Glaucous-winged Gulls from 12 families on Mandarte Island, British Columbia, from 23 June through 12 July 1973. Observations of 1.5 to 3.0 h duration were made from blinds at various times of day between 0600 and 2100 h for 13 periods in June and 16 periods in July. During each period three chicks were watched, each belonging to a different pair of parents. Ages of the chicks ranged from 2 to 32 days over the course of the study. The activity of the chicks and their distance from their nearest parent present on the territory were recorded at 1-min intervals. Five categories of behavior were noted: (1) Resting: The chick was inactive and in some cases may have been hiding. (2) Active I: The chick was less than 4 feet from its parent and was moving about. (3) Active II: The chick was moving about 4 or more feet from its closest parent. (4) Begging: The chick was soliciting food from its parent (see Tinbergen 1960 for a description of begging). (5) Feeding: The chick was obtaining food from its parent. June and July results presentation. THIS study compares the activity of Glaucous-winged Gull (Larus glaucescens) chicks after unsuccessful begging for food with their activity after having been fed. We wished to test the hypothesis that differences might exist in the behavior of hungry and well-fed chicks that would result in the hungry chicks wandering from their territories and thus being exposed to more frequent attacks by neighboring adult gulls. If hunger resulted in inappropriate behaviors by chicks, we could then link chick survival with the availability of food to foraging adults. METHODS We studied young Glaucous-winged Gulls from 12 families on Mandarte Island, British Columbia, from 23 June through 12 July 1973. Observations of 1.5 to 3.0 h duration were made from blinds at various times of day between 0600 and 2100 h for 13 periods in June and 16 periods in July. During each period three chicks were watched, each belonging to a different pair of parents. Ages of the chicks ranged from 2 to 32 days over the course of the study. The activity of the chicks and their distance from their nearest parent present on the territory were recorded at 1-min intervals. Five categories of behavior were noted: (1) Resting: The chick was inactive and in some cases may have been hiding. (2) Active I: The chick was less than 4 feet from its parent and was moving about. (3) Active II: The chick was moving about 4 or more feet from its closest parent. (4) Begging: The chick was soliciting food from its parent (see Tinbergen 1960 for a description of begging). (5) Feeding: The chick was obtaining food from its parent. June and July results were similar, and are combined in this presentation. RESULTS Intervals between a chick's feeding and its subsequent initiation of begging were longer during periods in which no parent returned to the territory than when begging followed the return of the parent. When begging was not preceded within an arbitrary 6-min period by the return of a parent, chicks initiated begging an average of 43 ± 24 min after their last feeding (N = 61). When a parental return was followed by begging within 6 min, the interval between the previous feeding and that begging was 31 .± 16 min (N = 16). This difference was statistically significant (t = 2.05, P < 0.05). The return of a parent to the territory was frequently followed by the initiation of begging by the chick. Of 71 parental returns watched, 65% were followed within 6 min by begging. The observed frequency of begging in the 6-min intervals following returns was 0.022. The expected frequency of begging following a parental return based on their joint probability was 0.0018 per 6-min interval, indicating that the return of parent probably stimulates a chick to beg. Activity patterns were different before and after begging (Fig. I). Before begging, resting predominated. If feeding followed begging, activity I close to the parent increased in proportion to resting while activity II remained at the same low level as before begging. When chicks obtained no food, begging was followed by an increase in activity II at distances greater than 4 feet from the nearest parent (Fig. I) . In some cases, especially after prolonged begging, the parent moved away from the chick; in other cases the chick moved away from the parent after ceasing to beg. The average distances of chicks from their closest parent are presented in Fig. 2. Before begging and after feeding, chicks were usually within 2 feet of their parents (Fig. 2). After unsuccessful begging this distance increased to 4 to 5 feet from the parent. Only after 10 or more minutes did the average distance between the chick and its parent begin to return to a distance similar to that of a chick that had fed successfully. Nine attacks on chicks by neighboring adult gulls occurred when chicks were at or beyond their territory boundaries. Eight of these attacks came after chicks were unable to obtain food from their parents subsequent to begging (P = 0.02, Binomial Test). The ninth attack was on a chick active (II) more than 4 feet from its parent after a successful feeding. The preponderance of attacks against chicks after they failed to obtain food is particularly striking. The total time in this category was only 26'% as great as the total time of observation subsequent to 526 HUNT AND McLOON [Auk,Vol. 92 successful feeding (Fig. 1). All nine attacks occurred when the chicks were active (II) 4 or more feet from their parents; chicks were noted in this category only 4% of the total time (Fig. 1). DISCUSSION Chicks were more active following begging (active II) and feeding (active I) than prior to begging (Fig. 1). Distances between the active chick and its parent were larger after unsuccessful begging than after feeding (Fig. 2), and resulted in increased exposure of the chick to attack by neighboring pairs. These results may explain why, in several species of gulls, chicks with relatively low growth rates have higher mortality rates than chicks with higher growth rates (Kadlec et al. 1969, Hunt 1972, Ward 1973, Hunt and Hunt 1975. Death from starvation is usually not the result of these low growth rates (Ward 1973, Hunt and Hunt MS). In one study chicks killed by neighboring gulls had significantly lower growth rates than chicks that survived (Hunt and Hunt MS) . Being underweight may lower the resistance of a chick to adverse weather, disease and attacks by adult gulls (Harris andPlumb 1965, Ward 1973). Our studies provide an explanation of why the underweight chick (i.e. those fed less often) may be more subject to attack in the first place. Fordham ( 1970) found that killing of chicks by adult Dominican Gulls (L. dominicanus) increased when the food supply to the colony decreased. Although he provided no data on growth rates, our findings support Fordham's suggestion that this increase in chick mortality was related to an increase in the chicks' wandering. A similar explanation is useful for understanding the results of Ward ( 1973). In comparing reproductive success of Glaucous-winged Gulls in colonies with differing access to food resources, he found lower growth rates and lower fledging rates on Mandarte Island where food was less available than on other islands. On Mandarte a larger percentage of the chick mortality was due to killing by other gulls, and we suggest that increased activity on the part of underfed chicks may have been partially responsible for these differences. This explanation may also account in part for the lower success of supernormal broods on Mandarte Island (Ward 1973). The return of a parent to the territory often stimulates its chicks to beg. Exposure of chicks to neighbors resulting from activity after begging may be minimized if parents time their returns to a frequency no less than the period between the feeding of a chick and its next spontaneous begging (about 45 min in this study). Frequent shorter 527 trips by a parent, as well as failure to provide food to a begging chick, may result in increased chick activity. Our results provide a link between food availability and densitydependent chick mortality without recourse to explanations dependent upon starvation. Decline in food availability will be reflected in the ability of parent gulls to provide begging chicks with food. \\'hen chicks fail to receive food, their increased activity will raise the probability of their being killed by neighbors. This probability of being killed is in turn a function of territory size (Hunt and Hunt MS) and should be greater in dense colonies (see also Ashmole 1963 for a similar argument concerning terns). Thus food will have two density-dependent actions, one related to the availability of food per individual, and the other related to the size of nesting territories and the chick activity discussed in this paper.

v3-fos

2020-12-10T09:04:22.832Z

1975-03-01T00:00:00.000Z

237232288

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Solid-Substrate Fermentor for Ochratoxin A Production A laboratory-scale fermentor designed for solid-substrate fermentation was constructed and tested. Its capacity to produce ochratoxin under varied conditions was determined with wheat as substrate. Ochratoxin yields of 2,000 to 2,500 μg/g of wheat were regularly obtained, and occasionally yields as high as 4,000 μg/g were obtained. The most critical factor in the fermentation was initial substrate moisture content; wheat tempered at 30 to 31% moisture produced the highest yields. Other variables tested were agitation and aeration rates, initial static culture time, and inoculum types and volumes. The demand for gram quantities of mycotoxins for testing purposes has brought into focus the need for larger, special production equipment and technology for its use. Heretofore, mycotoxins have been produced on solid substrate only in the laboratory, mainly in shakenflask culture (1, 3,5). In contrast to conventional liquid fermentations, many mycotoxins are produced on solid substrates, usually highmoisture grain, inoculated with a specific mold culture. Although grain fermentations may be conducted in laboratory equipment, no largescale, solid-substrate equipment is currently available or in common usage in the United States. In Japan solid-substrate fermentations are being conducted commercially in automated equipment (2). The main products are fermented foods and enzymes used in manufacture of soy foods. The substrate is cereal grain, usually wheat or rice. Apparently these fermentations do not require continuous agitation, and only occasional turning of the solid medium is involved. In the United States, the fermentation industry virtually centers around liquid culture in deep tanks. Many aspects of the process with liquid substrates are less involved than with solid media; e.g., effectual agitation may be accomplished with impellers, or sometimes the usual aeration will provide the necessary motion. A completely different approach is required for the mechanical movement of grain or other solid substrates to achieve the required agitation in a fermentation process. In this investigation, a laboratory-scale, solid-substrate fermentor was constructed and the production of ochratoxin (OT) was studied as a model system. OT was the mycotoxin of choice because of its relative ease of assay and extraction from fermented substrate. Experimental variables tested with the fermentor were initial substrate moisture levels, agitation and aeration rates, initial static culture time, and inoculum types and volumes. In addition, analyses were made periodically during the fermentation for time of OT optimal yield and for moisture and volume increases of the substrate. MATERIALS AND METHODS Fermentors. Originally we considered construction of a small, baffled, rotating drum fermentor similar to the laboratory-scale units developed for gluconic acid production (6) and later tried at the Northern Regional Research Laboratory in the early penicillin work. However, to explore the anticipated number of variables in a minimum of time, we constructed two fermentor units, each consisting of four simulated cross sections of a 13-in. (33-cm) diameter, baffled, stainless-steel drum (Fig. 1). Each section was 3 in. (7.6 cm) wide and had tight-fitting Plexiglas sides to permit observation during operation. The four drum sections were spaced on a common shaft of stainlesssteel pipe, and each was secured to the shaft with an Allen set screw in an aluminum hub. The shaft rotated in two half-bearings located near the ends. Power source was a Zero-Max variable speed drive (1 to 40 rpm) equipped with chain and sprocket drive. Fermentors were protected from heat generated by the power units with styrofoam insulation board shields. The individual drum sections were equipped with four baffles each to facilitate agitation. For the most effective agitation, it was considered desirable to have baffles of a size and shape that would permit all of the substrate to be elevated and subsequently dropped at some point during each revolution. Through experimentation, it was found that baffles 3 in. (7.6 cm) long and bent upward at a 300 angle in the direction of A sampling port for each fermentor consisted of a 1.25-in. (3.7-cm) round hole in the drum shell midway between two baffles. This port was used for charging the fermentor, inoculating, making water additions for moisture control, and withdrawing samples for analysis during the fermentation period. Tight-fitting rubber stoppers served as simple but effective closures for the ports. To test the effect of aeration on the fermentation by-product yields, the four-sectional fermentors were equipped to allow continuous passage of sterile air through each section. The shaft of stainless-steel pipe had four 3/16-in. (0.48-cm) holes drilled through the pipe walls to allow passage of air forced through the hollow shaft. A rotary joint was installed on each end of the shaft for connecting of air hoses. The fermentor units could be readily dismantled for cleaning and resterilizing. The power block assembly, mounted with bolts through a slotted base plate, could be moved backward so that the drive chain was easily removed from the sprockets. After removal of the rotary joints from the shaft ends, the assembly of four drum sections could be lifted as a unit off the half bearings. Loosening the Allen set screws allowed the individual sections to be slid from the shaft for cleaning and preparation for reuse. Sterilization was accomplished by reassembling the fermentor sections on their shaft and treating as a unit with ethylene oxide gas. Preparation of substrate. Since OT was the desired fermentation product, wheat was used as the substrate (3). Hard, red winter wheat (Parker) was used throughout these studies. To permit ready infection by mold hyphae, the wheat kernels were lightly cracked. Wheat fines were removed by screening. For each fermentor 1 kg of cracked wheat was tempered and sterilized in a 2,800-ml Fernbach flask. Tempering was accomplished by adding sufficient tap water to bring the wheat moisture content to 30 to 31%. The flasks were covered with metal foil to prevent evaporation and shaken occasionally to facilitate moisture distribution. After standing overnight at room temperature, any wheat masses were thoroughly broken up by shaking. For sterilization, the flasks were autoclaved 45 min at 121.5 C. The wheat was cooled overnight and transferred to the fermentors by pouring from the flasks through a wide-mouthed, sterile, stainless-steel funnel inserted in the sampling ports. Culture and inoculum. The culture used for OT production in these studies was Aspergillus ochraceus NRRL 3174, obtained from the Agricultural Research Service Culture Collection. Spores for inocula were produced either on malt extract agar in 6-oz (178 ml) culture bottles or on bread cubes by the method of Sansing and Ciegler (4). Spore suspensions from 2-week-old cultures in the culture bottles were prepared by adding 30 ml of sterile H2O and dislodging the spores with a wire loop; 5 ml of the suspension was added to each fermentor as inoculum. Sporulated bread cubes were used for inoculum either as dry cubes added directly to the fermentors or as an aqueous spore suspension made from the cubes. For the dry inoculum, four cubes were added to each fermentor. The aqueous bread cube spore suspension was prepared by adding 60 ml of sterile HO to a flask containing 20 of the cubes and agitating vigorously to dislodge spores; 5 ml of the resultant suspension containing about 108 spores/ml was added to each fermentor as inoculum. Fermentations were conducted in a 25 C constant temperature room. Sampling and analyses. Approximately 20 g of fermented wheat was removed daily from each fermentor for analysis. Samples were obtained by opening the sampling port while the fermentor was stopped, with the port situated near its lowest position of the cycle. Samples were tested for volume, moisture, and OT content. Temperature checks were also made with a sterile thermometer inserted into the wheat. Volume was ascertained by measuring a 10-g sample after lightly tamping in a graduated cylinder. Moisture content was determined by calculating the weight loss of a 10-g sample dried 24 h in a 110 C drying oven. Wheat samples were extracted and assayed for OT by the methods of Hesseltine et al. (3). Final quantitative determinations were made by visual comparisons, under ultraviolet light (wavelength, 366 nm), with varying amounts of pure OT-A standard. Starting fermentations in static culture. Theoretically, beginning the fermentation in static culture before starting regular agitation would allow initiation of mycelial penetration into individual wheat kernels without interruption from motion and friction created by agitation in a freshly inoculated fermentor. Static fermentation was accomplished by loosening the Allen set screw in the hub of one or more fermentors to allow the shaft to turn within the hub and the individual unit or units to remain stationary. Static culture for varying periods of time was tested by beginning the fermentation with three of the fermentors stationary and one revolving as a control. After 4, 6, and 8 days, agitation was started by tightening the Allen set screw of a particular fermentor. RESULTS AND DISCUSSION OT yields of 2,000 to 2,500 jsg/g of wheat were regularly obtained and occasionally yields as high as 3,000 to 4,000 ug/g were achieved with the four-sectional solid-substrate fermentor. These yields are well above those previously reported from shaken-flask culture. The highest reported yield from shaken flasks was 1,497 Mg/g of wheat (3). The OT peak was usually reached in 10 to 12 days (Fig. 2). A lag phase of 4 days was followed by a very rapid OT increase during the next 5 to 7 days. After the peak had been reached, there was usually a brief leveling off for 1 to 3 days, followed by a gradual diminution of yield. From Fig. 2, it will be noted also that during the OT production cycle there was a corresponding increase in both the wheat moisture content and volume. The moisture increase was the result of carbohydrate metabolism yielding H2O as one of the by-products. A slight moisture decrease occurred during the first 2 days, or early lag phase, since little H2O was produced before active metabolism had begun, and at the same time some substrate moisture was given up to the fermentor atmosphere. The wheat volume increase during the OT production phase closely paralleled the moisture increase. The volume usually increased 60 to 70% during the normal OT production cycle, and in prolonged experiments it increased by 100%. Individual wheat kernels swelled as the moisture content rose, taking on a "puffed wheat" appearance. The wheat expansion is a factor that must be considered in calculating initial substrate loads in relation to total fermentor capacity. Baffled fermentors can lose their agitation efficiency with initial overload-ing and subsequent substrate expansion during fermentation. The initial wheat moisture level is the most critical of all fermentation conditions. Highest yields were obtained with a starting moisture level of 30 to 31% (Fig. 3). Either higher or lower initial moisture content resulted in inferior OT production. Lower moisture resulted in a longer lag phase, a longer period to reach production peak, and a reduced yield. Higher moisture resulted in substrate adhering to fermentor walls and also rendered the substrate more vulnerable to bacterial contamination; yields were also inferior. If after the fermentation had begun it was found that the wheat moisture content was too low, it was both possible and practical to correct the deficit. Sterile water was added in increments of 5 ml or less to obviate sticking of wet substrate to the fermentor walls. Good OT yields were obtained when the substrate moisture was adjusted even though belatedly. It is apparent that the highest agitation rate, 16 rpm, resulted in the highest OT yields (Table 1) quired to reach the production peak than at lower agitation rates. Yields as high as 4,000 gg/g were obtained at 16 rpm, but 12 to 19 days were required at that speed. With agitation of 1 rpm, yields of 2,300 to 2,500 gg/g were obtained in only 8 to 9 days. Agitation rates of 6, 8, or 10 rpm demonstrated no advantage over 1 rpm, but shorter fermentation times were required to reach peak yields with all speeds under 16 rpm. At the low extreme, 0 rpm, the advantages of agitation are very dramatic; the yield was only 100 to 200 ,g/g with no agitation. Brief periods of agitation once or twice daily did not improve yields in the otherwise static fermentations. Likewise, starting the fermentation with static culture had no advantage. As initial static time was increased, progressively lower yields resulted, and the fermentation with no initial static time produced highest OT yields (Fig. 4). Agitation is obviously necessary and should be started as soon as inoculation is completed. Extensive aeration studies were not conducted because introduced air even at low rates was apparently deleterious to the fermentation. Yields with forced aeration were always inferior to those with no introduced external air. Of the several types of inocula used in the four-sectional fermentors, either of the two aqueous spore suspensions, whether from agar or from bread cubes, produced higher amounts of OT than the dry, sporulated bread cube inoculum. Of the two aqueous suspensions, that from sporulated bread cubes was preferred, because producing and harvesting the spores was less laborious than with the agar method. Typical of most mold fermentations, heat was generated during the OT fermentation. After a 4-day lag phase, there was a gradual rise of 1 to 2 C per day until the temperature leveled off at 33 to 33.5 C concomitant with peak OT production. The temperature might have risen considerably more, except that the narrow-width construction of the four-sectional fermentors allowed some heat dissipation. In larger vessels a greater heat buildup would be expected. High temperatures could greatly reduce product yields, and means for cooling would be necessary for conducting solid-substrate fermentations in larger units. Agitation serves a number of useful purposes in the OT fermentation. It effectively distributes spore inoculum. It maintains homogeneity throughout the fermentation period, preventing mycelial mass formation characteristic of static mold culture, and restricts growth to individual kernels. Agitation promotes aeration by exposing individual wheat kernels to the fermentor atmosphere momentarily during each revolution. One very important function of agitation is that of facilitating heat exchange and preventing localized overheating of substrate. The superior OT yields obtained with the four-sectional fermentor compared with those from shaken flasks might be attributed to the more efficient substrate agitation achieved in the baffled fermentor. In the shaken flask, wheat substrate seems only to slide around, and individual kernels probably surface only randomly for exposure to fermentor atmosphere compared with the regularity of the baffled fermentor. Also, the agitation rate can be more precisely controlled in the fermentor than in shaken flasks. The results obtained with OT-A as a model system in the four-sectional fermentor demonstrate the potential of solid-substrate fermentations in larger vessels for production of mycotoxins generally and possibly of other secondary metabolites. The concepts for fermentor design and operation resulting from these studies should be useful for scaling up equipment and conducting other solid-substrate fermentations.

v3-fos

2020-12-10T09:04:20.713Z

1975-03-01T00:00:00.000Z

237232515

s2

Oxidative Degradation of Squalene by Arthrobacter Species An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K2HPO4 to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity. Squalene is a naturally abundant acyclic triterpene, formation of which is thought to occur by the tail-to-tail condensation of two farnesylpyrophosphate (C,,) molecules via presqualenepyrophosphate (8,10), and is an important precursor of steroids and triterpenes. The mechanism of its cyclization to lanosterol via 2,3-oxidosqualene in mammalian livers has been reported by several workers (5,13). Zander et al. (14) have reported another nonoxidative cyclization of squalene in Tetrahymena pyriformis. By this organism, squalene is cyclized to form tetrahymanol by a hydration reaction. Recently, 12,13-dehydrosqualene formation by Staphylococcus aureus (11,12) and also by Halobacterium cutirubrum (9) has been reported, in which the molecule of squalene is dehydrogenated at its center of symmetry. In spite of these findings, no information on microbial degradation of squalene is available. We now wish to report the isolation and identification of the products of degradation of squalene by Arthrobacter sp. This organism is an isolate from soil capable of utilizing squalene and belongs to the genus Arthrobacter. MATERIALS AND METHODS Microorganisms and culture conditions. A strain of Arthrobacter sp. isolated from soil was used throughout this study. A detailed description of the bacterium will be presented elsewhere. The soil samples were inoculated into test tubes, each containing 3 ml of the selection medium consisting of 1.5% squalene, 0.2% yeast extract, and 0.4% NH4NO3, and incubated at 27 C for 3 days on a reciprocating shaker. After extraction of the culture broth with dichloromethane and evaporation of the solvent, the products were analyzed by thin-layer chromatography, using n-hexane-ether (95:5) as the solvent system. Single colonies were isolated from the culture broths that accumulated products of squalene degradation, as evidenced by thin-layer chromatography. A 0.5-ml portion of fresh seed culture was inoculated into 500-ml Sakaguchi flasks (a globular flask with flattened shoulder) containing 50 ml of medium. Medium A contained 2% corn-steep liquor and a specified amount of squalene and was adjusted to pH 6.9 with NaOH; medium B consisted of 2% glucose, 0.5% KNOI, 0.02% MgSO4 7H2O, 0.1% yeast extract, 0.1% KH2P04, and 0.5% squalene and was adjusted to pH 6.5. Flasks were incubated at 30 C on a reciprocating shaker. Assay procedures. Growth was monitored by absorbance at 550 nm after the removal of oily materials from the culture broth by extraction with 3 volumes of a solvent mixture (ethanol-butanol-chloroform, 10:10: 1), centrifugation of the cells at 12,000 x g for 10 min, and resuspension in water. The quantities of squalene and its degradation products were estimated by gas chromatography. Samples (50 ml) of culture broth were extracted three times with 30-ml portions of dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The oily residue was weighed and subjected to gas chromatography. The content of geranylacetone was estimated from the peak height by comparison with that of a known amount of an authentic sample. 400 Analytical methods. Gas chromatography was performed on a Hitachi gas chromatography 063 instrument equipped with a thermal conductivity detector. Separations were performed on a stainlesssteel column (100 by 0.3 cm) containing SE-30. Helium served as carrier gas at a flow rate of 21 ml/min. Temperatures of the column injection port and detector will be specified below. Infrared spectra were recorded on a Hitachi model 215 grating infrared spectrometer. Oily samples were run on neat liquid films between NaCl plates. The nuclear magnetic resonance (NMR) spectra in CDCl, were recorded with a JEOL model PS 100 spectrophotometer, with tetramethylsilane as the internal standard. Mass spectra were obtained with a Hitachi RMU-6E mass spectrometer. Chemicals. Squalene was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo) and used without further purification. Geranylacetone used as the authentic sample was a gift from Takasago Perfumery Co. Ltd. Isovaleric acid was prepared from #,#'-dimethylacrylic acid by catalytic hydrogenation. Other chemicals were obtained from commercial sources. RESULTS Biotransformation of squalene by Arthrobacter sp.: identification of geranylacetone. Geranylacetone was isolated from the culture broth as follows. After 38 h of culture in medium A supplemented with 242 mg of squalene per flask, the culture broths were combined (500 ml) and extracted three times with 300-ml portions of dichloromethane. The extracts were combined and dried over anhydrous sodium sulfate. After evaporation of the solvent, 10 ml of n-hexane was added to the residue, and the resulting precipitate was removed by filtration. The oily residue (1.740 g) obtained after evaporation of the solvent contained 55% geranylacetone and 35% unchanged squalene when analyzed gas chromatographically at the following temperatures: column, 310 C; injection port, 340 C; detector, 340 C. The yield, calculated on the basis of two molecules of geranylacetone from one molecule of squalene, was 55.87%. Pure geranylacetone (0.917 g) was obtained by distillation of the oily residue under reduced pressure. The sample thus obtained had a bp of 90 to 95 C at 7 mm of Hg. Elemental analysis showed: C, 80.21%; H, 12.03%; the calculated values for C13H220 were: C, 80.35%; H, 11.41%. The infrared spectrum of the sample was almost identical to that of authentic geranylacetone (cis-trans mixture) and had values of vn.axtl'm at 1,720, 1,440, 1,380, 1,360, and 1,155/cm. The 1,720/cm band is attributed to the >C=O group of the molecule. This observation was confirmed by mass spectral analysis (70 eV) where the parent ion was observed at m/e 194. Other peaks were observed at m/e 151, 136, 125, 107, 69, 43, and 41; m/e 151 was assigned as The NMR spectra of the compound in deuterated chloroform showed singlet signals at bppm of 1.59, 6H (cis-C,-CH3 and cis-C 0-CHO); 1.69, 3H (trans-C lo-CH3); and 2.11, 3H (CO-CH3) and a triplet band at 5.09 (olefinic protons at C5 and C9). Evidence for the trans geometric isomerism of the C,5-C. double bond was that the NMR signal of two methyl groups at C, and C,0 appeared at the same 6 value, 1.59 (2). Preparation of geranylacetone semicarbazone. To confirm the molecular formula, the semicarbazone derivative was prepared. After recrystallization from an ether-cyclohexane mixture, the semicarbazone had an mp of 94 to 95 C. Elemental analysis of the compound showed the following percent composition: C, 66.48; H, 10 The infrared spectrum of the sample was identical to that of the semicarbazone derived from authentic geranylacetone (cis-trans mixture) and had absorption bands at Vmaxfnujol values of 3,540, 3,300, 1,690, and 1,580/cm. The main mass spectral peaks observed had m/e values of 251 (parental ion), 182, and 69. On the basis of these data obtained from the isolated compound and its semicarbazone, the carbonyl compound produced from squalene by Arthrobacter sp. was identified as trans-geranylacetone. Time course of the production of geranylacetone. Figure 1 shows a typical time course of growth and geranylacetone formation in medium B. After a long lag period (12 to 20 h), cell growth and geranylacetone accumulation started. Geranylacetone formation was found to be parallel with the cell mass increase in the logarithmic growth phase and continued after the logarithmic growth phase at a higher rate than growth. The pH value of the culture broth remained constant during the culture period. The yield of geranylacetone obtained to squalene added was 16% (wt/wt), which was much smaller than that obtained in medium A (39%). Isolation and identification of carboxylic acids produced by Arthrobacter sp. Preliminary experiments had shown that several spots other than squalene and geranylacetone were observed when cells were cultured in medium A supplemented with 1% K2HPOi. For the isolation and identification of these products, the following experiments were carried out. Twelve flasks, each containing 75 ml of medium A supplemented with 1% K2HPO, and 0.65 g of squalene, were inoculated and incubated for 65 h. After acidification of the combined broths to pH 2 with 3 N HCl, the oil fraction was prepared according to the procedure described previously. Yield was 5.21 g. The oil was dissolved in dichloromethane and extracted with 10% sodium carbonate solution. The alkaline solution was acidified with 3 N HCl and extracted with dichloromethane. After drying over anhydrous sodium sulfate, the solvent was evaporated to give carboxylic acids in a yield of 0.54 g. The yield of neutral compounds that were not extracted with sodium carbonate solution was 4.3 g. The gas chromatogram of this neutral moiety showed that it contained 44% geranylacetone and 50% unchanged squalene. The carboxylic acids thus obtained were separated into five peaks by gas chromatography (temperatures: injection port, 250 C; column, 170 C; detector, 260 C). These peaks were designated A, B, C, D, and E in the order of retention time (Fig. 2). Fractions corresponding to peaks A plus B, C plus D, and peak E were collected at the detector outlet of the gas chromatograph instrument and used for identification experiments. Identification of isovaleric and f34Tdimethylacrylic acids. Infrared spectra of the mixture of acids A and B in film showed P,=, absorption at 1,640/cm. When the mixture was hydrogenated on palladium-charcoal (5%) in methanol under atmospheric pressure, a pure carboxylic acid, which was identical with peak A carboxylic acid by gas chromatography, was obtained. The infrared spectra of the carboxylic acid obtained by hydrogenation of the mixture coincided perfectly with that of isovaleric acid. This suggests that the carboxylic acid of peak B has a skeleton of isovaleric acid. Mass spectra of the A-B mixture showed two parental molecular ions at m/e of 102 and 100, which correspond to isovaleric acid (A) and carboxylic acid (B), respectively. The NMR spectra in CDC13 of the A-B mixture showed signals of (CH3)2-CHmethyl protons at 6 0.96 (doublet), CH3-Cmethyl protons at 6 1.90 and 2.15 (singlet), and an olefinic proton at a 5.62 ppm. From these data carboxylic acid B was identified as A,:'dimethylacrylic acid. The ratio of the quantities of isovaleric and f,f3'-dimethylacrylic acids estimated from the NMR spectra was 3:1. Identification of citronellic and geranic acids. The mass spectra of the C-D mixture (C==C). These data support the assignment stated above. To confirm the molecular structures of acids C and D, isolation of these two acids in the form of their methyl esters was performed. A mixture of carboxylic acids (A, B, C, D, etc.) (320 mg) was methylated with diazomethane in ether. From the reaction mixture, 214 mg of methyl esters was obtained by the standard method. The methyl esters of acids C and D were separated by gas chromatography (temperatures: injection port, 220 C; column, 150 C; detector, 215 C) (Fig. 3). Peaks C' and D' correspond to carboxylic acids C and D, respectively. Peak E' was a mixture of carboxylic acid esters which were not identified. Fractions corresponding to peaks C' and D' were collected separately and used for identification experiments. Identification of methyl (+ )-R-citronellate. The infrared absorption spectra of the methyl ester C' in film showed an ester carbonyl group absorption band at 1,740/cm and no strong absorption in the region of 1,600 to 1,700/cm, which indicated that the ester C' was an a,3- This ester showed a plain optical rotatory dispersion curve in the region of 250 to 600 nm. The optical rotation values obtained were [a]30025 = + 204°, [a ]3525 = +91°, and [a ]40025 = +68°(C = 0.0044 in methanol). This suggested that the ester is methyl (+ )-R-citronellate (6,7). Identiflcation of methyl geranate. The infrared absorption spectra of the methyl ester D' in film showed an ester carbonyl group at 1,720/cm and a strong absorption band of C=C conjugated with a carbonyl group at 1,645/cm. The mass spectra of the ester D' showed a peak of parental molecular ion at m/e 182 and other fragments at 151 (M+ -CH30), 123, 113, 83, 69, 55, and 41. These spectral data suggested that the ester D' is methyl geranate. DISCUSSION Arthrobacter sp. which was isolated from soil can oxidatively decompose squalene (C30) (I) into geranylacetone (C,,) (II). The yield of geranylacetone to squalene consumed was 56% on the basis that one molecule of squalene gives two molecules of geranylacetone. This fact suggests that squalene molecules in part were cleaved at the two sites shown by wavy lines in the structural formula in Fig. 4. In mammalian liver, squalene is first oxidized at its terminal double bond to give squalene-2,3-oxide (5, 8), which is then cyclized. In the case of Arthrobacter sp. the central part of squalene is attacked, and fission occurs symmetrically at C 1o=C11 and C 14==C 5, although the cleavage mechanism has not been elucidated. Another central attack of the squalene molecule to form 12,13-dehydrosqualene by S. aureus and by H. cutirubrum has been suggested (12,14). When medium A was supplemented with a high concentration of dipotassium phosphate, squalene was oxidized to carboxylic acids in addition to geranylacetone. The carboxylic acids comprised up to 10% of the total recovered VOL. 29,1975 oil. The main carboxylic acids were identified as isovaleric acid (V), #,3'-dimethylacrylic acid (VI), geranic acid (IV), and (+)-(R)-citronellic acid (III). Though several efforts to oxidize geranylacetone using the intact cells were not successful, it is probable that these carboxylic acids are derived from squalene by a "central attack" mechanism which is common to geranylacetone formation. The fact that all acids accumulated have beta-substituted methyl groups might suggest that this configuration is resistant to biological oxidation by this organism. The a,f3-saturated form was found to be predominant for both C5 and Clo acids. The oxidative cleavage of squalene by Arthrobacter sp. is a useful method of obtaining pure trans-geranylacetone. The supposed intermediate of the oxidative degradation of squalene by this organism, which retains a C30 skeleton, probably has functional groups such as hydroxyl groups in its central position. As has been suggested by others (1,3,4), flexible linear molecules, such as squalene, which have no specific groups could hardly be the object of selective chemical reactions, especially in central parts of the molecule. Thus, the application of this microorganism to introduce functional groups into central parts of the squalene molecule is interesting from the viewpoint of the synthesis of squalene derivatives.

v3-fos

2019-03-19T13:04:00.645Z

1975-05-01T00:00:00.000Z

82126294

s2

Occurrence of Helminthosporium species on cereals in Finland in 1971-1973. This study was carried out on Helminthosporium species found on cereals (Avena sutiva L., Hordeum vulgare L., Triticum aestivum L., Secale cereale L.) and couch grass (Agropycon repens (L.)PB.) The objective was to ascertain their distribution and general significance in Finland. The results were obtained from samples of cereals gathered in 2040 fields all over the country during the growing season 1971 1973. The samples of couch grass (approx. 170 samples) were collected in fields and the borders general significance in Finland. The results were obtained from samples of cereals gathered in 2040 fields all over the country during the growing season 1971 1973. The samples of couch grass (approx. 170 samples) were collected in fields and the borders of fields. The fungi in all the samples were examined by microscope, using the blotter method and with cultures. Many Helminthosporium species were found to be very common and abundant on cereals, especially in 1972, throughout the country and as far as Lapland. The imperfect stages (conidia) of the fungi occurred dominantly and in abundance, whereas the perfect stages of the fungi were found only occasionally at all times. Pyrenophora tritici-repentis (Died.) Drechs. on Agropyron repens was exceptional in this respect. The most widespread and common Helminthosporium fungi were the following species: H. avenae Eidam was found to be very common and abundant in more than 60 per cent of the oat fields and in nearly 90 per cent of the localities that were studied throughout the country. These figures apply to seedlings as well. H. gramineum Rabenh. ex Schlecht was found frequently in more than 30 per cent of the spring barley fields and in about 55 per cent of the localities that were studied. It was very common and abundant in Ostrobothnia and the northern parts of the country and was frequent on six-rowed barley, too. H. teres Sacc. was found to be common and abundant in nearly 60 per cent of the spring barley fields and in nearly 90 per cent of the localities that were studied. These figures apply to the seedlings as well and are especially representative of observations made in the western and southern parts of the country. The fungus was more common on six-rowed than on two-rowed barlev H. tritici-repentis Died, was found to be moderately common in southern and southwestern Finland as follows: in nearly 30 per cent of the spring wheat fields and in 40 per cent of the localities studied; in 17 per cent of the winter wheat fields and in 23 the winter rye fields; for both the latter mentioned moderate abundance was observed in more than 50 per sent of the localities studied. On Agropyron repens the fungus was very common and abundant in 50 per cent of the samples and in nearly 60 per cent of the localities studied. In addition, the perfect stage of the fungi, Pyrenophora triticirepentis (Died.) Drechs. with mature ascoma and ascospores was found in about 1/4 of the samples examined throughout the country. Introduction Several Helminthosporium species are known to be serious causers of leaf spot diseases on cereals cultivated all over the world (Sprague 1950). In Scandinavia these diseases have been known since the last century (Ravn 1901, Jorstad 1945, but due to the extensive use of mercurial fungicides they have continued to play only a minor role, except in the northern parts of the countries (Fritz 1966, Kolk 1966, Kolk and Karlberg 1973, Andersen 1955, Jorgensen 1969, Hansen and Magnus 1969, Overaa 1972. In recent years, however, there has been a marked increase in the prevalence of Helminthosporium species, especially on barley, e.g. in Norway (Hansen and Magnus 1969), Denmark (Smedegärd -Petersen 1971 and Finland (Blomquist 1970). There may be several reasons for this; the increased acreage devoted to cultivation of barley, the influence of new barley varieties and climatic conditions (Smedegärd Petersen 1972b, 1974, Mäkelä 1972. Their abundance may also be due to the use of undressed seed (Lihnell 1969, Blomquist 1970, Overaa 1972. In Finland there are thus far only a few studies about the Helminthosporium species causing diseases on cereals. However, these fungi have been known for a long time and they have continually caused serious damage, especially on barley (Liro 1917, Halkilahti 1971. In recent years there has been increased the interest in the Helminthosporium species of cereals (Mäkelä 1971, Rekola et ai. 1970, Halkilahti 1971. Cereals are grown on about 46 per cent of Finland's total field area (2 665 000 hectars). More than 90 per cent of the cereals area is covered by spring cereals (oats, barley, wheat); less than 10 per cent by winter cereals (wheat, rye). Oats (area 19 %) and barley (area 17 %) are among the most important cultivated plants in Finland and they thrive even in the northern parts of the country. Spring wheat (area less than 5 %), winter wheat (area 2 %) and winter rye (area 2 %) are grown in the southern and central parts of the country. The proportion of barley is showing an increase in recent years, 183 whilst the acreage devoted to wheat and rye is decreasing (Official statistics of Finland 1973). This study is part of a research project dealing with spot diseases on cereals, and specifically with the pathogens causing these diseases (cf. Mäkelä 1972Mäkelä 1974. The purpose of the present study is to clarify the occurrence of Helminthosporium species on cereals. The study is based on samples of cereals whicnwere gathered in farmers' fields throughout the contry over a three year period (1971)(1972)(1973). Materials and Methods The bulk of the cereal samples (Table 1) was gathered from southwestern and southern Finland; samples of barley and oats also came from northern Finland, particularly in 1973. Only rare samples were gathered from Uusimaa, South Savo and South Karelia in 1973, owing to exceptionally dry weather. The samples of barley were gathered in 803 felds from 251 localities. About 80 per cent of these were six-rowed varieties and about 20 per cent were tworowed varieties. The samples of oats came from 181 localities, tne total samples numbering 415. The samples of spring wheat were collected in 222 fields from 111 localities, the corresponding figures for winter wheat being 259 fields and 72 localities. The samples of winter rye came from 112 localities, the total samples numbering 341. The samples of Agropyron repens (c. 170 samples) were collected in fields and the borders of fields in about 80 localities throughout the country. The bulk was gathered at a growth stage when the grain was milky ripe, chiefly from late July to early August (20 July 14 Aug., 1971;16 July 8 Aug., 1972;21 July 14 Aug., 1973). The samples of winter wheat and rye as well as of Agropyron repens were gathered in May also. Furthermore, observations of appearance of diseases were made in fields during the entire growing season. As a rule it was attempted to take the representative samples from large areas of the field. In general the samples were garhered in the same field only once. In most cases the variety remained unknown. The (Kolkki 1966). The average monthly precipitation varies from 35 to 80 mm and is lower early in the growing season than it is in the autumn (Helimäki 1967) 184 185 The weather in spring and summer in 1971 was characterised by the alternation of one or two week periods of exceptional cold or heat. There was very little precipitation during the growing season ecxept for the early part of August, particularly in southern Finland. The weather during the growing season of 1972 as well as from May to August of 1973 was exceptionally warm; in July >t was even 2 -4°C higher than normal. Precipitation conditions varied greatly in different parts of the country. In 1972 there was little rain in May and in June, whilst in August there was quite a lot of rain in southern and central Finland. During the entire growing season 1973 the amounts of precipitation were smaller than normal. Uusimaa and Kymenlaakso in particular suffered from drought. The winter of 1970 1971 was long with abundant snowfall. The winters of 1971 1972 and 1972-1973 were much milder than normal (Meteorol. Yearb. Finland 1971. Results In the present study the old form of undivided genus Helminthosporium is used instead of genera Drechslera and Bipolaris (cf. Shoemaker 1959). The imperfect stages of Helminthosporium species are used instead of the perfect stages of Pyrenophora and Cochliobolus species because the former stage of the fungi appeared abundantly in Finland, whereas the latternamed stages are found rarely or not at all (cf. Mäkelä 1971 (Turner and Millard 1931, Dennis 1933,Muller 1963). The fungus is seed-borne and the most important source of infection is the resting mycelium on the grain (Turner andMillard 1931, de Tempe 1964). H. avenae is widespread but only important in cool and moist climates, which in fact offer the conditions that suit the crop best (Sprague 1950, de Tempe 1964). In Europe the fungus is known to be common and destructive in the wetter, northern and western areas of British Isles (Butler and Jones 1949). It occurs also in Germany (Muller 1963). In Scandinavia H. avenae has been known in Norway since 1891. There it has afflicted oat seedlings (Jorstad 1945). Today the fungus causes some damage to roots and culms of oats (Overaa 1972). In Denmark H. avenae has been known on oat plants and on seed since 1896 (Ravn 1901). It is very common but the fact that it causes leaf blotch on oats has been overlooked (Andersen 1955). Today the fugus is little known in Sweden (Fritz 1965, Kolk 1966. 186 In Finland, too, H. avenue is apparently considered to be of negligible significance because up to now it has not attracted attention. According to Halkxlahti (1973 a) H . avenue infection on samples of oat seed was common in the harvests of 1967 and 1972. The fungus was found on seed produced throughout the country as far as North Ostrobothnia. In field trials in 1968 and 1969 at Viikki, Helsinki, the crop losses caused by H. avenue on oats were 2 10% (Rekola et ai. 1970). H. avenue infects, besides oats (Avena spp.), also barley and other species of graminae (Sprague 1950, Braverman 1960, Shoemaker 1962, Ammon 1963. In this study H. avenue was found to be common on oats throughout the country as far north as Lapland (Inari) (Fig. 1). The fungus was found in the folowing localities; H. avenae was encountered in different years in an average 63 % (range 46 -74 %) of the oat fields (415 fields) and in 87 % of the localities (181 localities) examined (Table 1). The occurrence of the fungus was fairly uniform in different parts of the country ( Table 2). The fungus was found on seedlings, sometimes abundantly in, for instance peat soil in early summer. The first two or three leaves of an infected seedling show brown stripes with yellow-reddish margins (Fig. 2 A) (cf. Rekola et ai. 1970). Leaf damage was commoner, however, at maturity (cf. Jorstad 1945). Initially it took the form of spots with an reddish-orange margin surrounding a dead, brown area. The spots may join together ( Fig. 2 C) (cf. Muller 1963). In late summer the fungus was very common. The disease occurred equally throughout fields. H. avenae formed a very characteristic tufty, white cotton growth or coremium on leaves of oats in humid conditions (Fig. 2 (Drehcsler 1923, Sprague 1950). The fungus is seedborne and usually is overwintered by mycelium in the pericarp (Ravn 1901, de Tempe 1964. This disease is generally of limited importance (de Tempe 1964) but still causes crop losses in many countries, e.g. in the USSR (Shchekochikhina 1964, Rasvlev andKraotsova 1970) and in Czechoslovakia (Zekovic 1970). In Scandinavia H. gramineum has been extremely scarce in Denmark for a long time (Andersen 1955, Jorgensen 1969). The disease is found to some extent in Sweden (Kolk 1966) and in Norway (Hansen and Magnus 1969), though its economic significance is small. However, the disease has again occurred in greater abundance, particularly on six-rowed barley, in recent years in the central and northern parts of these countries (Linhell 1969, Overaa 1972, Kolk and Karlberg 1973. In Finland H. gramineum has long been common (Liro 1917) and continues to be so (Halkilahti 1971, Mäkelä 1972. According to field tests at the State Seed Testing Station, stripe disease has occurred in about 40 -5O % of all the samples of barley examined during the last thirty years (Halkilahti 1971) In 1970 and 1971 H. gramineum was also found in about 30 % of the barley fields located in the southern and central parts of the country (Mäkelä 1972) In this study H. gramineum was encountered on barley throughout the country as far as Lapland (Inari) (Fig. 3). The fungus was found in the following localities: H. gramineum was found in different years in an average 32 % (range 24 -4O %) of the fields (803 fields) and in 54 % of the localities (251 localities) studied (Table 1). The fungus was much commoner in six-rowed varieties (23-44 %) than in two-rowed varieties (18-29 %), an observation which is widely confirmed (Hansen and Magnus 1969, Zekovic 1970, Kolk and Karlberg 1973. Similar results were obtained during all the trial years. On the other hand the occurrence of H. gramineum varied greatly in different fields and in different provinces (Table 3). In the most important barleycultivating areas, in the south-western and southern provinces, the fungus was observed to occur the least frequently. In Ostrobothnia (EP, KP, PP) leaf stripe of barley was found more frequently than in the neighboring provinces, not including the northern parts of the country. Fields may be found where more than 1/3 of the barley plants have been destroyed by leaf stripe in the late summer (Fig. 5). Results of the State Seed Testing Station substantiate these observations (Halkilahti 1971(Halkilahti , 1973. Ascocarps, morphologically similar to Pyrenophora graminea or P. teres, were found only a few times on overwintered stubble of barley. The fungus causes net-blotch disease of barley. It occurs occasionally also on oats, wheat, rye and other grasses (cf. Shipton et al. 1973). The fungus can be seed-borne as conidia or mycelium. It may over-winter also as the perfect stage on culms straw and stubble and as sclerotia on the dead leaves and on crop debris (Webster 1951, Kenneth 1962, Smedegärd Petersen 1971. Seed infection is probably worse at low temperature (Ravn 1901). H. teres occurs in most barley-growing countries (Sprague 1950, CMI map no. 364, 1968 and has caused several kinds of damage to barley, particularly during the last years (Kenneth 1962, Evans 1969, Rintelen 1969, Melville and Lanham 1972. In Scandinavia H. teres is commonly found in Sweden (Frit 1966, Kolk 1966, Kolk and Karlberg 1973 and in Norway (Hansen andMagnus 1969, Overaa 1972), though its economic significance is small. Also in Denmark the disease has so far played a minor role (Andersen 1955). During the past five years, however, there has been a marked increase in the prevalence of H. teres (Smedegärd Petersen 1971 192 T a b l e 3. (1) In Finland the same trend is seen. The fungus is apparently considered to be of negligible significance because up to now it has not attracted attention. In 1970 and 1971, however, H. teres was found in over 50 % of the barley fields examined (Mäkelä 1972). In this study H. teres was encountered on barley throughout the country as far north as Kemi Lapland (Pelkosenniemi) (Fig. 4). The fungus was found in the following localities: H. teres was found indifferent years in an average 57 % (range 45 -67 %) of the fields (803 fields) and in 87 % of the localities (251 localities) examined (Table 1). The fungus was rather commoner in six-rowed varieties (47-66 %) than in two-rowed varieties (37-68 %). Also e.g. two-rowed barley cv Herta is known to be resistant to H. teres (McDonald andBuchannon 1964, Hansen andMagnus 1969). The occurrence of H. teres varied greatly in different fields and different provinces ( Table 3). The disease was observed to occur most commonly in the southern and southwestern parts of the country, which are the most important areas where barley is grown, and to decrease gradually towards the north. The fungus was found to be particularly rare in North Ostrobothnia and Lapland. This runs counter to H. gramineum, which was found to be commonest in the northern parts of the country and rarest in the most important cultivating areas (Table 3, Fig. 3). Results were similar during all the trial years. In 1973 H. teres was found to be comparatively rarer than in 1971 and 1972, especially in Uusimaa, South Karelia and South Savo, apparently owing to weather conditions. Also the occurrence of H. teres varied greatly in different fields. As a rule the disease was uniform in a given field. The fungus tainted the leaves of barley with barious kinds of spots, the commonest of which were net blotch and small leaf spot (Fig. 5). These often occurred together in the same sample (cf. Mäkelä 1972). This material does not furnish a basis for saying which type of symptom caused by H. teres is the commonest in Finland. Only in 1971 was the late summer sufficiently humid to study symptoms of the disease on barley leaves, whereas in 1972 and especially in 1973 barley ripened too rapidly. The spot type has been found to be somewhat Pycnidia were observed in less than 5 % of the samples examined (Mäkelä 1972). The perfect stage Pyrenophora teres has not been found with certainty in Finland (cf. p. 191). Helminthosporium spp Helminthosporium spp. on wheat and rye In this study there was found on wheat and rye a fungus (or fungi) of the Helminthosporium species which is morphologically similar to H. gramineum and H. teres. The fungus was observed on rye only three times in 1972 (U; Tuusula 2. 8., St: Loimaa mlk. 22. 7., ES: Sääminki 24.7.), and on winter wheat also three times (EH: Luopioinen 24. 7. 1972, St: Hämeenkyrö 1. 8. 1972, V: Muurla 25. 7. 1973). In addition Helminthosporium species were found on spring wheat in small quantities throughout the country up to North Savo ( The fungus was encountered in about 11 per cent of the spring wheat fields (about 220 fields) and in 18 % of the localities (111 localities) studied (Table 1). Conidia of the fungus (or fungi) were found only on ripening and withering leaves of rye and wheat on brown, necrotic spots usually in the company of other fungi (Fig. 2 H, I). Only rare isolates have been made from leaves of spring wheat. Besides barley, H. gramineum is known to occur in many localities, though with little importance (e.g. Pettinari 1955, Rasulev andKavtsova 1970) in inoculation tests also on rye (Rasulev andKavtsova 1970, Mäkelä 1972). Also H. teres has been found on wheat (cf. Shiptom et al. 1973), in inoculation tests both on wheat and rye (Mäkelä 1972). H. avenue has occurred sparsely on wheat and rye (Kolk 1966 The fungus causes leaf blight and root injury on wheat and Agropyron repens (L.) PB. but is frequently found on brown necrotic leaves of many grasses (Sprague 1950, Shoemaker 1962). It appears to be a worldwide pathogen on wheat, especially on spring wheat (Triticum aestivum L.) (Sprague 1950, Hosford 1971, Voitova 1971. In Europe the fungus has played only a minor role on wheat. It was first observed in Germany in 1935 (Raabe 1937), Switzerland in 1959 (Ammon 1963) and Austria in 1965 (Glaeser 1966). Only a little is known about H. tritici-repentis on rye (Secale cereale L.) in Poland (Garbowski 1932), the USA (Sprague 1950, Earhart 1952 and Canada (Shoemaker 1962). On the other hand the species has been reported to be common on Agropyron repens (L.) PB. throughout the world (Sprague 1950, Shoemaker 1960). Wheat Helmintkosporium tritici-repentis occurred on wheat in the southern parts of the country (Fig. 6). The fungus was common on winter wheat in the south- 1971-1973. 199 western parts of the country and on spring wheat also in South Savo and in South Ostrobothnia. These are the main wheat-cultivating areas in Finland (Table 4). The fungus was encountered in 27 % of the fields of spring wheat (222 fields) and in 40 % of the localities (111 localities) studied as well as in 17 % of the fields of winter wheat (259 fields) and in 53 % of the localities (72 localities) examined (Table 1) Perfect stage: V: Perniö, 24. 5. 1972 Spores of H. triti ci-rep entis were found in greatest abundance right often early spring (April) on winter wheat and after early summer (July) on spring wheat on ripening and whithering leaves (Fig. 10 G); namely, on brown necrotic spot. The perithecial stage was found on stubble in spring (Fig. 8). Winte rye Helminthosporium tritici-repenlis was encountered in the southern parts of the country up to Middle Ostrobothnia (Fig. 7). This is Finland's rye bowl. The fungus was found in about 1/4 of the rye fields studied (340 fields) and in about 1/2 of the localities (112 localities) ( Spores of H. tritici-repentis were fond on rye since early June. In ripening season they were observed chiefly on brown necrotic spots, streaks or necrotic areas these may extend the length of the blade -or the tip of the blade (Fig. 8 A). In addition to H. tritici-repentis several other parasitic species such as Septoria nodorum (Berk.) Berk, and other Septoria species, Puccinia, Fusarium species and Erysiphe graminis CD. were observed grow at the same time on the leaves of rye and wheat. Similar observations have been made elsewhere (Sprague 1950, Hosford 1971 Conidia of H. trilici-repenlis were long, straight, cylindrical and light yellowish-brown in colour (Figs. 8 and 10) (cf. Hosford 1972). They were morphologically similar on wheat and rye. The effect of this disease on the yield is not known. The fungus, however, is probably of small economic importance in spite of its commonness. Conidia of H. tritici-repentis was found to be most abundant on dead leaves in the early spring and the late summer; it was rather rare in the early summer. Conidia of the fungus observed on couch grass (Fig. 10 F) were similar to the conidia on wheat and rye (Figs. 8 and 10). Perfect stage, Pyrenophora tritici-repentis' ascomas with mature ascospores were found to be very common (about 1/4) on overwintered dead culms and leaves in early spring. May-June (Fig. 10) The fungus causes root rot, seedling blight and leaf spot on cereals, above all on barley and wheat as well as on dozens of species of grasses (Drechsler 1923, Sprague 1950). In the western hemisphere, where intensive cereal cultivation is practised and where temperatures are high, H. sativum is most important as a root rot or blight of wheat and barley (Sprague 1950). The fungus caused root rots of wheat also in zones with sufficient moisture in western and eastern Siberia and in the Soviet Far East (Korshunova 1968). In Europe, however, H. sativum commonly causes a leaf spot and seedling blight on barley and also, though less frequently on wheat (Muller 1956, Lange de la Camp 1958, de Tempe 1964. H. sativum survives in infected seed but can persist also in the soil (Muller 1956, de Tempe 1964, Evans 1969, Shchekochikhina 1971, Voitova 1971, Jorgensen 1974. The fungus occurs all over the world (Drechsler 1923, Sprague 1950, CMI map no. 322, 1967. In Europe, however, it is regarded as being of relatively little importance (Butler and Jones 1949, Muller 1956, de Tempe 1964. The fungus has been found on wheat and barley in Italy (Pettinari 1955), Holland (Vendrig 1956), Germany (Muller 1956), also on rye (Lange de la Camp 1958) and in Britain also on oats (Evans 1969). In Scandinavia H. sativum has been found sporadically in Denmark on the roots and stems of barley plants since 1930(Skov 1966, whereas it was very common on seeds of barley, wheat and oats (Andersen 1955). In recent years H. sativum has been recorded on barley seed (Jorgensen 1969(Jorgensen , 1974. The fungus has also been found rather commonly on barley straw and stubble in fields (SmedegArd Petersen 1972 a). The effect of such infections on the yield is not known. In Norway H. sativum was found for the first time on barley in 1960. As a leaf spot fungus it seems to be insignificant (Hansen and Magnus 1969). whereas it was found rather commonly on barley seeds as well as on oat seed, though somewhat rarely (Overaa 1972). In Sweden barley seeds were highly infected by H. sativum, while oats, wheat and rye to a lesser extent. The fungus had a distinct pattern of occurrence in different parts of the country (Fritz 1965, Kolk 1966. In Finland H. sativum was isolated from the leaves of barley from four localities for the first time in 1970. The fungus was also found on seeds of spring wheat and winter rye produced at Viikki (Helsinki) in 1969, as well as on many grasses (Mäkelä 1971). In the study carried out in 1970 and 1971 H. sativum was observed in about 15 per cent of the barley fields examined (180 fields). It was gathered in 90 localities in southern and central Finland. In the inoculation tests (H. sativum isolated from barley) all the cereals were attacked by the fungus (Mäkelä 1972). Barley H. sativum was found on barley throughout the country as far north as Kemi, Lapland (Pelkosenniemi) (Fig. 11). The fungus was found in the following localities: H. sativum was encountered in about 1/3 of the barley fields studied (about 800 fields) and in about 55 per cent of the localities (c. 250 localities) examined (Table 1). As a rule the fungus was commoner in tworowed than in six-rowed varieties ( Table 3). The fungus was observed to be most common in the southern and southwestern parts of the country. H. sativum was rarest in northern Finland (Table 3). As a rule conidia of H. sativum grew abundantly on the ripening leaves of barley without special lesions (cf. Muller 1956, Skov 1966. Sometimes the characteristic symptoms were found brown spots either with or without a Camp 1958, Mäkelä 1972). on the leaves having numerous darklight margin (Fig. 10) (cf. Lange de la Also in seedlings on spring barley in early summer (Fig. 10 H). brown stripes and spots were observed Wheat H. sativum was found on spring wheat (Fig. 12) in southern Finland up to Middle Ostrobothnia (Kärsämäki). It was also found in the experimental field in Lapland (Inari, Muddusniemi). The fungus was observed to be very rare on winter wheat, occurring only in Varsinais-Suomi and in Uusimaa ( Table 4). The fungus was found in the following localities: H. sativum was encountered in 9 per cent of the spring wheat fields (222 fields) and in 18 per cent of the localities (111 localities) examined and only in about three per cent of the winter wheat fields studied (about 260 fields) (Table 1). Also in seedlings of spring wheat brown tissues were observed in early summer, 1973 in one field in Peipohja (St). Rye On winter rye the fungus was observed only once in North Ostrobothnia (Muhos) (Fig. 12). Oats H. sativum was found on oats throughout the country up to North Ostrobothnia (Tyrnävä) (Fig. 13) H. sativum was found in per cent of the oat fields (415 fields) and in 17 per cent of the localities (181 localities) studied (Table 1). The fungus was found more frequently in Uusimaa than in the neighboring provinces (Table 2.) 209 In late summer H. sativum occurred on wheat and oats together with many other fungi e.g. H. avenae, H. trilici-repentis and Septoria species. The symptoms caused by H. sativum were rather unspecific (cf. Muller 1956, Mäkelä 1972. H. sativum was also found rather abundantly on grains of all cereals (barley, spring wheat, oats, rye) produced in fields in 1973 at Viikki (Helsinki) and at Maaninka (South Savo). Discussion This study was performed during the three year period 1971 1973. The meteorological conditions during the growing seasons were very similar. To be sure, there was great variation in the precipitation in different localities. The samples were gathered in different fields in different years and in part they came from different areas. The results are not given by year but are presented as a single unity. On the other hand they are grouped by biological provinces (Heikinheimo and Raatikainen 1971) which vary from each other in, for instance, climatic conditions, soil factors and, partially, in varieties (cv.). 1971-1973. 210 In comparing the results account should be taken not only of the above mentioned factors but also of errors that may possibly be due to the numbers of the samples. Nevertheless the possibility of error is reduced by the large number of sample localities as well as by the fact that for the most part the fungi determinations have been performed by the same individual, the researcher herself. Helminthosporium Halkilahti (1973 b), the main species of these, Otra, Pirkka and Porno were more frequently and more thoroughly inoculated by H. gramineum than the other varieties. Neglect in the dressing of seed has been more common in the central and northern parts of the country than in the south (Blomquist 1970). The same phenomena have also been observed in other Scandinavian countries (Lihnell 1969, Overaa 1972, Kolk and Karlberg 1973. H. gramineum also occurred on a mass scale in the USSR; the Archangel district, in particular, deserves mention (Shchekochikhina 1964). Furthermore, climatic factors play a considerable role in the abundant occurrence of H. gramineum in northern regions. This observation is supported by the fact that in this study H. teres occurred more commonly in the southern than in the northern parts of the country, whereas for H. gramineum the reverse was true. H. teres was the most common Helminthosporium species on barley. There are probably considerable crop damages, at least in those fields in which the fungus occurs abundantly already on the seedlings. The fungus caused premature destruction of the leaves and at the same time the ears remained underdeveloped (cf. McDonald and Buchannon 1964, Rintelen 1969, Smedegärd Petersen 1974. In Norway (Hansen and Magnus 1969) and Sweden (Kolk and Kalberg 1973), however, the disease is considered to be rather insignificant. The perfect stage of H. teres has not been found with certainty in Finland nor is there any evidence that the fungus persists in fields in any other form. This fact is nevertheless indicated by the extreme frequency and abundance of the fungus also in areas in which the seed is in general dressed. The fungus is known to be preserved in the straw, stubble and debris of barley on the ground surface in Britain (Evans 1969) and Denmark (Smedegärd Petersen 1972 a). Mercurial treatment is satisfactory for controlling the seed-borne inoculum (de Tempe 1964, Smedegärd-Petersen 1974. H. avenue was extremely common on oats through the country. Diseasesusceptible varieties were in part responsible for this. According to lahti's (1973 a) study of seed, Hannes, Ryhti, Sisu and Blenda, as well as Nip, which is widely cultivated in the northern parts of the country, are more frequently inoculated by H. avenae than are Pendek, Tiitus, Kyrö and Sörbo. In field experiments, too, (Rekola et ai. 1970) Hannes and Sisu were much more susceptible than Pendek. In this study the fungus was observed in certain cases to be particularly abundant on peat lands on both seedlings and on ripening leaves. In addition, it caused considerable crop losses (Hämeenlinna, cv. Hannes) (Butler and Jones 1949). In the other Scandinavian countries the fungus is considered to be of negligible importance (Andersen 1955, Fritz 1965, Kolk 1966, Overaa 1972. This may be due to differences in varieties (Halkilahti 1973 b) and climatic factors (Dennis 1933). Dressing is an important method of warding off seed-borne fungus (de Tempe 1964). In Finland, however, oats have never been dressed to the same extend as other cereals. The commonness of H. sativum on cultivated fields in Finland is surprising; it occurs as far north as Lapland (Inari 69 N, 27 E) though it is regearded as being a fungus of the warm, southern regions (Butler andJones 1949, Muller 1956). In recent years H. sativum has been observed also on numerous grasses throughout the country (cf. Mäkelä 1971). The occurrence of the fungus on all cereals as well as its frequency and abundance point to the fact that it has long been present in Finland. The exceptionally warm summers in these years have probably also contributed to this (cf. Muller 1956, Jorgenson 1974. The same conclusion is indicated by the fact that H. sativum occurred more frequently and more abundantly in the southernmost parts of the country and was considerably rarer in the northern parts. Typical leaf blotch caused by H. sativum (Lange de la Camp 1958) was rare on the barley leaves in comparison with the frequency of the fungus. The reason for this may be that the temperature was too low. This may also be a reason why so little is known about the occurrence of H. sativum in Finland (cf. Muller 1956). Virtually no true spores of the fungus turned up in the leaf samples. On the other hand, they developed rapidly and in abundance in the moist conditions that were employed in this study. The significance of the fungus as a crop-reducing factor is considered to be small in the Scandinavian countries (Fritz 1965, Hansen and Magnus 1969, Smedegärd -Petersen 1972 and elsewhere in Europe (Muller 1956, Lange de la Camp 1958, de Tempe 1964. In this study H. sativum apparently caused yield loss, at least in certain fields. In a number of cases the fungus was observed to damage the seedlings of barley, and spring wheat. According to JORGENSEN (1974), however, the seed-borne inoculum of H. sativum had little or no influence on the emergance of barley in the field. In preliminary seed studies the fungus was common in the seeds of all species of cereals, in which case the abundance of the fungus would seem to determine the degree of damage to the seedling (cf. de Tempe 1964). H. tritici-repentis was rather common in Finland in all the regions where wheat and rye are cultivated. Similarly, it was the most important disease 212 affecting couch grass (Agropyron repens). Couch grass is a weed that grows everywhere (Mukula et ai. 1969, Hulten 1971. and, accordingly, it may easily spread the disease to both rye (cf. Garbowski 1932) and wheat (Mitra 1934). The fungus is considered to be seed-borne on cereals and on wheat in particular (de Tempe 1964). In this study the fungus was not observed in seeds. On the other hand immature pseudothecia readily developed in abundance on the leaves of cereals in moist conditions. In nature they were observed rare with certainty. However, in the dead straws and leaves of couch grass in the spring there was also an abundance of ripe ascoma with matured ascospores. The fungus probably does not have much significance on cereals in spite of its commonness. The observations set forth in the above indicate that there are several rasons for the abundant occurrence of Helminthosporium fungi in Finland. First, the climatic conditions are favourable to the growth of these fungi because Helminthosporium species frequently occur as serious pathogens on plantations and on natural grasses throughout the country (Mäkelä 1971). The normal mean temperatures during the growing months (May-August) are rather low (approx. +9°+ lB°C ) even in the southernmost parts of Finland (Kolkki 1966). The most important Helminthosporium species occurring on cereals do, in fact, thrive in comparatively cool and moist conditions, particularly in their early stages of development (Ravn 1901, Dennis 1933, Shads 1934, McDonald and Buchannon 1964. In these conditions they are also most often preserved at the imperfect stage in seeds (Ravn 1901, Muller 1956, de Tempe 1964. In the present study, too, the Helminthosporium species occurred for the most part at the imperfect stage in the form of seedborne fungi, as is the case in the other Scandinavian countries, (except for part of Denmark). (Smedegärd Petersen 1971 and in Europe in general (Butler and Jones 1949, Muller 1956, de Tempe 1964. In very warm conditions the same fungi most often develope a perfect stage (cf. Kenneth 1962, Smedegärd -Petersen 1971. Warm weather is also favourable to secondary infection by windborne conidia (Dennis 1933, Shads 1934, Zekovic 1970. During the period of this study the growing seasons were warmer than usual. In the summer of 1972, when the temperatures for June and July were +2°+4°C higher than normal, with sufficient precipitation in July, Helminthosporium species occurred more abundantly than in other test years. The month of June was also very warm in 1973 but it was apperently too dry. Second, the majority of cereals that are cultivated in Finland are domestic varieties, most of which are extremely susceptible to inoculation by Helminthosporium. This is above all true of the most commonly cultiveted, six-rowed varieties of barley and oats (Halkilahti 1973(Halkilahti a, 1973. Third, undressed seed is used for the most part in Finland. According to Blomquist (1970), during the years 1953-1968, 77 % of the bread grain seed and 39 % of all the grain seed was treated with mercury disinfectants. Since 1969 only preparations of the alkoxyalkyl type have been available in Finland (Blomquist 1970). 213 Fourth, during the last decades the proportion of planting seed purchased in stores has increased and at the same time there has been an increase in the number of new varieties available. Being seed-borne fungi, Helminthosporium species have thus been able to spread more rapidly over large areas (cf. de Tempe 1964). Fifth, the shift to the exclusive cultivation of cereals instead of the former rotation with hay, especially in the southern and south-western parts of the country, has contributed to the spread of Helminthosporium species. The same can be said about the considerable increase in the acreage devoted to the cultivation of barley in recent years.

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Pathogenic Leptospiras Isolated from Malaysian Surface Waters Pathogenic leptospiras (1,424) isolated from natural waters and wet soils in Malaysia comprised 29 different serovars (synonym serotypes). All except two of the serovars had been found previously in Malaysia. The exceptional serovars were werrasingha, an Autumnalis serogroup member originally isolated in Ceylon, and a new serovar designated evansi. Serovar evansi had serological affinities with serovar ranarum which was isolated from the kidney of a frog in Iowa. The large variety of serovars found in jungle areas was consistent with similar previous findings of diverse serovar infections in troops who had operated in Malaysian jungles. McCrumb et al. (8) in a clinical and epidemiological study of 244 cases of leptospirosis in Malaysia directed attention to the jungle environment as "one of the most important natural foci of leptospiral infection." In 1961, Baker (5) initiated studies to determine the infectiousness of waters in the Malaysian rain forest. For this purpose, Baker and Baker (6) developed a sensitive hamster exposure method for the isolation of pathogenic leptospiras from natural milieu (6). The study was focused principally on the Gombak River and other adjacent streams or rivers located within an area of approximately 20 miles from Kuala Lumpur. Additional studies were carried out on jungle streams in the State of Pahang and in North Borneo, and on a ricefield and tin mining pool. During the course of these studies, approximately 1,424 pathogenic leptospiras were isolated from waters or wet shore soils. The serological characterization of the isolated strains is described in this report. The epidemiological aspects of the study will be presented separately. MATERIALS strains from the State of Pahang were derived mostly from shore sand along a jungle stream and river located near Kuala Lipis. Jungle streams in the western part of Sabah were the source of 62 leptospiral isolates. Nearly all of the isolates were recovered from hamsters dying from leptospirosis after their exposure to sample water or sample soil washings. The survey method is described elsewhere (6). Culture typing. The microscopic agglutination test was the basic means for identification of strains. The procedures for this test and also agglutininadsorption tests are described in detail elsewhere (2). Generally, strains employed as live antigens were tested for cross-agglutination reactions against the following eight screening serovar (synonym serotype) antisera: autumnalis, alexi, medanensis, javanica, bataviae, patane, mankarso, and pooled australis, grippotyphosa, and djasiman. On the basis of initial reactions, strains were subsequently tested against one or more serogroups to determine major antigenic affinities. These tests served to identify strains by serogroup. Representative strains of different patterns of cross-agglutination were then selected for comparative serological tests with type strains of serovars previously found in Malaysia. Isolates were assumed to be serologically homologous with type strains if their cross-reaction patterns were similar. Strains which differed from selected type strains in agglutinogenic characteristics were definitively identified by the use of appropriate agglutinin-adsorption tests (2). The identities of nine other representative isolates were confirmed incidentally for other purposes. The identification of strains was based to large extent on considerable background information obtained in previous studies of pathogenic leptospiras in Malaysia (1, Table 1). The wide variety of pathogenic serovars in jungle milieu was consistent with the broad array of serovar infections found in soldiers on jungle patrols (8). Within the State of Selangor, the ricefield and jungle areas studied had similar relative distributions of serovars. However, of 17 strains isolated in mining pools, 16 appeared to be related to serovar australis and one to serovar paidjan. The relative frequency of recovered serovar strains in the Pahang jungle superficially differed from those in the Selangor jungle. The variable findings may have reflected differences in size and types of samples and time of sampling. In the limited survey in Sabah mainly bataviae and autumnalis and a few icterohaemorrhagiae serovars were found.

v3-fos

2018-04-03T05:31:56.967Z

1975-01-01T00:00:00.000Z

43275196

s2

Effect of diet on enzymes of the brush border of the small intestine and kidney of rats. The effect of diets containing various amounts of casein and starch on enzymes bound to the brush border of the small intestine and kidney of rats were investigated with the following results. 1) Diets with low starch and high casein contents resulted in higher specific activity of leucineaminopeptidase in the small intestine than diets with high starch and low casein contents. Diets with high starch and low casein contents increased the specific activity of maltase. 2) Rat small intestine contains at least two isoenzymes of leucineaminopeptidase: one bound to the brush border and the other not bound to it but recoverable in the soluble fraction. Only the former was influenced by the diet. 3) The maximum velocity (Vmax) of leucineaminopeptidase bound to the brush border was twice as much in rats on a high casein diet as in those on a low casein diet, but the Michaelis constant (Km) was approximately the same in both groups of rats. 4) Leucineaminopeptidase and maltase activities in the kidney were not influenced by diet. Fractionation of the epithelial cells of the small intestine has indicated that the brush border membrane binds may hydrolytic enzymes, such as maltase , sucrase, lactase, dipeptidases, leucineaminopeptidase and alkaline phosphatase . It has been suggested that some of these enzymes play a fundamental role in the terminal digestion or absorption of nutrients (1,2). The effects of dietary con ditions on these enzymes, especially on disaccharidases , have been reported by several workers (3,4). DEREN et al. (4) demonstrated that administration of a diet containing carbohydrate to fasted rats caused a twofold increase in sucrase and maltase activities. They found that a diet containing sucrose caused a greater increase in sucrase activity than in maltase, whereas a diet containing maltose had the opposite effects, suggesting that these enzymes were specifically induced by the respective disaccharides. Alkaline phosphatase was also found to be affected 207 by the diet (5). However there are few reports on the effects of a diet on the peptide-hydrolyzing enzymes of the brush border. Leucineaminopeptidase, like disaccharidases, seems to take part in the final stage of digestion of foods, so it may also show adaptive changes to diet, especially when animals are fed on high protein diets. To study this possibility, rats were given diets with various starch and casein contents and the activities of maltase and leucineaminopeptidase bound to the brush border in the small intestine were measured. These enzyme activities were also measured in the kidney, where the epithelial cells lining the proximal tubules are very similar in structure and function to those of the small intestine (6, 7). Male Wistar DISCUSSION The present studies showed that in the small intestine the maltase activity in rats fed low casein and high starch diet was higher than that in rats fed high casein and low starch diet, whereas the leucineaminopeptidase activity in the former was lower than in the latter. These results confirm the reports that dietary carbohydrate influences intestinal disaccharidases (3,4), and also indicate that the intestinal leucineaminopeptidase is also influenced by the diet. These reverse changes in maltase and leucineaminopeptidase activities caused by the starch and casein contents seem understandable, since these enzymes are probably important for digestion of the diet. A similar pattern of response of trypsin, chymotrypsin and amylase to the diet was observed in rat pancreas (12) . It is interesting that leucineaminopeptidase bound to the brush border was influenced by the diet while the unbound enzyme, recovered in the soluble fraction was not . These findings suggest that the mechanisms of regulation of these two isoenzymes are different . The reverse changes of maltase and leucineaminopeptidase may be caused by some unknown specific factors functioning separately for each enzyme , and may represent changes in the numbers of molecules of each enzyme per cell rather than changes in the population of cells which bind the enzymes. The increased leucine aminopeptidase activity presumably represents increase in the quantity of the enzyme. The findings that the Km value of the leucineaminopeptidase was approxi mately the same in rats on low and high casein diets, while the Vmax value was twice as great in the latter is best explained as due to a twofold increase in the quantity of enzyme in rats on high casein diet. A similar explanation was proposed by DEREN et al. (4) for the dietary change in the intestinal disaccharidase activity . In preliminary experiments we observed that the changes of maltase and leucineaminopeptidase activities followed a similar time course and that the rise and thee fall in the respective enzyme activities were completed within two to three days and then remained unchanged. The time course of these changes seems to be nearly the same as the turnover time of intestinal cells (13) . Therefore, it is quite possible, as suggested by ROSENSWEIG and HERMAN (14), that the changes caused by dietary alterations are mediated through the crypt cells . However, DEREK et al. (4) showed that sucrase and maltase activities are inducible within 24 hr on feeding appropriate carbohydrate after 3 days fasting . Furthermore, recent studies (15) on the turnover of enzymes in the intestinal brush border demonstrated that disaccharidases are synthesized and catabolized during migration of epithelial cells and that individual brush border proteins do not necessarily have a uniform turnover rate. These observations suggest the alternative possibility that the enzyme activities bound to the brush border may be modified in the mature villus cell by dietary alterations. Further studies are needed on the actual mechanism of the dietary response of enzymes bound to the intestinal brush border. The present studies show that in contrast to the intestinal enzymes maltase and leucineaminopeptidase in the kidney are not influenced by the diet . Further more, as reported in the previous paper (8), brush border enzymes in the kidney do not show daily rhythmic changes like those in the small intestine . These find ings suggest that metabolic regulation of epithelial cells in the kidney is quite different from that of the cells in the small intestine.

v3-fos

2018-04-03T00:38:57.978Z

1975-09-01T00:00:00.000Z

13057125

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Degradation of 3-Hydroxybenzoate by Bacteria of the Genus Bacillus The pathway whereby certain bacterial strains of the genus Bacillus degrade m-hydroxybenzoate is delineated. Of 12 strains examined, nine were tentatively classified as representatives of the species Bacillus brevis, two of Bacillus sphaericus and one of Bacillus megaterium. All strains degraded m-hydroxyben-zoate via the same pathway. m-Hydroxybenzoate was hydroxylated to 2,5- dihydroxybenzoate (gentisate), which was oxidized by a gentisate 1,2-dioxygen-ase yielding maleylpyruvate. Maleylpyruvate was hydrolyzed without prior cis, cis to cis, trans isomerization yielding pyruvate and maleic acid. Numerous soils were examined by plate-count procedures and found to contain 10( to 106 aerobic sporeformers able to grow on m-hydroxybenzoate per g of dry soil. The pathway whereby certain bacterial strains of the genus Bacillus degrade m-hydroxybenzoate is delineated. Of 12 strains examined, nine were tentatively classified as representatives of the species Bacillus brevis, two of Bacillus sphaericus and one ofBacillus megaterium. All strains degraded m-hydroxybenzoate via the same pathway. m-Hydroxybenzoate was hydroxylated to 2,5dihydroxybenzoate (gentisate), which was oxidized by a gentisate 1,2-dioxygenase yielding maleylpyruvate. Maleylpyruvate was hydrolyzed without prior cis, cis to cis, trans isomerization yielding pyruvate and maleic acid. Numerous soils were examined by plate-count procedures and found to contain 10( to 106 aerobic sporeformers able to grow on m-hydroxybenzoate per g of dry soil. Bacteria of the genus Bacillus (bacilli) are an important component of the microflora of most soil and water environments and thus are potential agents ofbiological transformation and degradation of aromatic compounds that enter soil/water ecosystems. Unfortunately, little is known concerning the ability of Bacillus species to catabolize aromatic molecules. Certain Bacillus strains appear to degrade benzenoid compounds via reaction sequences similar to those described in other bacterial genera (1,2,9,17,(22)(23)(24)(25). However, recent investigations indicate that many bacilli may catabolize aromatic molecules via reaction sequences involving novel chemistry (4, 6, 10-12, 20, 21). The following describes my recent investigation into the catabolism of 3-hydroxybenzoate by various species of Bacillus. This investigation is part of a continuing project which has a goal of determining the mechanisms whereby bacteria ofthe genus Bacillus degrade aromatic molecules. MATERIALS AND METHODS Isolation, identification and growth of the microorganisms. Bacillus strains were isolated from pasteurized soil after selective enrichment on various aromatic compounds (Table 1). All strains were identified using the key and procedures of Gordon et al. (7). Stock cultures were maintained on brain heart infusion (Difco) slants that were stored at 4 C and subcultured biweekly. Microorganisms were grown in the minimal medium previously described (4), except that m-hydroxybenzoic acid replaced phydroxyphenylpropionic acid. One liter of medium contained in a 2-liter flask was inoculated with the growth of one stock slant and magnetically stirred at room temperature until cells reached early stationary phase. Cells were collected by centrifugation and washed by resuspension in 0.1 M potassiumsodium phosphate buffer, pH 7.2. This buffer was also used in all reaction mixtures. Preparation of cell extracts. Washed cell pastes were suspended in 2 to 3 volumes of buffer which contained 25% (by volume) glycerol. The resulting cell suspensions were passed through a French pressure cell (American Instrument Co., Silver Springs, Md.) at >10,000 lb/in2 applied with a hydraulic press (American Instrument Co.). Extracted cells were centrifuged at 26,000 x g for 20 min to give clear cell extracts containing 5 to 15 mg ofprotein/ml as determined by the method of Gornall et al. (8). All procedures were performed at 0 to 5 C. Enzyme assays. 2,5-Dihydroxybenzoate 1,2-dioxygenase (EC 1.13.11.4; gentisate 1,2-dioxygenase) was assayed by the procedure of Crawford et al. (5). Maleylpyruvate hydrolase was assayed by observing decrease in absorbance at 334 nm resulting from hydrolysis of maleylpyruvate to pyruvate and maleate (14). The hydrolase assay was performed after the gentisate 1,2-dioxygenase assay in the same reaction mixture while maleylpyruvate concentration was between 3 x 10-5 and 5 x 10-5 M. Emax of maleylpyruvate was assumed to be 10,800 (5) and 1 U of hydrolase activity is defined as the amount of protein required to hydrolyze 1 jLmol of maleylpyruvate per min. In no instance was there observed an increase in the rate of maleylpyruvate degradation by Bacillus extracts on addition of reduced glutathione (GSH). Fumarase (EC 4.2.1.2.) and maleate isomerase activities were assayed essentially as described by Scher and Jakoby (18). Spectrophotometric determinations of pyruvate formed enzymatically from gentisate were performed using lactate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) as previously described (3,4). Values shown in Table 2 are averages of two or more determinations, each initiated with a different concentration of gentisate. The second product formed on hydrolysis of maleylpyruvate is maleate (14; see below). Cell extracts of m-hydroxybenzoategrown Bacillus strains did not attack protocatechuate or catechol. Counting of bacterial populations. Total viable counts of soil were performed by dilution/plating of soil suspensions onto plate-count agar (Difco). Counts of total aerobic sporeformers were performed on the same medium after pasteurization of soil suspensions at 80 C for 15 min. Counts of total mhydroxybenzoate utilizers and of m-hydroxybenzoate-utilizing aerobic sporeformers were performed on minimal medium containing 500 mg of m-hydroxybenzoate per liter and 15 g of purified agar (Difco) per liter before and after pasteurization, respectively. Plates were incubated at 25 C. Thin-layer chromatography (TLC) and gaschromatography/mass spectrometry. Eastman chromatogram sheets (13181 silica gel; Eastman Ko- dak Co., Rochester, N.Y.) were used for analytical chromatography of aromatic compounds. Developing solvents were (A) benzene-methanol-acetic acid (45:8:2 by volume) and (B) benzene-ethyl acetate-80% formic acid (9:1:1 by volume). Aromatic compounds on plates were viewed under light of wavelength 253.7 or 375.0 nm. Gentisic acid exhibited a characteristic fluorescence when viewed on chromatograms under the former, but not the latter, wavelength of light. Organic acids were chromatographed on sheets of cellulose (Eastman, 13254 cellulose) using butanol-acetic acid-water (12:3:5 by volume; solvent C) or ethanol-ammonium hydroxidewater (16:1:3 by volume; solvent D) and were located by dipping chromatograms through AgNO3 in acetone followed by alcoholic NaOH (19). In solvent C maleic acid showed a spot of Rf 0.45, whereas fumaric acid showed a spot of Rf 0.80. Corresponding Rfs in solvent D were maleic acid, 0.68, and fumaric acid, 0.25. Analyses using an LKB-9000A gas-chromatograph/mass spectrometer were performed as previously described (16). Identification of maleate as an enzymic hydrolysis product of maleylpyruvate. The unsaturated, dicarboxylic acid formed on hydrolysis of maleylpyruvate by cell-free extracts of m-hydroxybenzoategrown Bacillus sp. was identified as maleate by the following procedures. A solution (total volume, 10 ml) containing 20 nmol of gentisate and approximately 50 mg of cell extract protein (prepared from m-hydroxybenzoate-grown Bacillus strain A2a or Cla) was incubated at room temperature with stirring for 3 h. The pH of the solution was adjusted to 4.0 and sufficient absolute ethanol was added to give a final concentration of 95%. Precipitated material was removed by centrifugation and the clear solution was concentrated to 1 to 2 ml by evaporation. Examination of this concentrate by TLC in solvents C and D revealed the presence of pyruvic acid (R. 0.6 and 0.7, solvent C) (19) and maleic acid (R. 0.45, solvent C; 0.67, solvent D), but no fumaric acid. When such enzymic reaction solutions were acidified to pH 2.0 and incubated at room temperature overnight prior to work-up, TLC revealed the presence of fumaric acid (Rf 0.8, solvent C; 0.24, solvent D), but no maleic acid. Control experiments indicate that, under the latter procedure, maleate is nonenzymically isomerized to fumarate. Materials. Enzymes and co-factors were purchased from the Sigma Chemical Co. All compounds listed in Table 1 as well as gentisic acid, organic acids, protocatechuic acid, and catechol were purchased either from the Sigma Chemical Co. or the Aldrich Chemical Co. Commercial compounds were examined for purity by TLC and recrystallized prior to use where necessary. RESULTS Soils sampled during this investigation typically yielded 108 to 1010 bacteria per g of dry soil, as determined by counting on plate-count agar. Of these approximately 10% survived pasteurization and represent the aerobic, sporeproducing component of the soil microflora. Of the total countable population of any particular soil, about 1% were able to grow on minimal media containing m-hydroxybenzoate as the only source of carbon and energy. Of the aerobic sporeformers in these soils approximately 0.1% were able to germinate and grow on mhydroxybenzoate plates. This represents, as an average for all soils examined, about 0.01% of the total viable count. Though this percentage seems small, it represents 104 to 106 aerobic sporeformers that are able to utilize m-hydroxybenzoate as a carbon/energy source per g of dry soil. Soils examined during this investigation were collected at numerous locations around downtown Albany, N. Y., and served as the source of all microbial strains used in this study. The Bacillus strains used during this investigation are listed in Table 1, along with their genus/species identification and the aromatic compounds upon which they were isolated. Results of enzymic assays of each strain, after growth on m-hydroxybenzoate, are summarized in Table 2. Cell extacts prepared from m-hydroxybenzoate-grown bacilli did not attack catechol or protocatechuate and none contained maleylpyruvate isomerase, maleate isomerase, or fumarase activities. Such extracts did readily degrade fumarylpyruvate. Succinateor glucose-grown cells lacked detectable amounts of aromatic pathway enzymes. The ring-fission product produced by oxidation of gentisate by Bacillus extracts showed spectral characteristics expected of maleylpyruvate; a Xmax in neutral or basic solution of 334 nm which is abolished upon acidification (5,15). It was possible to demonstrate directly the conversion of m-hydroxybenzoate to gentisate by using the Fe2" chelator a,a'-dipyridyl as an inhibitor of gentisate oxidation. Whole cells of Bacillus strain B9a were harvested after growth on m-hydroxybenzoate and allowed to oxidize m-hydroxybenzoate in the presence of a,a'-dipyridyl, as described by Hopper and Chapman (13). After a 3-h incubation, cells were removed by centrifugation. The supernatant was acidified to pH 2.0 and an excess of FeCl2 was added to trap a,a'-dipyridyl as its water-soluble iron complex. The cherry-red solution was extracted three times with ethyl acetate. Ethyl acetate extracts were combined, washed once with 0.1 M FeCl2 and twice with water, dried over anhydrous Na2SO4, and evaporated to yield a semicrystalline solid. This organic residue was examined by TLC using solvents A and B and shown to contain mostly gentisic acid and some residual m-hydroxybenzoic acid. Gentisic acid was unequivocally identified by its retention time and molecular ion (trimethylsilyl derivative, m/e = 370) as revealed by gas-chromatography mass spectrometry. Figure 1 illustrates the spectral changes ob- Downloaded from served at 340 nm during oxidation ofm-hydroxybenzoate by a supplemented extract of m-hydroxybenzoate-grown Bacillus strain C5f. Oxidation was dependent upon addition of reduced pyridine nucleotide to the reaction mixture. No changes in the ultraviolet spectrum of m-hydroxybenzoate were observed when NADH was omitted from the reaction mixture. The depicted spectral changes (Fig. 1) were also produced using other extracts prepared from strains chosen at random from the list in Table 1. The unsaturated, dicarboxylic acid formed on hydrolysis of maleylpyruvate by cell extracts of m-hydroxybenzoate-grown Bacillus sp. was identified as maleate (cf. Materials and Methods). DISCUSSION The data summarized above indicate that all Bacillus isolates examined degrade m-hydroxybenzoate by the reaction sequence shown in Fig. 2A. This gentisic acid pathway is a modified version of the sequence originally delineated by Lack (15; Fig. 2B) and has been observed previously only in two strains of Pseudomonas isolated by Hopper et al. (strains 2,5 and 3,5; references 13, 14 and personal communication). Spectral changes shown in Fig. 1 are consistent with requirements of the sequence of Fig. 2A. Thus on addition of m-hydroxybenzoate to a reaction mixture containing NADH and cell extract prepared from m-hydroxybenzoate-in-duced cells, one observes an initial decrease of A340 resulting from oxidation of NADH by an enzymatic hydroxylation of m-hydroxybenzoate forming gentisate. Gentisate formed is immediately oxidized by a gentisate 1,2-dioxygenase present in cell extracts, forming maleylpyruvate (Amax = 334 nm) which absorbs strongly at 340 nm. Its formation results in an increase of A 340. The final decrease in A340 reflects hydrolysis of maleylpyruvate by its hydrolase forming pyruvate and maleate. Hydrolysis of maleylpyruvate is not speeded on addition of GSH. When a,a'-dipyridyl is included in the reaction mixture, gentisate 1,2-dioxygenase is inhibited. Thus NADH oxidation is no longer masked by maleylpyruvate formation and A340 continues its initial decrease. It is conceivable that maleylpyruvate is isomerized to fumarylpyruvate by a GSH-independent isomerase prior to hydrolysis to pyruvate and a 4-carbon, dicarboxylic acid (particularly since cell extracts prepared from m-hydroxybenzoate-grown cells readily degrade fumarylpyruvate). This possibility is ruled out by our observation that maleic acid, rather than fumaric acid, accumulates from gentisate when the latter is oxidized by extracts prepared from mhydroxybenzoate-grown Bacillus A2a or Cla. Like the nonfluorescent pseudomonad of Hopper et al. (14), the bacterial strains examined here induce an apparently nonfunctional fumarylpyruvate hydrolase when grown on m-hydroxybenzoate. Hydrolysis of maleylpyruvate and fumarylpyruvate may be catalyzed by a single The enzymic specific activites and pyruvate yields shown in Table 2 are as expected for the pathway of Fig. 2A. Gentisate pathway enzymes, not present in glucoseor succinategrown cells, are induced to high levels during growth on m-hydroxybenzoate. Also, one molecule of gentisate yields one molecule of pyruvate (Table 2) in a GSH-independent, enzymic reaction. Actual isolation of gentisic acid as a metabolite of m-hydroxybenzoate after inhibition of cells with aa'-dipyridyl is direct evidence of gentisate participation in the catabolic pathway. Most of the Bacillus strains examined are tentatively classified as isolates of B. brevis (nine strains); however, strains of B. sphaericus (two strains) and B. megaterium (one strain) are also represented. Thus, it seems unlikely that degradation of m-hydroxybenzoate via the GSH-independent, gentisate pathway will be of taxonomic value in distinguishing species of Bacillus. It may, however, be possible to identify a small group of species with the taxonomically valuable characteristic of ability to grow on m-hydroxybenzoate. Our results indicate that the GSH-independent gentisate pathway may be of general occurrence in the genus Bacillus, rather than the GSH-dependent pathway. Investigation of many additional strains of Bacillus species that utilize gentisate as a catabolic intermediate will be necessary to determine whether or not this is a valid generalization. As far as I know this is the first demonstration of the presence of a gentisate pathway among bacteria of the genus Bacillus (other than a preliminary report by Crawford and Chapman [R. L. Crawford and P. J. Chapman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, 03, p. 1921) as well as the first report ofdegradation of m-hydroxybenzoate by microorganisms of this classification.

v3-fos

2018-04-03T04:56:33.754Z

1975-02-01T00:00:00.000Z

40859015

s2

Ecology of Soil Arthrobacters in Clarion-Webster Toposequences of Iowa Toposequence variations in soil properties were characterized and related to variations in populations of total isolatable bacteria and arthrobacters. Increases in soil NO,-N, available phosphorous, NO-N-producing power, Arthrobacter counts, and the percentage of the total counts represented by arthrobacters were correlated with decreases in soil acidity. The total bacterial counts were not correlated with soil acidity but were associated with percentage of soil organic matter and percentage of clay. The percentage of the total counts represented by arthrobacters was lowest at the summit position and increased downslope to the highest value in the toeslope position. Factor analysis of the data revealed that 67 to 81% of the total variance exhibited by all variables per site-sampling period could be accounted for by soil acidity, soil structure, soil fertility, soil moisture, and bacterial factors. A selective medium was developed for soil arthrobacters and tested on a wide variety of central Iowa soils to determine its potential as a medium for enumeration as well as isolation. The medium developed in this study was found to be superior to the other available direct-isolation media for soil arthrobacters. Various studies have shown that members of the genus Arthrobacter are often among the more numerically predominant bacteria routinely isolated from soils (15,25). These soil arthrobacters are nutritionally very diverse (20,27), and many isolates can be found that exhibit the ability to degrade various pesticides (9,14,24). Very little work has been done, however, to determine possible correlations between variations in soil properties and variations in any particular group of soil bacteria. Soil pseudomonads have been found associated with slightly acid rhizosphere soil samples, whereas arthrobacters have been associated with slightly alkaline non-rhizosphere soil samples (22). Increased numbers of arthrobacters have been associated with soil samples adjusted to higher moisture contents, whereas pseudomonads have been predominant in soil samples adjusted to lower moisture contents (21). Topography is a very complex soil formation factor that could affect the bacterial populations by influencing certain soil properties through climate or drainage-related functions (11). Proceeding downslope from the shoulder position to the toeslope in Clarion-Webster toposequences of Iowa, the percentage of soil 'Present address: Department of Microbiology, Oregon State University, Corvallis, Ore. 97331. organic matter increases to a maximum while the mean particle size decreases to a minimum. The thickness of the A-horizon and the depth to carbonates or mottles decreases as the slope gradient becomes steeper (29). The toposequence soils in Clarion-Webster toposequences represent a gradation in textural classes; several studies have associated nematode populations with texture variations (18,19), but no attempts have been made to do this with bacterial populations. Soil organic matter levels are interrelated with other soil properties, and little is known about the effects of this relatively stable soil property of bacterial populations. Arthrobacters have normally been isolated from soils by using either enrichment techniques or by randomly picking and identifying isolates from media used to determine total counts (15). Mulder and Antheunisse (16) developed a selective procedure for arthrobacters involving two separate media where the identification was based on observation of a morphological cycle possessed by members of this genus. Their method was not intended to serve as a means of enumerating Arthrobacter populations and, because of the lack of a suitable enumeration procedure, one was developed in our study. This study investigated variations in total isolatable bacteria and Arthrobacter populations in two toposequences in the Clarion-Webster soil association area in Iowa. Toposequence variations in soil properties were characterized in relation to their effects on the total bacterial and Arthrobacter populations. MATERIALS AND METHODS Site location and description. The two toposequences were located in the Clarion-Webster soil association area in north-central Iowa and are described in Table 1. Site I was in a corn-soybean rotation from 1963 to 1968 and in continuous corn from 1968 to 1973, whereas site II was in a cornsoybean rotation from 1963 to 1973. During this interval site I received no lime treatments, whereas site II received the appropriate amount of lime to maintain the soil pH at 6.9. Sample collection and sampling periods. Four adjacent rows of corn that extended parallel to the toposequence transect were chosen at each sampling site, and nine core samples were removed, three each from the middle of the furrow between adjacent rows of corn. Three sampling sites were chosen in each of the four soil types comprising the toposequences. The core samples were obtained and processed individually on 30 August at site I (soil temperature, 33 C) and 25 October at sites I and II (soil temperature, 26 C). All core samples were taken from the Ap-horizon at a depth of 10 cm at both sampling sites. Total bacteria analyses. All core samples were placed in plastic bags and transported to the laboratory, and platings were performed on the same day that each core sample was obtained. A 5.0-g sample was aseptically removed from the previously unexposed center of each core sample and suspended in 495 ml of sterile 0.5% peptone broth. Each sample was then agitated in a Waring blender for 3 min at low speed, serial dilutions in 0.5% peptone broth were Harps, 0%, Harps, 0%, none none made, and 0.1-ml portions of appropriate dilutions were spread over the surface of sterile media in petri plates. Total counts were made from a medium containing 0.1% peptonized milk (Difco), 0.1% yeast extract (Difco), 0.01% Acti-Dione (Upjohn Co.), and 1.5% agar. The pH was adjusted to the pH of the particular soil being plated and plates were incubated at 25 C for 10 days, after which colonies were counted. All platings were done in triplicate. Arthrobacter selective medium and analyses. Seventeen Arthrobacter named strains from the American Type Culture Collection (ATCC, Rockville, Md.) and 20 Arthrobacter, 6 Bacillus, 6 Micrococcus, 4 Nocardia, 4 Streptomyces, 4 Flavobacterium, and 6 Pseudomonas soil isolates were screened on 31 dyes, 13 antibiotics, and 11 assorted compounds to determine possible selective properties for the arthrobacters. The soil isolates were taken from the medium used to determine total counts and were identified to the genus level according to procedures outlined by Buchanan and Gibbons (5). All 67 cultures were tested on a wide range of concentrations of each of the 55 potential selective agents to detect any differential as well as selective properties. The screening was performed by incorporating the various concentrations of the potential selective agents in either Trypticase soy agar (BBL), peptonized milk agar, or nutrient agar (Difco). The three basal media were tested at a variety of concentrations with additions of various amounts of yeast extract as well as with the potential selective agents. Those agents that were heat sensitive were filter-sterilized and added aseptically to the cooled, autoclaved media. The media were adjusted to a variety of pH values ranging from 5.0 to 8.5. The cultures were transferred to the surface of the media with a multipoint inoculation device (7). All plates were incubated at 30 C for 72 h, after which plates were examined. Those compounds that exhibited either selective or differential properties for the arthrobacters were retested on various concentrations of the three basal media at varying pH values. A total of 720 different variations were examined to determine the best possible combination of a basal medium plus yeast extract plus various concentrations of different selective ingredients. The best selective medium had the following composition: 0.4% trypticase soy agar, 0.2% yeast extract, 2.0% NaCl, 0.01% Acti-Dione, 150 pig of methyl red (Harleco) per ml, and 1.5% agar. The methyl red was filter-sterilized and added aseptically to the autoclaved, cooled medium (see Results and Discussion). Soil samples were diluted and plated, and plates containing the selective medium were incubated at the temperature used for the total count medium. The pH was adjusted to the pH of the particular soil being plated. The selective medium was tested, on a variety of soils, to determine what percentage of the isolates were arthrobacters by subculturing and microscopic examination of all the colonies on various randomly selected plates for each soil type. The colonies were transferred to trypticase soy agar plus 0.2% yeast extract and examined microscopically for possession of a rod-to-coccus morphological cycle, snapping divi-ECOLOGY OF SOIL ARTHROBACTERS sion, pleomorphism, and V-forms (5). The same procedure was also performed on plates containing the total count medium and the media developed by Mulder and Antheunisse (16). For enumeration of the arthrobacters in the four soils examined in the ecological survey ( Table 1), 78% of the counts on the selective medium was taken as the Arthrobacter counts. Soil analyses. After the bacterial analyses were performed, eight portions were taken from each core sample, two each for the following determinations: soil NO,-N (4), soil NH4-N (2), NO.N-producing power (26), and soil moisture (28). One particle size analysis was performed on each core sample by using a modified pipette method (28). The remainder of each core sample was air-dried and screened through a 4.0-mm sieve, and two replicate determinations for all procedures were made on each core sample. Soil pH, total exchangeable bases, exchangeable hydrogen (10), soil organic matter (6), available phosphorous (17), and soluble salts and saturation percentage (3) determinations were then performed. Statistical analyses. Simple correlation matrices were computed, and a preliminary set of factor-loading values for the factor analysis was computed from these matrices by using the principal components method (8). These factor-loading values were subjected to a varimax rotation (12) to maximize the factor loadings without changing the specific variance of each variable. The linear factor analysis model (23) used for each of the 16 variables was z l = a F, + aF. + aF3 +cE,. This model equation expresses each variable, z, in terms of three factors, F1 to F,, and an error factor E. The factor loadings, a and c, indicate the extent to which each factor participates in the test. The specific variance of the error factor for each variable indicates how much of the variation exhibited by the variables is not explained by the three factors. This particular factor analysis model was used because the results of a test of significance for the total number of factors indicated that there were not more than three factors involved at any one site-sampling period (13). In using this model we assumed that the sample size of 108 toposequence samples per site-sampling period was large enough to avoid sampling error. To insure this, only factor-loading values larger than 0.50 or smaller than -0.50 were considered significant correlational values. RESULTS AND DISCUSSION Of all the compounds tested as possible selective agents, only a few exhibited any selective properties for the arthrobacters. The combination of Acti-Dione at 0.01% and NaCl at 2.0% effectively inhibited all fungi and most streptomycetes, nocardia, and gram-negative bacteria. The methyl red at 150 ,g/ml inhibited other gram-positive bacteria (bacilli and micrococci) but did not affect the arthrobacters. The pH of the medium, between 5.0 and 8.5, did not affect its selectivity, and the combination of trypticase soy agar at 0.4% and yeast extract at 0.2% gave the highest yield of arthrobacters with the addition of the selective ingredients over the other basal media (data not shown). In testing the selective medium (Table 2), the percentage of the colonies identified as arthrobacters was much higher (74%) than that of either the total count medium (14%) or the nutritionally poor medium (24%). From the soils tested, approximately 25% of the colonies on the selective medium were not arthrobacters, and microscopic examination was necessary to distinguish them. In examining the selective medium for enumeration potential ( Table 3) the percentages of arthrobacters from the selective medium were close to or slightly higher 29,1975 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from than the percentages from the total count medium. Because of this close agreement, it was decided to use the selective medium for enumeration purposes by taking a percentage of the colonies growing on the plates as being the arthrobacter counts and comparing these with a Percentage of arthrobacters for the NPM and SM were obtained by using the numbers of arthrobacters from both of these media as determined from the data in Table 2. These figures were then compared with the total counts for each soil type to obtain the percentage of the total counts represented by arthrobacters for each of the media. AND HOLT APPL. MICROBIOL. the counts from the total count medium to arrive at the percentage of arthrobacters contained in any one sample. The nutritionally poor medium (Table 3) was not suitable for enumeration purposes. Further tests on the four soils used in the ecological survey (data not shown) indicated that 78% of the counts on the selective medium was a suitable figure for determining the arthrobacter counts from the respective soils. The largest total bacterial and Arthrobacter populations occurred at the toeslope position of both toposequences during each sampling period. The smallest total bacterial and Arthrobacter populations occurred at the backslope position and increased down to the toeslope and up to the summit position ( Table 4). The percentage of the total counts represented by arthrobacters was lowest at the summit and increased downslope to the highest percentage in the toeslope position. Pronounced changes in soil variables accompanied these variations in bacterial populations at each toposequence (Table 4). However, due to the higher pH caused by the limed conditions in the soils at site II, the variation in most of the variables was not as great as for either sampling period at site I. Proceeding from the summit to the toeslope position, there were increases in soil pH, percentage of clay, percentage of silt plus percentage of clay, soluble salt levels, percentage of organic matter, soil NO,-N, NO-N-producing power, available phosphorous, total exchangeable bases, percentage of moisture relative to field capacity, and percentage of moisture relative to percentage saturation. There were decreases downslope in the exchangeable hydrogen and soil NH,-N (Table 4). Due to the interpretational method of factor analysis, each of the factors was arbitrarily named, depending upon which variables appeared to be consistently interrelated ( Table 5). The soil fertility factor name was chosen because NO-N is the end product of nitrification and therefore is a useful indicator of the ability of the soil to supply plant-available NOU-N. The other factors were named according to the obvious combinations of variables composing the various factors. More of the variation in the soil fertility, acidity, structure, and bacterial variables was accounted for in factor analysis than variation soil moisture variables, which was indicated by higher specific variance values of the soil moisture variables compared with the other variables (Tables 6-8). At site I during both sampling periods (Table 6, 7), the soil acidity factor was positively correlated with soil NO-N, NO-Nproducing power, available phosphorous, Arthrobacter counts, and the percentage of the total counts represented by arthrobacters, and negatively correlated with soil NH,-N. The soil structure factor was negatively correlated with percentage of moisture relative to field capacity, total bacterial counts, and Arthrobacter counts. The absence of a soil fertility factor at site I on 30 August (Table 6) was probably due to interference by the roots of the corn plants with the soil fertility variables (uptake of available P and NO-N). By 25 October (Table 7) the roots were dead, the interference was removed, and a soil fertility factor, which was positively correlated with percentage of moisture relative to percentage of saturation, was generated. At site II (Table 8) the soil acidity factor was positively correlated with the same variables as at site I, but the degrees of correlation were not as great. The soil structure factor was not correlated with any variables, whereas the soil fertility factor was negatively correlated with the percentage of moisture relative to field capacity and positively correlated with the percentage of moisture relative to percentage of saturation, total counts, and Arthrobacter counts. At site I, increased soil acidity resulted in decreased soil NOO-N and increased soil NH,-N (Table 5). This was a measure of the lessened activity, due to acid sensitivity, of the nitrifying bacteria Nitrosomonas and Nitrobacter (1). The increased soil acidity was responsible for the decreased Arthrobacter counts and percentages of the total counts represented by arthrobacters. The total bacterial counts were influenced strongly by the soil structure factor (percentages of clay and organic matter) and, to a lesser degree, by the soil acidity factor. The Arthrobacter counts were positively correlated with acidity, but the degree of correlation was not as great (Tables 6-8) as was that of the percentage of the total counts represented by arthrobacters. This was due to the effects of the soil organic matter and clay content on the Arthrobacter counts, especially at the shoulder position (Table 4), whereas these variables did not significantly affect the percentage of the total counts represented by arthrobacters. The same relationships were found at site II, but the significant correlations were not as great due to the decreased variation in many of the variables caused by the limed conditions. During the 30 August sampling period at site I, 76.80% of the total variance removed by all factors was accounted for, whereas 80.92% was accounted for in the 25 October sampling period site I (Table 9). At site II 67.84% of the total variance was accounted for. The increased contribution of the soil acidity factor at site I accounted for the greater total percentage of the variance removed as compared with site H. The 20 to 30% of the total variation unaccounted for represented other unmeasured and/or unknown environmental factors. Factor analysis of the data from the toposequence soils examined in this study indicated that the arthrobacters in these soils were acid sensitive and their numbers decreased in a cause-and-effect relationship with increasing acidity. At site I on 25 October (Table 4), the percentage of the total counts represented by arthrobacters was 4.68% at the summit (pH 5.79) and increased to 23.39% at the toeslope as the acidity decreased (pH 7.42). At site H on 25 October (data not shown), the variation was less due to the limed conditions since, at the summit, 14.81% of the total counts were arthrobacters (pH 6.91) and increased to 20.26% at the toeslope as the acidity decreased (pH 7.38). The total bacterial counts were not correlated with soil acidity during any of the site-sampling periods, which probably indicated that as the acidity increased and the Arthrobacter counts decreased, the numbers of some type of acidtolerant bacterium were increasing. This study demonstrated that the distribution and abundance of certain types of bacteria (in this case, arthrobacters) were, to a large extent, determined by certain ecological variables. If the assumption is valid that microbial populations are selected by their environments, then the methodology applied in this study might find additional uses in determining which environmental variables most strongly influence the distribution of any of a wide range of microorganisms.

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2018-04-03T02:00:25.021Z

1975-07-01T00:00:00.000Z

31862430

s2

Effects of trace metals on the production of aflatoxins by Aspergillus parasiticus. Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low micrograms-per-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 mug/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 mug of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 mug or less of the metal per ml, salts of iron, manganese, cooper, cadmium, trivalent chromium, silver, and mercury partly or completelyinhibited aflatoxin production, without influencing mat weight. Cottonseeds, peanuts, corn, and other agricultural commodities important in the food and feed industries may become contaminated with' aflatoxins, but the basic factors that influence aflatoxin production have not been completely studied (4). The toxin is a metabolite of Aspergillus flavus and of the very closely related species A. parasiticus. Effects of growth media on aflatoxin production by A. flavus have been studied mostly in comparisons during culture on various seeds and other natural substrates (4). More limited work has involved defined media containing various sources of carbon and nitrogen (3). Mateles and Adye (10) deleted molybdenum, boron, copper, iron, zinc and manganese, singly and in combination, from their growth medium and concluded that ". . . zinc is required for the production of aflatoxin. The slightly reduced yields obtained when iron or manganese were deleted were due to a visible reduction of growth, which was not observed when zinc was. deleted." Omission of copper, molybdenum, and boron did not reduce aflatoxin production. Lee et al. (5) also reported that zinc was specifically required for production of aflatoxins by A. flavus. An approximate maximum in mat weight was obtained with 0.4 ,ug of zinc per ml, whereas aflatoxin increased from a low level at this concentration to a maximum at about 2.0 jsg of zinc per ml. Davis et al. (2) concluded, "The influence of minerals on aflatoxin synthesis may be indirect 'Present address: U.S. Food and Drug Administration, Washington, D.C. 20204. through their essentiality for growth or more directly on the process of toxin synthesis per se. The influence of zinc and iron appeared to be indirect, whereas that of molybdenum appeared direct." Zinc and iron, at 0.5 to 1 ug of the sulfate per ml, stimulated both aflatoxin production and growth, i.e., mat weight; they also inhibited aflatoxin production, but not growth, at 10 ttg/ml. A minimum of 0.5 g of MgSO, per liter appeared essential for maximum production whereas a fifth of this amount was sufficient for maximum growth. The purpose of this work was to determine in synthetic culture whether salts of certain metals at low parts-per-million levels could influence the production of aflatoxins by A. parasiticus and, if so, whether this effect would be accompanied by changes in the overall growth of the fungus as measured by mat weight or in the finalplH of the growth medium. MATERIALS AND METHODS Organism. The isolate used was A. parasiticus NRRL 2999, from the USDA Northern Regional Research Laboratory, Peoria, Ill. The trace element composition of the basal medium was as indicated in the tables (see Tables 1-4). To make stock solutions of the standard trace metal salts, we dissolved FeSO4 .7HO, CuSO, .5HO, MnSO. and Ca(NO)2-4H20 in water. Zinc glycerophosphate was used to avoid zinc precipitation. Thus, 90 mg of this salt was dissolved in a minimum amount of glacial acetic acid and water, the solution was adjusted to pH 5.2, and the volume was made up to 25 ml. One ml of the zinc glycerophosphate solution in 1 liter of basal medium provided a final concentration of 1 Ag of zinc per ml. Cadmium, chromium, silver, cobalt, nickel, lead, and aluminum were added as nitrates, mercury as mercuric chloride, and ruthenium as the chloride. The complete salt medium was adjusted to pH 5.2 with glacial acetic acid. All water was passed through an ion-exchange column. Thirty milliliters of medium was dispensed into each of several 250-ml flasks. The flasks, previously acid washed, were stoppered with cotton plugs and autoclaved 15 min at 15 lb. of steam pressure. After cooling, they were inoculated by needle with spores of the fungus and incubated at 30 C without shaking for time periods as specified in table headings (see Tables 1-4). The treatments were replicated six times in individual flasks, except that Cr3+ and Ag+ treatments were replicated five times. Assays. After incubation, the final pH of the filtrate was measured, and the culture fluid was filtered through Whatman no. 1 paper. The mycelial mats were washed with 4 ml of distilled water, and the wash water was combined with the filtrate. The mycelial mats were dried for 3 days at 40 C and weighed. Aflatoxin analyses were performed on both filtrates and mats. A 25-ml portion of the pooled filtrates was extracted with 20 ml of CHCl, for 30 min in a 50-ml glass-stoppered centrifuge tube. Then it was centrifuged for 10 min at 2,000 rpm, and the aqueous phase was removed. The CHCl, phase was passed through a column of anhydrous sodium sulfate, and the column was rinsed with an additional 20 ml of CHCl. The chloroform extracts were evaporated to dryness, and the residues were saved for thin-layer chromatography (TLC). The combined mycelial mats from each treatment were placed into a blender cup and blended for 5 min at full speed with 50 ml of acetone-water (70:30). A 10-ml portion was transferred to a 50-ml glass-stoppered centrifuge tube, and 2 ml of lead acetate plus 14 ml of water was added to the tube. The precipitate that formed was sedimented by centrifugation for 10 min at 2,000 rpm. The supernatant was extracted with 20 ml of CHCl,, phase separation was aided by centrifugation, and the aqueous phase was discarded. Then the CHCl, phase was extracted with 2 ml of 0.1 N NaOH, shaking for 1 min. The sodium hydroxide was removed after centrifugation, and the CHCl, phase was passed through a column of anhydrous sodium sulfate. The column was rinsed with 20 ml of CHCl,, the combined CHCl3 effluents were evaporated to dryness, and the residue was saved for TLC. The residues were dissolved in 200 ml of CHCl,, and aliquots were applied to silica gel-prepared TLC plates (Schleicher and Schuell no. 1500). The plates were developed in unlined, unequilibrated tanks with 150 ml of acetone-chloroform (1:9). Developing time was about 45 min for a solvent movement of 12 cm. Quantitation was by comparison with reference standards of aflatoxins on the same TLC plate. Aflatoxin production was calculated as follows: Nanograms per flask = Nanograms aflatoxin in medium (= total milliliters of medium x ng/ml) number of replicates Nanograms aflatoxin in mats + (= total wet weight in grams x ng/ml number of replicates A test showed that the trace elements used in the culture flasks did not interfere with aflatoxin analyses. To each of a series of test tubes, a 10-ml sample of control medium with 240 ng of aflatoxin B1 per ml was transferred. Then each tube was spiked with a trace element at a concentration of 5 tig/ml. The trace elements used were iron, copper, zinc, manganese, cadmium, cobalt, lead, mercury, aluminum, and nickel. After extraction of the aflatoxin as described above, 10-ul amounts from each tube were spotted on TLC plates next to 10-Al aliquots of extract from the control medium with aflatoxin and no added trace elements. The plates were developed as described above. The intensities of the fluorescent spots from the control medium extract could not be distinguished from those from the spiked media extracts. Table 1 shows results with progressive additions of zinc, manganese, iron, and copper salts to the basal medium with a constant level of each of the remaining elements tested. At a zero-added level of zinc (Table 1), aflatoxin production was very low, mat weight was low, and final pH of the medium was low. RESULTS At zinc levels of 1 to 25 jg/ml, aflatoxin production was much higher, with a moderate depression at 25 yg/ml, whereas both mat weight and final pH were higher and nearly constant. The fact that at the 1 gg/ml level of added zinc the aflatoxin production was increased to about 1,000 times that with no zinc whereas mat weight was increased only 2.5 times suggests, in accord with previous reports (5,6,10), that zinc has a stimulating influence on aflatoxin production apart from its essentiality for overall growth. The ratio of aflatoxins in the mat to aflatoxins in the medium is not recorded here, because it did not vary consistently in this or other experiments. Thus, we used only the mat-plus-medium summation figures in all tables. In general, aflatoxin B1 > G1 > B2 > G2 throughout all zinc concentrations, a relationship also seen in later experiments with most other trace elements. With added manganese from 0 to 25 gg/ml (Table 1), aflatoxin production increased to its highest level at the 5 (Table 1), aflatoxin production was relatively high, mat weight was somewhat lower than at higher iron concentrations, and the final pH was 4.5. With iron in the range of 1 to 25 ,g/ml, aflatoxin production decreased progressively with increasing iron concentration while mat weight was constant around 240 mg and final pH was nearly constant around 7.0. At all levels of added copper from 1 to 25 ug/ml (Table 1), a depression of aflatoxin production occurred, even though mat weights and the final pH were unaffected. Thus, zinc, manganese, iron, and copper all depressed aflatoxin production at 25 gg/ml or at some ower level, but copper depressed it most. The aflatoxin-producing response of A. parasiticus to zinc (Table 1, experiment 1A) was verified in two similar experiments ( Table 2, experiments 1B and 1C). In them, the zinc-aflatoxin relation was much like that in experiment 1A; the response of the fungus to successive increments of zinc in the levels of 0 to 10 ug/ml was much greater in aflatoxin production than in growth, and the optimum zinc concentration for aflatoxin production was higher than that for growth. In the three experiments, zinc caused 30to 1,000-fold increases in aflatoxin levels with increases of less than threefold in growth. The decrease in level of aflatoxin between 7 and 14 days ( Table 2), was in accord with the literature (1,4). An extra confirmatory experiment on effects of zinc was run with half-strength amounts of each of four major salts as the only deviation from experiment 1A (Table 1). In this experiment, the total aflatoxin produced in micrograms at 0, 1, 5, 10, and 25 jg of zinc per ml was 0.47, 30.5, 126.2, 155.6, and 23.5 with mat weights of 100, 221, 223, 211, and 208 mg, and final pH values of 3.1, 2.8, 3.0, 3.4, and 6.0, respectively. These results, although marked by low pH, continued to indicate that zinc stimulates aflatoxin production more than mat weight. That is, between 0 and 5 Ag of zinc per APPL. MICROBIOL. on March 22, 2020 by guest http://aem.asm.org/ Downloaded from ml, the total aflatoxins increased about 268fold, whereas mat weight only about doubled. As in the experiments reported above, the level of zinc for maximum aflatoxin production was higher than that for maximum growth. The maximum aflatoxin level was at 10 jg of zinc per ml. Cadmium at all levels added from 1 to 25 tig/ml depressed production of aflatoxin greatly, but without influencing mat weight or final pH (Table 3). Thus, its effects were similar quantitatively to those of copper. Trivalent chromium-depressed aflatoxin production at metal levels of 1 to 10 jg/ml, but was without major effect on mat weight or final pH (Table 3). TABEz 2. Effect of addition of zinc glycerophosphate to a basal medium on the production of aflatoxins, mat dry weight, and final pH of medium after 7-and 14-day incubation with A. parasiticus 2999a Silver up to 1 ug/ml depressed aflatoxin production without affecting mat weight or final pH (Table 3). At 5 gg/ml, it reduced aflatoxin to nondetectability and depressed mat weight greatly. Thus, at unusually low levels it effectively depressed aflatoxin production and growth. The effects of various metals at 5 gg/ml on aflatoxin production are shown in Table 4. Mercury depressed aflatoxin level greatly without decreasing mat weight or lowering the final pH, while aluminum and ruthenium also depressed aflatoxin production somewhat. Nickel, cobalt, and lead had little or no effect on any of the three measured test results. DISCUSSION The results here described agree with the conclusion of previous workers that zinc stimulates aflatoxin production (2,5,6,10). Also they agree with earlier findings that some trace metals at low concentrations may inhibit aflatoxin production (2). No certain explanation, however, can be offered for the mechanism of the effect shown here by the various metallic ions on the production of aflatoxins. Four of the more potent metals in the inhibition of aflatoxin production in the present work, i.e., copper, cadmium, silver, and mercury, are also fungicidal at higher concentrations. Each of these four is known for its reaction with sulfhydryl groups, and such reactions have been suggested as a cause of their fungicial action (9). In data similar to those reported here for the aflatoxins, Takao (11) has reported inhibition of riboflavin synthesis by copper, mercury, and silver. Aflatoxin contamination of the seeds at harvest appears to be very rare in the U.S. cotton crop and is localized especially in very limited areas. A. flavus, it might be reasoned by some individuals, is a ubiquitous fungus which infects cotton seeds very nearly universally before harvest but produces aflatoxins only in the presence of suitable amounts of trace metals. Actually, temperature seems to us to be a factor of dominant importance and mineral supply to the cotton plant of uncertain significance. Infection of the seeds at harvest with A. flavus is of very limited occurrence across the cotton belt and geographical localization of the boll rot seems most strongly associated with very high temperatures in the affected areas and with the fact that A. flavus is a fungus adapted to growth at high temperatures (7,8). TAmz 4. Effect of addition of salts of several metals at 5 Ag of metal per ml to a basal medium on the production of aflatoxins, mat dry weight, and final pH of medium after 7-day incubation with A. parasiticus 2999

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2020-12-10T09:04:22.678Z

1975-01-01T00:00:00.000Z

237231610

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Spoilage Association of Chicken Breast Muscle The ability of pure cultures of bacteria isolated from spoiling chicken breast muscle to produce strong off-odors was tested by using sterile breast muscle sections. The incidence of organisms capable of producing strong off-odors and changes in flora during storage of naturally spoiling muscle at 2 C was traced, and the relationship between bacterial type and off-odor production was noted. The ability of pure cultures of bacteria isolated from spoiling chicken breast muscle to produce strong off-odors was tested by using sterile breast muscle sections. The incidence of organisms capable of producing strong off-odors and changes in flora during storage of naturally spoiling muscle at 2 C was traced, and the relationship between bacterial type and off-odor production was noted. Studies with sterile muscle sections allow assessment of the ability of pure bacterial cultures to produce off-odors associated with spoilage. Studies have been described recently in relation to the spoilage of porcine muscle (4) and fish muscle (5). The work described traces the flora of chicken breast muscle during lowtemperature storage and mhakes use of sterile sections to characterize organisms capable of producing strong off-odors on this substrate. MATERIALS AND METHODS Origin and isolation of strains. The breast skin of chilled, eviscerated chicken was removed, and the underlying muscles (pectoral proper and supra coracoid) were excised in approximately 5-g portions. These were stored in sterile petri dishes at 2 C. The muscle was not homogenized in order to preserve the cellular and physical integrity of the substrate. Three portions (15 g total) were selected at random at each sampling time (0, 4, 8, 12, and 16 days). Samples were homogenized in 135 ml of saline peptone diluent by using a Colworth Stomacher (A. J. Seward and Co. Ltd., London). Serial dilutions were prepared and plated in nutrient agar (Lab-Lemco Production of off-odors on chicken breast muscle. A modification of the technique described by Gardner and Carson (4) was used to excise sterile sections (about 2 g) from chicken breast muscle. The muscle was not roasted with a bunsen burner since this would have cooked into the depth of the tissue. Sections were stored in sterile screw-capped bottles. A number of checks for sterility were employed. (i) Sections were stored at refrigeration temperatures for at least 14 days before use and examined visually and I Present address: Faculty of Agricultural Science, University of Tasmania, Hobart, Tasmania 7001. 44 olfactorily for growth before use. (ii) To provide an index of the efficiency of excision of each batch of sections, 10% were selected at random as sterility controls; each of these was incubated at 22 C after addition of 5-ml amounts of nutrient broth. After incubation of the pure culture inoculum on the muscle section, a streak plate was prepared and colonial purity was checked. The 250 cultures isolated from naturally contaminated chicken breast muscle stored at 2 C were grown in nutrient broth for 48 h at 22 C, harvested by centrifugation (4,000 x g for 20 min), and washed twice in sterile phosphate buffer (0.1 M, pH 6.8) to give a final concentration of approximately 5 x 104 cells/ml. Portions of washed-ceU suspensions (0.1 ml) were inoculated onto muscle sections to give approximately 2.5 x 103 cells/g and incubated at 2 C for 14 days. Sensory examinations were carried out at 7 and 14 days. Analysis of head space vapors. An objective description of the odors produced by inoculation of the sterile muscle sections was provided by gas chromatographic analysis of the head space vapors. The instrument was a Phillips PV 4000 series gas chromatograph fitted with a Tenax GC column (Field Instruments Co. Ltd., Tetrapak House, Richmond, Surrey) 3 m in length with an internal diameter of 2 mm. The gas (oxygen-free nitrogen) flow rate was 20 ml/min, the injection port temperature was 240 C, and the oven temperature was 200 C. Characterization of isolates. The scheme of Shewan et al. (11) was used to identify the isolates. Eighteen-hour nutrient agar slope cultures were Gram stained (12), and motility was examined by the hanging-drop technique. Motile cultures were stained for flagella by the Liefson technique as cited by Norris and Swain (10). Oxidase reaction (8), mode of attack on carbohydrates (6), and fluorescent-pigment production (7) were also examined. RESULTS Numbers and incidence of different bacteria during spoilage of naturally contaminated muscle. Counts on nutrient agar at each sampling time are as follows. On days 0, 4, 8, 12 and 16 of storage, 1.97 x 103, 6.80 x 106, 3.20 x 108, 1.25 x 10k, and 1.44 x 109 organisms were recovered, respectively. All of the 250 strains isolated from naturally contaminated muscle stored at 2 C were gram-negative, motile rods which included representatives of Pseudomonas groups I, II, and IV (11) and an enteric type. The salient features of each group are shown in Table 1. Table 2 illustrates the distribution of these types (as a percentage of the flora) during the period of storage. The initial flora consisted almost exclusively of Pseudomonas group I organisms. The proportion of these decreased steadily with time, being replaced by Pseudomonas type II. Although the proportion of Pseudomonas group I strains fell consistently during storage, the actual numbers increased until the 12-day stage. Pseudomonas group IV organisms were also recovered as a small fraction of the flora up to 8 days, but not after 12 or 16 days of storage. These types, however, should not be discounted as insignificant; in terms of numbers, group IV types represented 2% of 3.2 x 108 organisms per g at the 8-day stage, i.e., a count of 6.4 x 106/g. A number of enteric organisms were also recovered after 8 days (10%) and 12 days (2%). Incidence of off-odor producers. The incidence of organisms producing strong off-odors when inoculated onto sterile breast muscle sections is shown in Table 3. Initially, 15% of the isolates produced strong odors, and this increased steadily to the 16-day stage when 45 of the 56 isolates (80%) were in this category. Within both major groups there appears to be a progressive selection for organisms able to produce strong off-odors ( Table 3). Types of off-odor produced. Three distinctive types of odor were recognized. These were described as "sulfide-like, fruity, and evaporated milk." Because of the difficulties m interpretation of subjective descriptions of odors, an objective analysis of the smells was provided by gas chromatography (Table 4). It is interesting to note that all of the peaks produced by the sulfide-like samples were also present in the fruity samples-the former showed a number of additional peaks. Sulfide-like odors rose to a peak (22% of samples) with the strains isolated after 8 days of storage, fruity types remained uniformly low throughout, and strains giving the evaporatedmilk odor increased steadily during storage, but particularly rapidly between the 12-and 16-day stages. Relationship between taxonomic position and off-odor produced. Within each of the Pseudomonas groups were found strains that caused no detectable off-odor and each of the three types of odor described above. Expressed as an overall percentage, a greater proportion of Pseudomonas type II (73%) caused strong offodors than Pseudomonas type I (22%). This, however, may simply be a reflection of the selection for Pseudomonas type II during the period of storage. At the point where a general off-odor was first detectable on the naturally spoiling sample (8 days at 2 C), Pseudomonas type I represented 56% of the flora (1.79 x 108/g), and Pseudomonas type II represented 32% of the flora (1.02 x 108/g). Thirty-three percent of the Pseudomonas I strains isolated at 8 days produced strong off-odors when inoculated onto sterile muscle sections. The corresponding figure for Pseudomonas II 8-day isolates was 47%. The predominant odor produced by the Pseudomonas II organisms was the evaporatedmilk type. This was particularly evident with those strains isolated during the latter part of the storage period. Forty-one of the 54 Pseudomonas II strains isolated after 16 days of storage at 2 C produced this odor when inoculated onto sterile muscle sections. All of the enteric types produced strong off-odors, whereas only sulfide-like odors were recorded for Pseudomonas type IV. (The number of representative strains of the latter two groups was low.) DISCUSSION It is generally accepted that off-odors and other changes associated with the spoilage of poultry meat stored at chill temperatures are caused by the growth of a few groups of psychrophilic bacteria (2,3). In this study, inoculation of certain strains onto sterile breast muscle sections produced strong off-odors which may be indicative of the ability of these strains to contribute to the spoilage of naturally contaminated muscle. Adams et al. (1) traced the incidence of spoilers during the course of spoilage of fish press juice. These normally remained less than 20% of the population. Similarly, Herbert et al. (5) showed that, although organisms capable of causing strong off-odors on cod muscle increased considerably in numbers during chill storage, they never accounted for more than 10 to 20% of the total flora. This is not the situation with chicken breast muscle, where there is a consistent increase in the fraction of the population able to cause strong off-odors. Indeed, it appears that within both numerically domir.ant groups there is a selection for these types as storage progresses. The preponderance of Pseudomonas group I and II strains on chicken breast muscle and the faster growth of the Pseudomonas type II agree with the results of Barnes and Impey (3). These workers inoculated breast and leg muscle with a mixture of Pseudomonas I and II, P. putrefaciens, and Acinetobacter strains in proportions similar to those expected on a carcass immediately after processing. P. putrefaciens and Acinetobacter types were not recovered from breast samples after 4 days at 1 C, although both increased considerably on leg samples. The nonpigmented pseudomonad, although initially present as only 2% of the flora, rapidly became predominant on breast samples. The differential effect on the growth of the various types was attributed to the influence of the relatively low pH of chicken breast muscle (5.7 to 5.9). No P. putrefaciens or Acinetobacter strains were recovered in this study. Lerke et al. (9) characterized the spoilers of sole press juice and found an extremely close correlation between taxonomic position and spoilage ability. Herbert et al. (5) linked fruity odors to P. fragi (group II) and sulfide-like odors to P. putida (group I) and III/IV types including P. putrefaciens. The results above indicate that off-odor producers and strains that cause no detectable organoleptic change on sterile chicken breast muscle occur within the same taxonomic group. It may be concluded that the flora of naturally spoiling chicken breast muscle at 2 C is dominated by Pseudomonas groups I and II, with Pseudomonas IV and an enteric type also present. The Pseudomonas II strains are most favored by the conditions and increase rapidly to become the major portion of the flora. During spoilage there is a selection for types able to produce strong off-odors when inoculated onto sterile muscle sections. ACKNOWLEDGMENTS The technical assistance of W. E. Espie and his staff is acknowledged. I am grateful to J. T. Patterson and P. A. Gibbs, Queen's University, Belfast, for advice during the preparation of this manuscript and for assistance with gas chromatography.

v3-fos

2020-12-10T09:04:12.727Z

1975-08-01T00:00:00.000Z

237230605

s2

Effects of a Commercial Malathion Preparation on Selected Soil Bacteria The aromatic petroleum distillates present in a commercial preparation of malathion insecticide had a greater effect on viability of pure cultures of soil bacteria than did the malathion, whereas neither component had an effect on the population of the natural soil community. The effects of a pesticide on soil microorganisms can be investigated by field studies or laboratory testing. Field studies generally are concerned with changes in the total viable population of the microflora or variations in the total metabolic activities per unit of soil (5,6) and therefore not always capable of delineating qualitative changes resulting from interactions of the pesticide and specific soil microorganisms. These are more easily detected by laboratory investigation using limited numbers of test organisms and a controlled environment. In laboratory studies the pesticide tested is normally in the purest state obtainable because of the possibility that solvents, diluents, carriers, or synergists may produce effects of their own (1,3). Reports concerning these effects, however, are comparatively rare. A commercial malathion solution (ACME 50% malathion [0,O-dimethyl-S-(1,2-biscarethoxy)ethylphosphodithioate] spray, Sherwin Williams Co.} was separated by simple distillation below 155 C into a malathion fraction and a solvent fraction (Aromatic petroleum distillates [APD]). The APD came over as a clear liquid with a boiling range of 135 to 155 C. The malathion residue was a dark-brown liquid which was identified by gas chromatographic comparison with a 98.9%-pure malathion furnished courtesy of American Cyanamid Co., Princeton, N.J. The two fractions were tested for their effects upon a soil community and selected soil bacteria growing as axenic cultures. For testing the effects of the malathion fraction or the solvent fraction (APD) on the soil community, 0.44 ,l/g of soil (computed from the manufacturer's recommended dosage) of malathion or APD as a water emulsion was used. An aliquot of soil treated with water served as a control. No differences in growth were noted for the 2-or 24-h postaddition populations plated on Martin agar medium (8), Jensen agar medium (7), or soil extract agar (2) when compared to the control. For testing the effects of malathion, APD, or components of APD on axenic cultures, these reagents were added to a log-phase culture to give a final concentration of 400 gl/l. Cultures were incubated at 24 or 30 C and 160 or 200 rpm. The complex medium used was tryptoneglucose-yeast extract broth (BBL); the simple medium used was a mineral salts-0.5% succinate broth (4) with 0.01% yeast extract added. Viable populations were determined before and 1 h after addition of the reagents by plating on tryptone-glucose-yeast extract agar plates. When the malathion and APD fractions were added to the cultures growing in either the complex or simple medium, the malathion fraction had no bacteriocidal effect, and the APD bacteriocidal effect varied depending on the organism and the medium in which it was grown and tested (Table 1). Of five aromatic hydrocarbons shown by Walker and Ahlberg (9) to be present in the 135 to 155 C boiling-point range corresponding to that of APD, three (isopropylbenzene, o-xylene, and m-xylene) exhibited a major bacteriocidal effect on Arthrobacter crystallopoietes in simple medium, and two (ethylbenzene and p-xylene) showed a minor effect (Table 2). In the axenic studies the solvent fraction (APD) of the malathion insecticide spray exhibited a greater bacteriocidal effect for the bacteria tested than did the malathion fraction. The effect was variable depending on the organism tested and the medium used. This variation in the effect of the APD on bacteria growing in two media of different complexity reaffirms that the choice of medium for laboratory testing of effector compounds on soil microorganisms will influence the results. Also solvents, diluents, carriers, and other nonprinciple components of commercial products must be considered in the evaluation of a product's impact on an ecological system. This work was done while G. J. S. was a trainee on Public Health Service research training grant GM 00926 from the Institute of General Medical Sciences.

v3-fos

2018-04-03T02:26:42.884Z

1975-06-01T00:00:00.000Z

33773927

s2

Violet Red Bile 2 Agar for Stressed Coliforms' Counts on a new, autoclave-sterilizable violet red bile (VRB-2) agar were compared with counts on freshly boiled VRB agar. Yields on VRB-2 agar averaged 217, 180, 130, and 112% of counts obtained on the control medium for samples of water, cottage cheese, frozen vegetables, and raw milk, respectively. The general principle used for the development of VRB-2 agar could be applied to many other kinds of selective plating media. When describe The second paragraph of Materials and Methods inadvert-ently omitted the manuscript: "Two media were used for the VRB-2 procedure. Plates containing inocula were poured with a basal medium that contained all the ingredients of VRB agar (Difco Manual, Difco Laboratories, Detroit, Mich.), except bile salts and dyes. The sterile overlay consisted of VRB agar containing double the usual concentrations of bile salts no. 3, neutral red, and crystal violet. Thus, upon equilibrium in the petri dish, the composition of VRB-2 agar was identical to that of conventional VRB agar. Several other modifications of formulas, in which either the bile salts or dyes alone were omitted, were not as satisfactory as VRB-2 agar; the data are not presented here." When a bacterial population is exposed to environmental stress, some cells may become injured. A substantial volume of data exists in the literature showing that injured cells may not grow after exposure to secondary stresses, such as high temperatures (4) or the presence of inhibitory agents (8,9). If a period of recovery is afforded the injured cells before exposure to a secondary insult, however, many of the injured cells become refractive to one or more secondary stresses (7,8). In this report, we describe a modified violet red bile (VRB) agar procedure for the enumeration of coliforms. The formula of the medium was altered so that recovery of injured cells could occur before the cells were exposed to selective inhibitory agents that prevent repair and subsequent cell proliferation. The modified medium is termed VRB-2 agar. MATERIALS AND METHODS The plating procedures used were essentially those described in Standard Methods (2). Exceptions were that seven plates were used per dilution, 10-ml pipettes were used to measure 1-ml samples and 1-ml pipettes were used to measure 0.1-ml samples, and incubation was at 30 C for 30 to 48 h. Cottage cheese and vegetable samples were prepared in dilutions of 10-I and homogenized for 30 s and 2 min, respectively, before further dilutions were made. Both basal and overlay VRB-2 agars were sterilized, in 100-ml aliquots, at 121 C for 10 min. Conventional VRB agars were either boiled and cooled before use or were sterilized at 121 C for 5 min. Both sterile VRB and VRB-2 agars were remelted at 121 C for 5 min before use. RESULTS AND DISCUSSION VRB-2 agar was first tested by using water samples (Table 1); the water samples were from widely dispersed locations on the Des Moines River and three locations from streams in Ames, Iowa. When recoveries on freshly boiled VRB agar were set at 100%, autoclave-sterilized VRB agar yielded an average of only 24% recovery (last column, Table 1). Surface-plating on autoclave-sterilized VRB agar also resulted in low recoveries, which showed that the temperature of the culture medium (5) was not the primary or sole cause of low recoveries on the autoclavesterilized medium. In all samples except one (sample 12, Table 1) VRB-2 agar permitted yields of 100% or greater. The relative quantities of base and overlay agars were not critical (Table 1). Average percentage yields were greatest when equal quantities of base and overlay agars were used, but this was probably a result of dye concentration. Plates that received relatively large quantities of overlay (VRB-2A) were dark, making typical colonies difficult to discern; plates that received relatively low quantities of overlay (VRB-2C) did not contain sufficient dye for adequate colonial differentiation, and some colonies might have been overlooked. An experienced worker can readily estimate the quantities of base and overlay agars when pouring plates without resorting to pipettes or automatic equipment. 'ND, Not determined; insufficient data. and inhibitory agents that prohibit cell recovery when the conventional VRB plating procedure is used. Table 2 shows the results obtained when boiled VRB, autoclave-sterilized VRB, and VRB-2 agars were used for coliform counts of frozen vegetables and cottage cheese. Overall, the VRB-2 agar was more productive than the other two methods examined, and in several instances the greater productivity of VRB-2 agar was outstanding. Higher yields, when compared with those with use of boiled VRB agar, were obtained by using VRB-2 agar in six of nine vegetable and five of seven cottage cheese samples. Furthermore, there were occasions when samples of these foods yielded too few colonies to attain statistical validity (data not shown); in each instance, the VRB-2 agar was the higher yielding of the three treatments examined. Two samples of raw milk also were studied; VRB-2 agar gave counts that were 113 and 112% of counts obtained on boiled VRB agar; yields on autoclave-sterilized VRB agar were only 57 and 51% of the boiled VRB agar counts. Colonies appearing on VRB-2 agar from samples of raw milk and water were confirmed by streaking them on eosin methylene blue agar, followed by Gram stains and tests for gas production in lactose broth. Of 80 colonies from milk samples, 73 grew on eosin methylene blue agar, were gram-negative rods, and produced gas in lactose broth. Similar percentages of isolates from water were confirmed as coliforms. Only 50 colonies from cottage cheese were examined; all grew on eosin methylene blue agar and were gram-negative rods. No isolates from frozen vegetables were studied, nor did we identify any of the isolates as to species. Therefore, we do not know whether VRB-2 agar permitted growth of additional strains or recovered more of the same groups that grew on boiled VRB agar. Confirmation of the studies reported herein is needed, of course, before VRB-2 agar can be recommended for routine use. For this reason, our report might seem premature. We wish, however, to emphasize the principle of the method, i.e., plating in or on a nonselective medium that is layered above or below a selective medium. This principle is not new. It probably originated with Eijkman (3), who used separate layers of medium for cell growth and detection of lipolytic activity. Similar proce-VRB-2 AGAR FOR STRESSED COLIFORMS dures for the detection of lipolysis have been used by other investigators (1,5,6). Rose and Litsky (7) incubated membrane filters on a pad containing an enrichment medium so that cell recovery occurred before selective ingredients had diffused from a base layer of agar, through the pad, and to the cells on the filter surface. We have extended the studies of Rose and Litsky (7) by pointing out that direct-plating procedures also can be modified to facilitate the recovery of stressed cells. Such modifications can be effected without alteration of the overall media formulations or drastic changes in normal plating procedures. We have not yet tested boiled VRB-2 agar to discover if even higher yields can be obtained. Autoclave-sterilized media are most applicable to routine use, but boiled VRB-2 overlay agar may effect even greater recoveries than are reported here.

v3-fos

2016-05-04T20:20:58.661Z

1975-04-15T00:00:00.000Z

172468

s2

Comparison of sire breeds for crossbred lamb production from new zealand romney ewes Results were reviewed from studies involving comparisons of the performance of different breeds of sheep and their crosses particularly in relation to body size, reproductive and maternal performance, carcass attributes and aspects of mineral metabolism. Crossbreds offer a wide range of performance specifications for an almost infinite variety of farming systems and market requirements, but in most cases heterosis appeared to play only a relatively small part in determining crossbred performance in respect of ‘ single ’ traits, altough at times this part may be important. Among the examples cited, heterosis had a large effect only in determining levels of calcium and copper in blood of crosses among the particular breeds examined. In general, estimates of heterosis were found to vary greatly not only for different traits but from cross to cross when the ‘ same ’ trait was considered. This suggests that the magnitude of heterosis is not readily predictable for specific breed combinations. However, the value of crossbreeding in a farming system should not be assessed only in relation to single traits, but should be considered for combination of traits and for the profitability of the system. There is insufficient evidence on the magnitude of’ profit heterosis ’. Among the examples cited, heterosis had a large effect only in determining levels of calcium and copper in blood of crosses among the particular breeds examined. In general, estimates of heterosis were found to vary greatly not only for different traits but from cross to cross when the ' same ' trait was considered. This suggests that the magnitude of heterosis is not readily predictable for specific breed combinations. However, the value of crossbreeding in a farming system should not be assessed only in relation to single traits, but should be considered for combination of traits and for the profitability of the system. There is insufficient evidence on the magnitude of' profit heterosis '. Je 50 Muttertiere aus je 10 Halbgeschwistergruppen von 4 (Meiereihof) bzw. 6 (Oberer Lindenhof) verschiedenen Kreuzungskombinationen auf der Basis von Merinolandschafen wurden mit je 10 Böcken aus 4 englischen bzw. Französischen Fleischrassen über 3 bzw. 5 Deckperioden angepaart. Alle in diesem Zeitraum geborenen Lämmer (durchschnittlich 40 je Untergruppe) wurden einer Intensivmast zugeführt und versuchsmässig ausgeschlachtet. Die Aufzucht der Lämmer erfolgte im Meiereihof bei ausschliesslicher Frühjahrslammung herkömmlich an der Mutter, auf dem Oberen Lindenhof bei mehrmaligem Ablammen je Jahr mit Hilfe von Milchaustauschern. COMPARISON OF SIRE BREEDS 2 . Progeny of long-wool sires, and particularly the Lincoln, clip more wool at post-weaning shearing than of the Down breeds, which exhibit quite small variation. 3 . Sire breeds vary in average liveweight growth of their progeny, the Suffolk, Hampshire and Dorset breeds producing the heaviest and the Merino and Romney the lightest lambs. 4 . Within any breed wide differences exist between progeny growth rates of the best and poorest sires, emphasizing the great importance of sound selection of rams and of adequate genetic sampling in breed comparisons. 5 A 3 x 3 x 2 factorial experiment is being carried out and involving 3 breeds of sire ; Southdown, Suffolk and Cotswold to a common dam line, the Finnish Landrace x Dorset Hoyn . Two planes of nutrition are used, ad libitum and restricted i. e. restricted to grow at z/ 3 the average growth rate shown by those comparable lambs on ad lib. intake. Entire males and females are compared. All progeny were individually penned after weaning at r8 p. 100 midparent weight (i. e. approx. 5 weeks old) and fed on 8 7 p. 100 rolled barley diet containing r 5 p. 100 crude protein in the dry matter. Lambs were slaughtered at 40 , 50 , 6 0 and 70 p. l oo midparent weight. The left side of each lamb was physically dissected into lean, bone, submuscular and intramuscular fat and waste. Scatter diagrams were drawn and within trial and group relationships between weight of carcass tissue and carcass weight were judged to arithmetically linear. However marked differences in slopes and some differencies in intercepts were noted. Multiple regression was used to quantify the interaction found by use of a linear model. The resulting coefficients gave a quantitative partition of the treatment and interaction effects on slope and intercepts. Significant differences in intercept were attributable only to breed of sire, in particular the Cotswold sired lambs within lower levels of fat and higher levels of lean tissue. The effects in slope were more diverse and more important with significant effects for breed, sex and interaction between breed and sex, sex and nutrition and between breed, sex and nutrition. In practical terms, Southdown crosses appear to be unsuitable for this intensive production system. The Cotswold is well suited to sire lambs to be slaughtered at weight between 30 and 4 o kg live weight. A greater sex differential within the Suffolk crosses suggested that female lambs are unsuitable, whereas male lambs for which growth is restricted may be taken to comparatively heavy weights to produce carcases of about 25 kg. It is necessary to measure the merit of crossbreeding for meat production from sheep in terms of yield of meat per hectare and the acceptability of the carcase to the consumer. The efficiency of meat production is governed by the reproductive rate of the flock and the rate of growth of the lambs, both in relation to the size of ewe which in turn governs stocking rate. Acceptability of the carcase depends on its weight and basically on its component muscle : bone ratio and fat percentage.

v3-fos

2020-12-10T09:04:12.665Z

1975-10-01T00:00:00.000Z

237233356

s2

Gas Chromatographic Presumptive Test for Coliform Bacteria in Water A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively. A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively. Detection and enumeration of coliform bacteria of fecal and nonfecal origin are among the most commonly used microbiological indicators of quality in food and water. Whereas there are numerous analytical methods for detecting coliform bacteria quantitatively, the primarily used techniques require up to 2 days before results are available. Such delays impose obvious health hazzards, and, in the case of food, economic burdens since microbiological results often determine safety for human consumption or storage time. There is, then, need for techniques which make possible rapid assessment of the microbiological quality of water and foods. In an earlier report from this laboratory the detection of Escherichia coli by gas chromatography was described (3). The method was based on the Eijkman concept and detection was accomplished by analyzing for the presence of metabolically produced ethanol in cultures incubated at 44.5 C. According to the Eijkman concept coliform bacteria can be differentiated on the basis of growth at elevated temperatures. Coliform bacteria of fecal origin grow and ferment lactose at 44 to 46 C and are usually + + --or -+ --IMViC types, whereas coliform bacteria from other sources are rarely able to grow in the 44 to 46 C temperature range (4). In this report we describe the application of the Eijkman principle to a gas chromatographic method for the presumptive detection and estimation of coliform bacteria. By incubating cultures inoculated with water samples at 37 or 44.5 C total coliform and fecal coliform concentrations can be estimated. MATERIALS AND METHODS The pure cultures used in this study were a laboratory strain of Escherichia coli B and coliform bacteria isolated from the effluent of the local sewage treatment plant. In experiments involving water samples, the samples were inoculated directly into culture medium, and the indigenous coliforms were allowed to grow out. The growth medium was M-9 salt mixture (6) supplemented with 1.0% lactose, 0.1% Casamino Acids, and 0.1% yeast extract at pH 7.2. The medium was made double strength and sterilized by membrane filtration. For experiments 1 ml of medium was inoculated with an equal volume of water sample, and the cultures were incubated in water baths at 37 or 44.5 C. Best results were obtained when the cultures were stirred with small Tefloncoated magnets and an immersible magnetic stirrer. Coliform concentrations in water samples were determined by the multiple-tube most-probable number method (MPN) in lauryl sulfate tryptose broth (LST) or by the membrane filter procedure (MFC) with M-endo broth both as described in Standard Methods (1). The presence of coliforms was verified by plating positive cultures on EMB agar and by IMViC tests. Gas chromatographic analyses of ethanol in the cultures were done with a Varian model 1200 gas chromatograph equipped with a hydrogen flame ionization detector. The column was 5 feet by 1/8 inch outer diamter (ca. 152 by 0.32 cm) aluminum tubing packed with 80/100 mesh porapak QS (Waters Associates) with nitrogen at 30 cm3 per min as the carrier gas. Other operating parameters were a column temperature of 170 C, injector temperature of 190 C, and the detector at 200 C. The electrometer was operated at 1 x 10-10 A full scale on a 1-mV 10-inch (ca. 25 cm) recorder. The instrument was standardized daily by injecting 2 jAI of a 0.01% aqueous ethanol solution. Detector response was linear between 0 and 0.01% ethanol (Fig. 1). Analysis of cultures was accomplished by injecting 2 ;L of culture directly onto the column. The retention time for ethanol was 45 s. Between each analysis the column was purged with 5 ILI of water. RESULTS An important consideration in high-sensitivity gas chromatography is a sample free ofinter- U O la fering compounds contributed by the culture medium. In preliminary experiments it was found that autoclaving the culture medium resulted in thermal degradation of lactose and yeast extract which resulted in numerous volatile compounds which chromatographed with metabolically produced ethanol. Filter sterilization minimized interfering compounds contributed by the medium although as shown in the 0-to 420-min panel in Fig. 2 some background peaks were present. The compounds associated with these peaks were found to be associated with lactose; fortunately, the peaks did not interfere with ethanol analysis (Fig. 2). As indicated, one of the chromatographable medium compounds was identified as acetic acid. Also shown in Fig. 2 are the chromatographic changes in the culture medium caused by coliform metabolic activity. It can be seen that concomitant with ethanol formation the acetic acid peak also increased in size. However, acetic acid formation was not used as an indicator of coliform bacteria since it was difficult to reliably detect small increases in peak size. Occasionally a water sample contained microorganisms which grew in the test cultures but did not produce ethanol or other detectable chromatographic changes in the medium. Subculture showed these organisms were non-lactose Chromatographic changes in I ml ofculture medium inoculated in 1 ml ofa water sample initially containing approximately 80 coliform bacteria per ml. Incubation temperature was 37 C. The water sample contained fecal and nonfecal coliforms. VOL. 30, 1975 fermentors. Apparently utilization of other medium constituents did not produce metabolic end products which were detectable under the analytical conditions used in this study. Similarly, when lactose was omitted from the culture medium no chromatographic changes in the medium as a result of microbial activity were detected in 24-h cultures incubated at 37 or 44.5 C. Whereas our initial research indicated that laboratory cultures of E. coli produced ethanol from lactose fermentation, it was of interest to determine if this was also true for coliform bacteria isolated from contaminated water. Water samples taken over a 10-day period were inoculated directly into duplicate LST broth tubes, and the tubes were incubated for 24 h at 37 and 44.5 C. Coliform bacteria were isolated from positive tubes on EMB agar, and the isolates were IMViC typed. The coliform-isolates were inoculated into the M-9 lactose medium described above and incubated at 37 and 44.5 C. Results summarized in Table 1 show that at 37 C all the organisms grew and produced ethanol. At 44.5 C all the + + --group and 16 out of 18 of the -+ --group grew and produced ethanol. Only 8 out of 68 coliform isolates with --+ + characteristics grew and produced ethanol at 44.5 C. The data in Table 1 show that relatively few --+ + coliform bacteria grew at 44.5 C in the M-9 lactose medium. However, when temperature-tolerant organisms with nonfecal IMViC classifications are present in water samples a positive ethanol test results. Consequently, in gas chromatographic experiments ethanol-positive 44.5 C samples were streaked on EMB agar and colonies with the typical sheen tested on citrate agar or IMViC typed. Only when a positive culture was shown to contain citrate-negative coliforms or coliforms with + + --or -+ --characteristics was the test considered positive. In practice we did not find any 44.5 C positive samples which did not contain fecal coliforms, although mixed flora composed of both fecal and nonfecal types were not uncommon. The kinetics of formation of ethanol were the same for pure cultures of E. coli and for cultures inoculated with water samples (Fig. 3). However, as would be expected the time period between inoculation of the cultures and the formation of detectable amounts of ethanol was dependent on the initial coliform concentration of the inoculum. Under the analytical conditions used in the study the minimum detectable ethanol concentration in cultures was 1 to 2 ng. Theoretically the minimum detectable ethanol concentration was 0.1 ng; however, we were not able to attain this level of sensitivity due to base line instability. To determine the relationship between the initial coliform count and the incubation time required for ethanol formation, 1-ml aliquots of diluted water samples were inoculated into equal volumes of 2 x culture media and incubated at 37 or 44.5 C. Total coliform concentrations in water samples were determined by MPN tests in LST broth at 37 C after 48 h and fecal coliform concentrations were determined by the MFC method at 44.5 C after 24 h. Typical 37 C results ( b Tubes were inoculated with 1 ml of each dilution. c Ethanol was determined by gas chromatography. Cultures contained 1 ml of water sample and 1 ml of 2 x M-9 lactose medium. ments (Fig. 4) show that initial coliform counts were exponential functions of the incubation times required for ethanol formation. Coliform detection required less time at 37 than at 44.5 C for reasons which are discussed below. With respect to sensitivity, at 37 C it was possible to consistently detect differences in the incubation time required for ethanol formation when the initial number of organisms differed by a factor of two or more at initial counts of 10 per ml or higher. When the initial count was less than 10 coliforms per ml of sample, variability tended to reduce reliability. Detection of small numbers of fecal coliforms at 44.5 C was more variable than total coliform detection at 37 C. In part this was due to longer lag times at 44.5 C. In addition we found that when the initial fecal coliform count was less than 10 per ml the cultures frequently failed to grow at all. Apparently, fecal coliforms in water samples have difficulty in adapting to 44.5 C; however, we found that once adapted to this temperature the organisms grew at the same rates as at 37 C. It is worth noting that in the experiments summarized in Fig. 4 the presence of coliform bacteria was confirmed in 79 out of80 water samples positive for ethanol. As might be expected, coliforms in water samples cultured at 37 C were usually mixed IMViC types, whereas at 44.5 C fecal IMViC types predominated. At 37 C the tests could be terminated after 12 h ofincubation since cultures negative for ethanol after 12 h were negative for coliform bacteria at 24 and 48 h. At 44.5 C the cutoff time for ethanol formation was 14 h. DISCUSSION The gas chromatographic procedure described here could serve as a useful presumptive test in cases where a specific coliform limit is established. For example if the limit was 10 coliforms or less per ml then a test culture Relationship between the initial coliform concentration in water samples and the incubation time required for ethanol formation. Cultures contained 1 ml of water sample and 1 ml of2 x medium. Total coliforms were determined by the MPN test in LST broth at 37 C, and fecal coliforms were determined by the MFC test in M-Endo broth at 44.5 C. Samples for gas chromatography were taken at 30min intervals. containing 1 ml of the sample should not contain detectable ethanol after 9 h of incubation at 37 C or 11 h at 44.5 C. Cultures which were positive for ethanol could be confirmed by conventional methods. If greater sensitivity or shorter detection times are required samples can be concentrated by centrifugation or membrane filtration. We have obtained the same detection times with concentrated samples as with unconcentrated samples both at the same initial coliform density. Shorter detection times would also be possible by operating the gas chromatograph at maximum analytical sensitivity. If this had been possible coliform detection times would have been shortened by 1 h or more. With respect to rapidity the gas chromatographic method detection times are comparable with those reported for radiorespirometry (2,5) and calorimetry (5). At present, incubation at 44.5 C is not completely selective for fecal coliform bacteria since mixed coliform populations were occasionally isolated from water samples cultured at 44.5 C in M-9 lactose medium. This was also seen when samples were cultured in LST broth for MPN determinations at 44.5 C. It is not known ifthe nonfecal coliform organisms were fermenting lactose and producing ethanol at 44.5 C or growing synergistically with fecal types. An advantage of the method described here is that coliform analyses can be done without sample preparation. Direct analysis of culture medium is possible since the column packing has ideal characteristics for separating polar compounds such as water and alcohols. Columns are relatively unaffected by accumulation of nonvolatile culture medium constituents or bacterial cell components since peak shapes and retention times were the same after 2,800 analyses on the same column. We are aware of the possibilities of automating the gas chromatographic procedure and this problem is currently under investigation. We are also investigating application of the method to determine coliform contents and total bacterial counts in foods. The results of these studies will be reported later.

v3-fos

2018-04-03T00:50:21.606Z

1975-10-01T00:00:00.000Z

26809816

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Psychrophilic Microorganisms from Areas Associated with the Viking Spacecraft Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture and assembly areas of the Viking spacecraft. Populations ranging from 4.2 x 103 to 7.7 x 100/g of soil were isolated from the 15 soil samples examined. Temperature requirements were determined, and those growing at 3 C, but not at 32 C, were designated as obligate psychrophiles in this investigation. Populations ofsoil bacteria, including aerobic sporeformers, ranging from 1.5 x 102 to 9.8 x 105/g were capable of growth at 3 C, but not at 32 C. Bacterial isolates were identified to major generic groups. No psychrophilic sporeformers were isolated from soil from the manufacture area, but psychro- philic sporeformers ranged from 0 to 6.1 x 103/g from soil from the assembly area. and biochemical testing. From these re- sults, the temperature studies, and colonial characteristics, the organisms were identified to major generic groups. The isolates were stained for their Gram reaction, tested for motility by phase-contrast microscopy, tested for oxygen requirements, and subjected to numerous biochemical tests (4). Orga- nisms thought to be sporeformers were grown on AK-2 sporulating agar (BBL) at either 7 C (10 to 14 days) or 24 C (2 to 3 days). These were then stained to demonstrate production of spores. Mwcrococcaceae were identified according to the method of Baird-Parker (1). The gram-positive rods were identified as sporeformers (Bacillus) or non-sporeformers. On the basis of the tests performed, the latter group was designated as the Corynebacte-rium-Brevibacterium group. The gram-negative rods were placed into one of two groups. The nonpig-mented ones were placed into the Alcaligenes-Acine- tobacter group and the pigmented ones into the Fla-vobacterium-Cytophaga group, although the taxo- nomic relationship of this group is still uncertain (16). The fungi were identified to genus according to the methods of Barnett and Hunter (2) and Barron (3), and the yeasts were identified following the method of Lodder (10). This was performed with the assistance of John Brandsberg, Center for Disease Control, Kansas City, Kansas. One purpose of the United States planetary quarantine policy is to determine guidelines for the prevention of contamination of Mars with terrestrial microorganisms which might grow in the Martian environment (6). For this reason, it is essential that various groups of microorganisms associated with planetary vehicles destined for Mars be studied in this respect. A great deal of previous research has been conducted on organisms isolated from the manufacture and assembly areas of interplanetary spacecraft, but most of these studies dealt primarily with mesophilic bacteria and the heat resistance of mesophilic sporeformers. The Viking spacecraft will be decontaminated by means of dry heat (13). The standard procedures for the microbiological assay of such spacecraft environments call for the exclusive use of 32 C as the incubation temperature (11). This procedure limits the assay to detection of mesophilic organisms, which make up the majority of microorganisms found on spacecraft surfaces (15). Although it is generally agreed that psychrophiles may not be the most heat resistant of the microorganisms, they should not be excluded from investigations related to planetary quarantine, because these may include organisms with the physiological characteristics to grow in the hostile environment of Mars (7). Also, it is known that some Bacillus spp. and Clostridium spp. can grow at low temperatures (9,17), and sporeformers are the more heat-resistant microorganisms. It is recognized that there are numerous definitions of psychrophilic organisms, including those based on optimum growth temperature and those based on possible growth ranges (8). This present study is not intended to debate the definition of psychrophiles but to demonstrate that the present incubation temperature of 32 C used in the microbial monitoring of spacecraft environments might be excluding propulations ofmicroorganisms of significance to planetary quarantine. The Viking spacecraft is scheduled to be launched to Mars in 1975; consequently, the primary objective of this investigation was to determine the presence and concentration of psychrophilic microorganisms in various environmental areas associated with the spacecraft. MATERIALS AND METHODS Selection of samples. Soil samples were obtained from three sites where the Viking spacecraft is manufactured (Denver, Colo., M), and 12 sites where the spacecraft is housed in preparation for launching (Cape Canaveral, C). Surface soil samples no deeper than 6 inches (ca. 15 cm) were taken from areas around main entrances through which dust contamination might enter the buildings. Isolation of microorganisms. Immediately after being returned to the laboratory, each sample was thoroughly mixed, and 10-g portions of each were decimally diluted in 1.0% peptone to a final dilution of 10-6 prior to plating. The first bottle (90 ml) in each dilution series contained glass beads, the bottle was sonicated for better dispersal of soil particles (14). Subsequent dilution tubes (9 ml) were mixed on a Vortex mixer to assure thorough mixing. Amounts of 0.1 ml were transferred to the surface of Trypticase soy agar (Baltimore Biological Laboratories [BBLI) and Mycophil agar (BBL), pH 4.0, and spread with glass spreading rods. Because all isolations included incubation of samples at 7 C, media used throughout this investigation were stored at 7 C for at least 24 h prior to use. To prevent possible damage to psychrotolerant organisms by addition of molten agar, the spread-plate technique was employed in all counts. All manipulations were performed in a laminar flow cabinet (Envirco MiniBench, model MBO-48, Albuquerque, N.M.). Duplicate plates were prepared for aerobic, anaerobic, and fungal counts. The plates were placed in the 7 C incubator (Freas model 805) immediately after inoculation, and only the anaerobic plates were allowed to reach room temperature during the manipulations. The anaerobic plates were rechilled after inoculation, placed in Brewer Anaerobic Jars with GasPaks and anaerobic indicators (BBL), and plated in the 7 C incubator as soon as anaerobic conditions were achieved as shown by the indicator. A freshly inoculated Trypticase soy agar slant of Alcaligenes fecalis (NASA standard test organism, Center for Disease Control, Phoenix, Ariz.) was placed in each anaerobe jar as a biological indicator of anaerobiosis. In no case did this control organism grow in the anaerobe systems. All incubators were monitored with maximum-minimum registering thermometers (Taylor model 5458), which were checked daily. Slight increases in temperature occurred only during times when samples were being added to or removed from the incubators. Temperature studies. Colonies from plates having 30 to 300 colonies after 14 days of incubation were transferred to Trypticase soy agar slants for incubation at 3 C (10 to 14 days), 24 C (3 to 5 days), and 32 C (48 h). After growth had occurred, the results were recorded, and organisms showing growth at 3 C, but not at 32 C, were classified as psychrophilic, according to the definition used in this investigation. Identification of isolates. All isolates from the temperature studies were examined individually by staining and biochemical testing. From these results, the temperature studies, and colonial characteristics, the organisms were identified to major generic groups. The isolates were stained for their Gram reaction, tested for motility by phase-contrast microscopy, tested for oxygen requirements, and subjected to numerous biochemical tests (4). Organisms thought to be sporeformers were grown on AK-2 sporulating agar (BBL) at either 7 C (10 to 14 days) or 24 C (2 to 3 days). These were then stained to demonstrate production of spores. Mwcrococcaceae were identified according to the method of Baird-Parker (1). The gram-positive rods were identified as sporeformers (Bacillus) or nonsporeformers. On the basis of the tests performed, the latter group was designated as the Corynebacterium-Brevibacterium group. The gram-negative rods were placed into one of two groups. The nonpigmented ones were placed into the Alcaligenes-Acinetobacter group and the pigmented ones into the Flavobacterium-Cytophaga group, although the taxonomic relationship of this group is still uncertain (16). The fungi were identified to genus according to the methods of Barnett and Hunter (2) and Barron (3), and the yeasts were identified following the method of Lodder (10). This was performed with the assistance of John Brandsberg, Center for Disease Control, Kansas City, Kansas. RESULTS Population studies. Viable counts of microorganisms growing at 7 C from soils from the manufacture and assembly areas of the Viking spacecraft are presented in Table 1. Viable counts from Athe manufacture area are approximately 2 logs higher than those from the assembly area. In all but one sample (C-4) the aerobic bacterial counts were the highest, with sample C-4 containing a higher population of anaerobes. In 10 of the 15 samples the anaerobic counts were higher than the fungal counts. Temperature studies. One of the means of grouping the various isolates for identification included their ability to grow at 3 C (10 to 14 days), 24 C (3 to 5 days), and 32 C (48 h). Organisms showing growth at 3 C, but not at 32 C, in the designated time are defined as psychrophiles. Many of these did show growth at 24 C ( Table 2). Since many investigators prefer a more rigid definition of psychrophiles, results in Table 2 also show the percentage of organisms that grew at 3 C but not at the other two temperatures. Even though the total population is higher in the samples from Denver, the percentage of psychrophiles is higher in the samples from Cape Canaveral. This is espe- cially true in the group that includes organisms growing at 3 C but not at 24 or 32 C. The results given for percentage of obligate psychrophiles from these soil samples may appear high, but it must be remembered that they are based upon counts in which the organisms were orginally isolated at 7 C. Identification. Anaerobic isolates from the manufacture area were stained and found to consist of approximately 70 to 80% sporeformers and 20 to 30% gram-positive non-sporeforming rods. Samples from Cape Canaveral yielded almost 100% sporeformers. During further testing, it was determined that these were facultative aerobes belonging to the genus Bacillus. It was also determined that many of the aerobic isolates were facultative. From these results, it was decided to exclude the anaerobes from further investigation because obligate anaerobes were not demonstrated. The fungi were indentified to genus, and these results are presented in Table 3. Molds were not identified to species. The yeasts from the manufacture area were all determined to be Cryptococcus albidus. Those from Cape Canaveral were identified as either C. albidus, Cryptococcus laurentii, Rhodotorula rubra, or Rhodotorula minuta. The aerobic bacteria were identified to major generic groups including Micrococcus, Bacillus, Corynebacterium-Brevibacterium, Alcaligenes-Acinetobacter, andFlavobacterium-Cytophaga. Different isolates within each major group were recognized, and representatives of these have been lyophilized. The results of these determinations are presented in Table 4. The fungi isolated on these plates were not identified but were included in the counts. Samples from the manufacture area appear to contain primarily members ofthe Corynebacterium-Brevibacterium group and members of the genus Micrococcus, especially subgroup 8. Of interest is the fact that these samples contain only low percentages of aerobic sporeformers. In contrast to this, samples from Cape Canaveral contain primarily members of the genus Bacillus and the Corynebacterium-Brevibacterium group. Percentages given in Table 4 are calculated from the aerobic counts of Table 1. The actual number of Bacillus per gram of soil is similar when the manufacture area samples are compared to the Canaveral samples. Even though the percentage ofBacillus in the aerobic population of the manufacture area samples is quite low, M-3 contained the largest population of aerobic sporeformers. DISCUSSION Attempts to prevent the contamination of interplanetary spacecraft intended to enter the atmosphere of Mars have at least a twofold purpose. One is to prevent the contamination of the Martian surface with terrestrial organisms which might alter the state of the planet; the other is to assure that terrestrial contaminants do not interfere with the life detection experiments on the spacecraft (6). It is accepted that the conditions on the Martian surface are not such that mesophilic organisms will be exposed to a temperature approaching 32 C (12), and the Viking Lander Biological Instrument will be maintained at a temperature of approximately 15 C (H. P. Klein, personal communication). Standard NASA procedures for the microbial monitoring of spacecraft specifies incubation at the single temperature of 32 C (11). The majority of contaminants found in the spacecraft are mesophilic organisms (15), but the use of this single temperature could possibly exclude organisms better adapted to grow in the cold environment of Mars or in the Viking Lander Biological Instrument. This present investigation has demonstrated the presence of relatively large soil populations of organisms which would not be detected by the present microbial monitoring procedures for spacecraft environments. This estimate might be low because of the use of a single isolation temperature of 7 C. Of the populations just described, the sporeforming rods are probably the most important group, because the Viking spacecraft will be subjected to dry-heat decontamination prior to launch. The soil samples from Denver showed no psychrophilic sporeformers, whereas the samples from Cape Canaveral showed an average of 2.1 x 103 psychrophilic sporeformers/g of soil. These results demonstrate the presence of a population that may have been excluded by present monitoring procedures.

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Incidence of Listeria monocytogenes in Nature During a research project on the occurrence of Listeria monocytogenes 194 strains were isolated in southern West Germany during the years 1972 to 1974: 154 from soil and plant samples (20.3%), 16 from feces of deer and stag (15.7%), 9 from old moldy fodder and wildlife feeding grounds (27.2%), and 8 from birds (17.3%). The highest number of isolates was obtained from uncultivated fields. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the. first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources. In spite of the growing literature on Listeria monocytogenes the question whether this organism is primarily soil born or originates from animals excreting L. monocytogenes with feces (3,4,6,8,13) remains unresolved. Therefore, this study has focused on the occurrence of L. monocytogenes in plants, soil, and feces of wild animals and birds. MATERIALS AND METHODS Samples of plants and soil from various parts of southern West Germany to be tested for L. monocytogenes were collected from: (i) cultivated fields (maize, wheat, oats, barley, potatoes, etc. and pastures and meadows), (ii) uncultivated fields (fields and meadows that had lain fallow for years); (iii) forests, wildlife feeding grounds, and mud from creeks, rivers, and ponds); (iv) feces and residues of fodder from wildlife feeding grounds. Each sample of plant or fodder specimen from wildlife feeding grounds was collected with sterile rubber or disposable plastic gloves or with sterile scissors and was packed in plastic bags. The soil samples were obtained from the surface of the ground, as well as from a depth of 10 cm, using small sterile shovels. It was taken into consideration that soil samples from the surface are regularly contaminated with plant residues and that contamination could not be avoided when soil samples were collected from the depth. Shredded plants, soil samples (15 to 20 g), or feces (1 to 10 g) were suspended in 50 to 400 ml of enrichment broth and grown in 500-ml glass bottles. The following enrichment media were employed: (i) potassium thiocyanate medium (according to Lehnert [19]) and potassium thiocyanate medium substituted with 10 ug/ml of Acriflavin (Serva) per ml (K. Hbhne, Ph.D. thesis, Justus-Liebig University, Giessen, West Germany, 1972); (ii) tryptose broth (Difco); and (iii) brain heart infusion broth (Difco). For medium (i) the seeded containers were maintained at 22 C for 7 days. After 2 and 7 days, samples were plated on four tryptose agar plates (Difco) with 40 #g of nalidixic acid (Serva, Heidelberg; subsequently called TN medium) added per ml. This TN medium was incubated at 37 C for 24 h and for an additional 24 h at 22 C. Then the growth was checked for typical colonies with a microscope by use of the Henry illumination technique (1,2). Using enrichment media (ii) and (iii), cultures were maintained for 12 months at 4 C. After 1, 3, 5, and 12 months, one loopful of the material was plated on four TN agar plates. In addition, a sample from each culture was transferred into Stuart's liquid with the aid of an alginate tampon and incubated at 22 C for 1 week. Subsequently, this tampon was suspended in 10 ml of Ringer solution and one loopful was plated on four TN agar plates (5). Additionally, brain heat infusion (5.0 ml) was inoculated with 0.1 ml of material from the brain heart infusion enrichment broth, incubated at 22 C for 2 weeks in the dark, and streaked on four TN agar plates (15). All colonies suspected of being Listeria were examined for catalase activity and motility and tested further as described by Seeliger (11). The virulence of the isolated Listeria strains was tested in an outbred strain of mice (NMRI/Han) weighing 18 to 20 g. Four mice each were injected with 0.5 ml of an 18-h glucose broth culture intraperitoneally. A strain was considered to be virulent if the mice died within 3 weeks and Listeria could be isolated from the organs at necropsy. The determination of serovars was done according to the procedures outlined by Seeliger (11). RESULTS Cultivation in the enrichment broth of Lehnert (9) was found to be an excellent method for 29 the isolation of Listeria. The same medium substituted with acriflavin yielded even better results, but the time of its use was too short for a definite evaluation. L. monocytogenes occurs in a high proportion of plants, soil samples, and feces of deer and stags as well as of birds ( Table 1). The highest positive results were obtained from the surface of soil specimens and plants, particularly in fields that had lain fallow for years and were overgrown with grass and small shrubs ( Table 2). Only a few strains of L. monocytogenes could APPL. MIcaomOL be isolated from the depth of soil from uncultivated fields. Faded and decayed grass was a direct indicator of the presence of Listeria. The same Listeria serovar was found repeatedly at the same place at half-year intervals. In some instances two different serovars were detected in the same sample. Positive findings were irregular at the same site. From a meadow near Kaiserstuhl that had not been used as pasture land for some years two strains were isolated in the autumn of 1971, none in the spmmer of I I I I 1 2 a New antigen combinations, not yet designated (see Table 3). 1972, and again two in the autumn of 1972. Somewhat unexpected was the isolation of L. monocytogenes from the leaves of shrubs 50 cm above ground, in addition to those on the ground. L. monocytogenes could be isolated from samples of mud in relatively great numbers. It appears to survive and to multiply particularly well in a moist environment. The lowest number of positive results was obtained from fields and meadows used for agricultural purposes. An entirely different distribution of L. monocytogenes was noted in the upper Black Forest, where it was isolated exclusively from the surface of the soil of wildlife feeding grounds. On the other hand, the incidence of Listeria in the deciduous forests of the foothills and in the valleys, as well as in the plain, was rather scattered. The low incidence of L. monocytogenes in upper Black Forest was associated with low pH values of the local soil (sometimes below 3.5). A high percentage of samples collected in a small forest north of Salem, near the Lake of Constance, yielded Listeria. This forest is surrounded by cultivated fields, and the pH value of the soil samples was found to be between 4.8 to 7.6. In this area Listeria was cultured from 22 out of 46 specimens in a single wildlife feeding ground, in its vicinity as well as at a distance of 200 m. A great number of Listeria isolates were obtained from feces and old moldy specimens of fodder collected from wildlife feeding grounds; 17.3% of the birds examined were Listeria positive. In a pheasant and a partridge, septicemia was noticed, whereas two blackbirds, a sparrow, a cnaffinch, a hawk, and another songbird harbored the organism only in the intestinal tract. Among the isolated strains, serovars 1/2b and 4b were found to be predominant ( Table 1). The serovars of 20 of the Listeria strains (Table 3) belonged to antigen combinations not listed on the extended scheme of Seeliger (12). Six of these strains, including two serovars carrying the 0 antigen XV, showed no hemolysis on sheep blood agar. The colonies of the other isolated serovars exhibited varying patterns of hemolysis, i.e., from very pronounced betahemolysis to hemolysis that was hardly visible or even completely lacking. Sometimes hemolysis first became visible in subcultures. Only 37 out of 103 strains tested were virulent for mice, and they were found practically in all areas. Most isolates of serovar 4b, but only two of serovar 1/2b, were virulent (Table 4). (16) indicating that L. monocytogenes has a saprophytic life. The present study confirms that Listeria can be isolated frequently from old, faded, or moldy plants. Although it was recovered during all seasons, there was a slight increase during autumn. In this context it may be mentioned that the isolation of L. monocytogenes from the above sources was independent of the incidence of listeriosis among domestic animals in the same areas. The existence of the various serovars with and without hemolysis does not allow any definitive conclusion at this time as to whether or not certain nonhemolytic bioor serovars are merely saprophytes and perhaps belong to subspecies different from L. monocytogenes. The occurrence of Listeria in wildlife feeding grounds and in the feces of wild animals raises the question whether L. monocytogenes is an inhabitant of the normal intestine and whether there is a cycle between animals and the soilplant environment (14), as suggested by Kampelmacher and van Noorle Jansen (7), who stated that L. monocytogenes might have a cycle similar to that of Salmonella. It is rather difficult to evaluate the role of birds with respect to the spread of Listeria in nature. Birds carry L. monocytogenes in the intestinal tract and are thus obviously able to spread it, as is suggested from its isolation from shrub leaves. One may also speculate whether L. monocytogenes survives the winter in a manner similar to that of insect-spread enterococci (10). It seems unlikely that birds and other wild animals are the essential or only source responsible for the distribution of Listeria in nature. The high incidence of Listeria in plant and soil samples would indicate that, according to the present concept, it is primarily saprophytic. A

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Basis for the Resistance of Several Algae to Microbial Decomposition The basis for the resistance of certain algae to microbial decomposition in natural waters was investigated using Pediastrum duplex, Staurastrum sp., and Fischerella muscicola as test organisms. Enzyme preparations previously found to convert susceptible algae into spheroplasts had no such effect on the resistant species, although glucose and galacturonic acid were released from P. duplex walls. Little protein or lipid but considerable carbohydrate was found in the walls of the refractory organisms, but resistance was not correlated with the presence of a unique sugar monomer. A substance present in Staurastrum sp. walls was characterized as lignin or lignin-like on the basis of its extraction characteristics, infrared spectrum, pyrolysis pattern, and content of an aromatic building block. Sporopollenin was found in P. duplex, and cellulose in Staurastrum sp. Cell walls of the algae were fractionated, and the fractions least susceptible to microbial degradation were the sporopollenin of P. duplex, the polyaromatic component of Staurastrum sp., and two F. muscicola fractions containing several sugar monomers. The sporopollenin content of P. duplex, the content of lignin or a related constituent of Staurastrum sp., and the resistance of the algae to microbial attack increased with age. It is suggested that resistance results from the presence of sporopollenin in P. duplex, a lignin-like material in Staurastrum sp., and possibly heteropolysaccharides in F. muscicola. The basis for the resistance of certain algae to microbial decomposition in natural waters was investigated using Pediastrum duplex, Staurastrum sp., and Fischerella muscicola as test organisms. Enzyme preparations previously found to convert susceptible algae into spheroplasts had no such effect on the resistant species, although glucose and galacturonic acid were released from P. duplex walls. Little protein or lipid but considerable carbohydrate was found in the walls of the refractory organisms, but resistance was not correlated with the presence of a unique sugar monomer. A substance present in Staurastrum sp. walls was characterized as lignin or lignin-like on the basis of its extraction characteristics, infrared spectrum, pyrolysis pattern, and content of an aromatic building block. Sporopollenin was found in P. duplex, and cellulose in Staurastrum sp. Cell walls of the algae were fractionated, and the fractions least susceptible to microbial degradation were the sporopollenin of P. duplex, the polyaromatic component of Staurastrum sp., and two F. muscicola fractions containing several sugar monomers. The sporopollenin content of P. duplex, the content of lignin or a related constituent of Staurastrum sp., and the resistance of the algae to microbial attack increased with age. It is suggested that resistance results from the presence of sporopollenin in P. duplex, a lignin-like material in Staurastrum sp., and possibly heteropolysaccharides in F. muscicola. The cell wall probably is a major determinant of the resistance or susceptibility of algae to microbial decomposition in natural ecosystems inasmuch as it represents a key obstructing structure which must be disrupted, either mechanically or enzymatically, before an attacking organism has access to the contents of the algal protoplast and causes death of the organism. Although considerable work has been done to determine which components of the cell walls of fungi render these organisms refractory to microbial decomposition (3,5), little is known about the components of algal cell walls which protect them from possible attack in nature. In a study of the susceptibility of several algae to microbial degradation in natural waters, it was observed that Staurastrum sp., Fischerella muscicola, and Pediastrum duplex were particularly resistant to attack under conditions where other algae were readily destroyed and their contents liberated (Gunnison and Alexander, Limnol. Oceanogr., in press). The present investigation was initiated to determine which components in the cell walls were respon-I Present address: U. S. Army Corps of Engineers, Waterways Experiment Station, Vicksburg, Miss. 39180. sible for the resistance to microbial degradation. GUNNISON AND ALEXANDER amino acid, lipid, uronic acid, and sugar content of the walls were determined in the same manner as described for susceptible walls (Gunnison and Alexander, Can. J. Microbiol., in press). The walls were fractionated by a procedure modified from that of Boroughs and Bonner (6). A 50-mg sample of walls was treated for 2 h with 20 ml of 0.2% ammonium oxalate at room temperature, and the residue (designated I-1) was removed by centrifugation. The soluble fraction was designated S-1. Fraction I-1 was washed with distilled water and then treated with 20 ml of 0.05 N HCl at 85 C for 4 h. The insoluble residue was removed by centrifugation and discarded, the solubilized material being designated fraction S-2. A separate 100-mg portion of walls was incubated for 4 h at 4 C in 20 ml of 4% NaOH, and the soluble fraction (S-3) was separated from the unextracted material (fraction F-2) by centrifugation. Fraction F-2 was then suspended for 4 h in 17.5% NaOH at 4 C, and the suspension was separated into a soluble (S-4) and an insoluble fraction (I-3) by centrifugation. Fractions S-1, S-2, S-3, S-4, and I-3 are stated to contain pectates, protopectin, polyuronide hemicelluloses, noncellulosic polysaccharides, and a-cellulose, respectively (6). All materials were extracted twice in 20-ml volumes of extraction solution. Each insoluble residue was collected by centrifugation at 31,000 x g and washed once in 20 ml of distilled water. The two extracts and the water used to wash the insoluble residue derived from a given extracting solution were pooled. The extracts were taken to dryness in a rotary evaporator (Rinco Rotavapor). Fraction S-1 was then dissolved in 8.0 ml of distilled water. Prior to concentration, fractions S-2, S-3, and S-4 were neutralized, and after concentration, they were suspended in distilled water, dialyzed against distilled water at 4 C for 12 h, reconcentrated, and then again suspended in distilled water. Fraction I-3 was washed 10 times in distilled water and then lyophilized. For analysis of the fractions, either 10 ml of a soluble fraction or 10 to 15 mg of fraction I-3 was hydrolyzed in a sealed ampoule for 12 h with 2 ml of 6 N HCl at 105 C. After removal of excess HCl at 50 C under vacuum, the hydrolyzate was examined for total carbohydrate, reducing sugars, and sugar monomers, the latter by thin-layer chromatography (Gunnison and Alexander, Can. J. Microbiol., in press). Protein content was assessed after hydrolysis of the fraction for 60 min with 1 N NaOH at room temperature followed by neutralization with HCl; protein was determined by the Folin phenol method (13). A lignin-like material was extracted from Staurastrum sp. walls and from pine wood sawdust by the H2SO4 extraction method of the Technical Association of Pulp and Paper Industries (18). Sporopollenin was prepared from P. duplex by the sequential extraction method of Zetsche and Vicari (23). For infrared analysis, 2 to 4 mg of the material was made into a pellet with 40 mg of KBr, and the spectra were obtained with an infrared spectrophotometer, model IR-10 (Beckman Instruments, Fullerton, Calif.). The quantity of apparent lignin and sporopollenin in the wall preparations was established by determining the residue remaining after the extraction. For pyrolysis-gas liquid chromatographic analysis, 100 Ag of the lignin preparation was applied to a platinum pyrolysis coil; the latter was subsequently fired at 800 C in a model A-25 Pyrolyzer Accessory (Wilkens Instruments, Walnut Creek, Calif.). Separation of the resulting products was accomplished by gas chromatography using a stainless-steel column (3 mm diam by 2 m long) packed with Chromosorb W, 60 to 80 mesh (Johns Mansville, New York), as a solid support for the SE-52 liquid phase (Applied Science Laboratories, State College, Pa.). The products of pyrolysis were carried onto the column with N2 flowing at a rate of 20 ml/min. The column was held at 50 C for the first 6 min after pyrolyzer firing, and it was then subjected to a programmed temperature rise of 8.6 C/min until 185 C was reached. A flame ionization detector was used to monitor the exit of products from the column. Analysis of the fused lignin for phenylcarboxylic acids by thin-layer chromatography was performed on precoated plates of silica gel containing a fluorescent indicator (Eastman Kodak Company, Rochester, N.Y.) using benzene-methanol-acetic acid (90:16:8) as the solvent system (17). The solution of fused lignin was analyzed for catechols by gas chromatography using a modification of the procedure of Helling and Bollag (10) in which 10% DC 200 (Applied Science Laboratories) on Chromosorb W, 80 to 100 mesh (Johns Mansville), was used as the stationary phase. For gas chromatography, the injector, column, and detector temperatures were 260, 230, and 290 C, respectively. The flow rate of the carrier gas, N2, was 60 ml/min. To obtain 14C-labeled walls, the cultures were harvested after 14 days, and the algal cells collected from 8 liters of medium were placed in each of two 2-liter Erlenmeyer flasks. To these flasks was added 1.5 liters of the culture supernatant which had been adjusted to pH 8.3, and 0.5 ml of NaH14CO (New England Nuclear, Boston, Mass.) having an activity of 1 mCi/ml was then introduced. The algal suspensions were incubated on a rotary shaker at 150 rpm at 22 C under light at 5,400-lux intensity, and the labeled cells were harvested after 7 days, washed in 0.01 M phosphate buffer (pH 7.0), and lyophilized. Cell walls and wall fractions were prepared as described above. To determine the resistance of labeled walls and wall fractions to microbial attack, 50 mg of walls, 10 to 90 mg of a wall fraction, 21 mg of lignin, or 18 mg of sporopollenin was suspended in distilled water to a final volume of 10 ml. To each of two 250-ml Erlenmeyer flasks were added 5.0 ml of a suspension of the walls or an isolated fraction, 35 ml of the salts solution, and 0.25 g of soil (Williamson silt loam) contained in 2.5 ml of distilled water. In some instances, 2 ACi of [I4C ]glucose rather than walls or a wall fraction was used as the sole carbon source. The flasks were incubated at 25 C on a shaker operating at 120 strokes/min. Samples were taken at regular intervals, and the loss rate of labeled carbon was determined by a modification of the method of (11). For this purpose, labeled CO2 that remained in the reaction flask at the sampling time was driven off by acidification with 0.5 ml of 2 N HCl, and 0.50-ml amounts of the acidified reaction mixture were placed in scintillation vials with 10 ml of scintillation solution (7). The radioactivity was then determined in a scintillation counter, model Mark II (Nuclear Chicago, Chicago, Ill.). RESULTS Enzymatic digestion of cell walls. The sources of the various enzyme preparations used have already been described (Gunnison and Alexander, Can. J. Microbiol., in press). Chitinase, ,B-glucuronidase, hemicellulase, lipase, lysozyme, Pronase, and lytic enzyme preparations of Streptomyces isolates G4 and G7 had no effect on walls of these three resistant algae. Similarly, cellulase (free of polygalacturonase activity) was without effect on walls of F. muscicola and Staurastrum sp., and the polygalacturonase preparation did not digest F. muscicola walls. On the other hand, carbohydrate was released from the walls of P. duplex by cellulase and polygalacturonase preparations. Most of the solubilized carbohydrate appeared to be glucose, although the Bitter and Muir (4) test showed the presence of some galacturonic acid (Table 1). Essentially all of the wall material solubilized enzymatically could be accounted for as glucose and galacturonic acid. The polygalacturonase preparation had cellulase activity as determined by the formation of glucose from cellulose, but it released no sugar from ,B-1,2 glucan, ,B-1,6 glucan, laminarin, or starch. The polygalacturonase preparation also released small amounts of carbohydrate from Staurastrum sp., but neither reducing sugars nor specific products were found in the hydrolyzates. By contrast, incubation of these two enzyme preparations with intact algae, using the methods previously shown to lead to sphero- plast formation with susceptible species (Gunnison and Alexander, Can. J. Microbiol., in press), did not result in the conversion of Staurastrum sp. or P. duplex cells into spheroplasts. Composition of the wails. The amino acid content of walls of two of the algae is given in Table 2. No amino acid was dominant, and the quantitative composition differed among the two species. It is interesting that the results for the green alga, P. duplex, were quite similar qualitatively and quantitatively to those found in Cylindrospermum sp. (Gunnison and Alexander, Can. J. Microbiol., in press), a bluegreen alga that is decomposed readily by microorganisms. Amino acids were not detected in Staurastrum sp. cell walls, but it is possible that levels below the sensitivity of the ninhydrin method used (15) were present. The protein, carbohydrate, uronic acid, and lipid composition of the cell walls is presented in Table 3. The wall of Staurastrum sp. is dominated by carbohydrate and uronic acids, and no lipids were found. Carbohydrates are also prominent in the walls of F. muscicola and P. duplex, but their abundance is not as great as in Staurastrum sp. The data for carbohydrate and uronic acid content of F. muscicola are anomalous because of the high recoveries, and it is likely that interfering substances affected the analytical determinations. A comparison of the sugar and uronic acid monomers found in acid hydrolyzates of the cell walls is presented in Table 3. Glucose was abundant in the three organisms, but different though F. muscicola and P. duplex contained this sugar. Mannose was present only in two of the organisms. These data provide no insight into why the algae are resistant since a unique composition is not indicated. Composition of wall fractions. The values obtained from chemical analyses of the wall fractions are presented in Table 4. It is evident that only a small percentage of the Straurastrum sp. wall was solubilized by the ammonium oxalate, HC1, and NaOH treatments which were used to obtain fractions S-1, S-2, S-3, and S-4. Fraction I-3, which had residual wall constituents not solubilized by NaOH, contained 78% of the Staurastrum sp. material which was originally subjected to the alkali treatments, and a large portion of this residue was not hydrolyzed by treatment with 2 N HC1. Uronic acids made up a significant part of fractions S-1, S-2, and S-3 of Staurastrum sp., and these may be in the form of pectic substances since the only uronic acid found was galacturonic acid. A lignin-like material was found in fractions I-3 and S-4, the quantity of this substance in the two fractions being equivalent to 5.6 and 0.3%, respectively, of the wall weight. This lignin-like substance was not observed in the other fractions or in the Most of the material subjected to fractionation appeared in either the HCl-soluble S-2 or the NaOH-insoluble I-3 fractions (Table 4). These two fractions were quite similar in the kinds and amounts of materials which they contained, although alkaline hydrolysis of the two fractions followed by the Folin protein determination showed that fractions S-2 and I-3 had 0.2% and 3.1%, respectively, of the wall weight in the form of protein. The major components in hydrolyzates of fractions S-2 and 1-3 were reducing sugars. Alkaline hydrolysis followed by protein determination (13) showed that; fractions S-1, S-3, and S-4 contained an amount of protein equal to 0.2, 0.1, and 0.2%, respectively, of the total wall weight. Fractionation of the walls of P. duplex yielded results similar to those expected in the fractionation of walls of higher plants, for which the method was devised (6). Galacturonic acid was an important component of fractions S-1 and S-2, whereas the only reducing sugar in S-2 was xylose. Because the anthrone test that revealed the abundance of soluble materials in S-2 is primarily a test for carbohydrates, xylose was probably the principle monomer responsible for the 6.7% value. Fractions I-3 and S-4 contained sporopollenin in amounts equal to 2.4 and 1.2% of the wall weight, but none of the other fractions and neither of the other two algae contained sporopollenin. After hydrolysis, fraction S-3 was found to have a reducing sugar component (fucose) in nearly four times the abundance of the uronic acid (galacturonic acid). Fraction S-4 had the monomeric composition which would be expected of a polyuronide hemicellulose, whereas fraction I-3 consisted almost entirely of glucose. Part of fraction I-3 could not by hydrolyzed with acid, a substantial portion of this unhydrolyzed material probably being sporopollenin. Alkaline hydrolysis of the fractions followed by the Folin protein test showed that fractions S-1, S-2, S-3, S-4, and 1-3 of P. duplex contained 0.1, 0.3, 0.2, 0.1, and 0% of the total wall weight as protein, respectively. X-ray diffraction analysis. To provide further evidence for the occurrence of cellulose, X-ray diffraction analysis was performed on dried slide mounts of the I-3 fractions of the algae and of MN cellulose 300 (Brinkman Instruments, Westbury, N.Y.) using the method of Gunnison and Alexander (Can. J. Microbiol., in press). The X-ray patterns are depicted in Fig. 1. Authentic cellulose showed broad peaks with maxima at 15 and 22 degrees 20 and a small deflection having its maximum at 34 degrees 20. 1-3 fractions of F. muscicola and P. duplex gave none of these patterns, thus indicating that cellulose was either not present or was masked by other components in these preparations. The I-3 fraction of Staurastrum sp. exhibited a 22-degree peak in the same region as the cellulose standard, suggesting that this polymer was present. Resistant components of cell walls. Sporopollenin was isolated by the same procedure from the spores of Lycopodium clavatum (obtained from J. Russell, Cornell University), the "classic source of sporopollenin" (8), and from the walls of P. duplex. The infrared spectra of these preparations and of the acetolyzed derivative of P. duplex cell walls prepared by the method of Atkinson et al. (2) are given in Fig. 2. The spectra of the sporopollenins obtained from the walls of P. duplex and from L. clavatum are nearly identical. Almost the same spectrum was obtained using acetolyzed P. duplex walls. Since sporopollenin is the only known polymer of biological origin which can withstand the acetolysis process (8,21) and because the infrared spectrum of P. duplex is the same as that of L. clavatum, the preparation derived from P. duplex is considered to be sporopollenin. Analysis of the walls obtained from a 21-day-old P. duplex culture showed they contained 3.3% sporopollenin. Lignin-like material was obtained from Staurastrum sp. in an amount equal to 5.6% of the wall weight. The pyrolysis-gas chromatograms of lignin from Staurastrum sp. and pine wood meal are shown in Fig. 3. The patterns are nearly identical, except that the Staurastrum sp. preparation yielded a smaller quantity of products per unit of material pyrolyzed and only the algal preparation showed a small peak at about 4 min after initiation of pyrolysis. Infrared spectra of the two lignins were nearly identical except for a small shift of pattern between 1,400 and 1,800 cm-1 and slight differences in the peaks occurring in the region between 800 and 1,400 cm-1 (Fig. 4). The authentic and the algal lignins were subjected to alkaline fusion by a slight modification of the method of Rassow and Zickmann (20). To 2.5 g of KOH and 2.5 ml of distilled water in a nickel crucible were added 10 mg of the preparation and 90 mg of zinc dust. The mixture was heated on a steam bath either for 10 min or until the KOH had dissolved. The mixture was brought to 250 C in a furnace, at which time 5.0 ml of a 50% KOH solution in distilled water and an additional 0.75 g of zinc dust were added. Heating at 250 C was continued for 1.5 h, after which the mixture was cooled to room temperature and dissolved in 40 ml of distilled water. The alkali-fused lignin was acidified to pH 2.5 with 6 N HCl, and the mixture was dried on a rotary evaporator and then dissolved in 5.0 ml of distilled water. Residual KCI was removed by filtration. Using the procedures described above for analysis of the fused lignin, it was observed that protocatechuic acid and the products of alkaline fusion of the authentic and the algal lignins had RF values of 0.39 by thin-layer chromatography and retention times of 39 s by gas chromatography. Thus, the Staurastrum sp. preparation apparently possessed a phenyl structure, a characteristic of lignins (20). Decomposition of labeled walls and wall fractions. The degradation of Staurastrum sp. is presented in Fig. 5. Although the lignin preparation obtained from this alga was subject to microbial decomposition, it was attacked more slowly than the original walls and all of the other fractions. The lignin-like material accounted for less than 30% of S-4; its presence may explain the slower rate of attack on S-4 than the other fractions, but the degree of protection is obviously not great. 1-3 was readily metabolized, possibly the result of its content of cellulose and the lack of influence of the small quantity (less than 4%) of lignin-like material present. Fractions S-1, S-2, and S-3 were rapidly degraded, and less than 25% of the original 14C label remained after 24 days. Since the quantity of '4C left in the fractions after 24 days was directly correlated with their content of apparent lignin, it is possible that the constituents metabolized in the first week are largely wall material could be obtained. The cells were Ys then suspended in 100 ml of buffer. One-half of )osition of labeled Stauthe suspension was used to prepare algal lawns ractions. as described by Gunnison and Alexander (Limpolysaccharides, leaving a refractory lignin-rich substance behind. The decomposition of F. muscicola walls and wall fractions is depicted in Fig. 6. It is evident that S-1, S-3, and S-4 were rapidly degraded, each losing more than 50% of the original "C' label in 24 days. The rates of decomposition of fractions S-2 and I-3 were quite similar, and they were much more resistant to decomposition than the other fractions. The unfractionated wall material was especially refractory, and more than 80% of the original label was still present after 24 days. It is noteworthy that the rates of decomposition of S-2, I-3, and unfractionated walls were quite similar after the first few days, suggesting a possible role for components of S-2 and I-3 in shielding the walls from microbial attack. The rate of metabolism of [14C ]glucose was determined to illustrate the rate of oxidation of a good carbon source for microorganisms. The decomposition of labeled walls and wall fractions of P. duplex is shown in Fig. 7. The sporopollenin component was not attacked at all in the 24-day interval since all of its 14C label was recovered at the end of the test period. Fraction S-4 was more extensively degraded, but 50% of its original label was still present after 24 days. Unfractionated walls were even nol. Oceanogr., in press), whereas the remainder was lyophilized and used to prepare cell walls. The lawns were examined for their susceptibility to microbial attack by the method of Gunnison and Alexander (Limnol. Oceanogr., in press), and the lignin content of Staurastrum sp. walls and the abundance of sporopollenin in P. duplex walls were determined. The data demonstrate a relationship between the lignin content of Staurastrum sp. walls and the decomposability of the alga ( Table 5). The correlation is not linear because the major increase in lignin content occurred between 5 and 12 days, during which time the alga remained moderately susceptible, whereas the lignin level increased only slightly between 12 and 21 days during which period the cell became markedly resistant. The results with the sewage inoculum, in which a decrease in resistance occurred between day 5 and day 12, are anomalous. Studies of the decompositon of P. duplex cells of various ages strongly suggest a relationship between the sporopollenin content of the wall and the decomposability of the alga. Although the 5-day-old cells were highly to moderately susceptible to degradation, 12-day-old cells were quite resistant (Table 5). During the 3-day period, the sporopollenin content of the walls rose from 1.2 to 4.2%. No difference in resistance was observed between the 12-and 21-day-old cells, and the sporopollenin content during this period remained reasonably constant. DISCUSSION Cellulase and polygalacturonase preparations act on Chlamydomonas reinhardtii and Ulothrix fimbrata, two algae that are readily susceptible to attack in natural waters, and the preparations extensively degrade the algal walls so that the cells are converted to spheroplasts (Gunnison and Alexander, Can. J. Microbiol., in press). By contrast, the cellulase and polygalacturonase preparations exhibited low activity against the resistant algae. These data thus correlate with the resistance of the organisms in freshwaters (Gunnison and Alexander, Limnol. Oceanogr., in press). The observation that moderate digestion of P. duplex walls was affected by the two enzyme preparations, as contrasted with the small influence on intact cells, suggests that enzymatic hydrolysis of the surface of this organism is facilitated by the exposure of areas of the wall other than the outer surface. Inasmuch as such exposures probably occur in nature only when the cell is perforated mechanically or when autolysis occurs, the polysaccharidase activity probably is not often important in governing the persistence of the alga. Although the polygalacturonase preparation was not free of cellulase, the release of galacturonic acid from P. duplex walls which were incubated with this preparation suggests that the walls contain a polygalacturonic acid, the latter possibly serving to hold together either fibrils or layers of cellulose. The failure of lysozyme to digest walls of these resistant species is in contrast with the susceptibility of Cylindrospermum, a lysozyme-sensitive blue-green alga quite susceptible to microbial decomposition in freshwaters (12; Gunnison and Alexander, Limnol. Oceanogr., in press). The walls of Staurastrum sp. contain a component which appears to be similar if not identical with lignin, a polyaromatic known for its resistance. The slow decomposition of the purified microbial lignin or lignin-like material, as compared with the rate of degradation of the other fractions or the unfractionated walls, suggests that the polyaromatic may be responsible for the slow rate of decompositon of the alga in nature. The absence of a linear correlation The data strongly suggest but do not show unequivocally that the material isolated from Staurastrum sp. walls is in fact lignin. The limited amount of information on the possible occurrence of lignin in algae is usually interpreted to mean that it is not present (17), although some data exist to the contrary (9). On the other hand, lignin in a group as primitive in the evolutionary sequence as the algae might differ substantially from the polymer characteristic of higher plants. F. muscicola was resistant not only in natural waters but its walls also were totally unaffected by the enzymes used. Nevertheless, three of the cell wall fractions were easily degraded, so that they probably do not contain the substance conferring resistance. Fractions S-2 and I-3 decomposed quite slowly and at approximately the same rate, and hence they may contain the refractory constituent. S-2 and I-3 had approximately the same monomer composition, and it thus seems reasonable to believe that resistance of the walls and of the alga are related to constituents of fractions S-2 and I-3. Although these fractions were metabolized to a slight degree before the rate of decomposition declined to almost that of the original walls, this initial attack may have been at the expense of inaccessible components made available by the fractionation procedure. Because acid hydrolyzates of these two fractions contained several different sugars in similar amounts as well as galacturonic acid, it is tempting to postulate that the resistance is attributable to the presence of a heteropolysaccharide. Sporopollenin but none of the other fractions of the walls of P. duplex was completely refractory to microbial attack. Thus, sporopollenin seems to be responsible for the resistance of the walls and presumably the persistence of the alga. Evidence exists that the sporopollenin is localized in the outermost layers of the wall of Pediastrum (2,16). Silica is also present on the surface of Pediastrum walls (14), and the silica may likewise protect other components of the wall and thus shield the cell contents from microbial attack. In view of the digestion of the walls and the release of glucose by the cellulase preparation, cellulose is apparently present in P. duplex. This polymer may be located in fraction I-3, which yielded large quantities of glucose on acid hydrolysis. The results demonstrate that the biochemical bases for resistance of at least certain algae to microbial attack in natural habitats can be attributed to discrete structural entities. In addition, resistance of the wall to decomposition has an important effect on the rate of mineralization of its several constituents. Why polyaromatics and heteropolysaccharides may contribute to the longevity of the algae has already been discussed (1). Whether these constituents are important to the survival of other species and if different wall components account for the resistance of other algae to microbial decomposition require additonal investigation.

v3-fos

2020-12-10T09:04:20.469Z

1975-08-01T00:00:00.000Z

237234128

s2

Total Rinse Method for Microbiological Sampling of the Internal Cavity of Eviscerated Broiler Carcasses A method is described for spray-rinse sampling the entire visceral cavity of broiler chicken carcasses for microbiological analyses. The method is designed to provide a more comprehensive sample than swabbing or excision of small areas. During a microbiological study of the internal cavity of eviscerated broiler chicken carcasses it became desirable to develop a new method for sampling the total cavity surface. Sampling the internal cavity is inconvenient because of limited accessibility due to the size and location of the eviscerating cut. Also, irregularity of the cavity surface in the backbone area causes difficulty in obtaining representative samples with common methods. Patterson (6) and Baldock (1) have reviewed numerous methods for sampling internal and external poultry carcass surfaces. These include swabbing areas of various sizes and locations with moistened cotton or alginate swabs, excising small quantities of surface tissue which are subsequently blended or shaken, spray rinsing several square-centimeter areas, and rinsing parts or whole carcasses. These methods, with the exception of the total bird rinse, may not provide a representative microbiological assessment because of the limited areas sampled and the known variations of contamination of different parts of a carcass (4). We have devised a spray-rinse method for sampling the total internal cavity of broiler carcasses in order to achieve a more representative analysis of its microbiological condition. MATERIALS AND METHODS Rinse sampling procedure. Most sampling was performed in a commercial poultry processing plant on freshly eviscerated carcasses taken directly from the line immediately after the final wash. A carcass was placed on its back, and excess neck skin was removed by using sterile forceps and scissors. Next, a polystyrene disposable funnel was introduced, stem first, through the opening cut in the abdominal wall, passed through the visceral cavity with the aid of a sterile disposable pipette, and forced into the neck opening so that the stem protruded beyond the opening (Fig. 1). A tight seal was formed between the funnel and neck area muscles so that the funnel served as a rinse collection device when the bird was hung by the hocks on a shackle. The visceral cavity was then spray rinsed with two 50-ml quantities of sterile 1% (wt/vol) sodium citrate which drained through the funnel and was collected in a sterile milk dilution bottle. Spraying was done with a sterile disposable 50-ml syringe equipped with a 14-gauge stainless-steel cannula that was curved to about a 45-degree angle and crimped at the end to give a fan-type spray pattern. During rinsing the spray was directed into all parts of the cavity with as much pressure as could be applied by hand. A fresh syringe and funnel were used for each carcass, and the cannula was dipped in 95% ethanol and flamed before each use. Swab sampling procedure. Carcasses were sliced longitudinally through the breast with an alcoholflamed knife and spread open to avoid contact with visceral cavity surfaces. The entire visceral cavity surface of each carcass was swabbed with three sterile alginate swabs moistened with sterile 1% sodium citrate. Each swab was vigorously rubbed over an area of about 150 cm' according to the "total objective swab (TOS)" method described by Mossel and BUchli (5). All three swabs were deposited in 30 ml of sterile 1% sodium citrate and shaken to dissolve the alginate. Artificial contamination procedure. Recovery efficiency of sampling methods was determined using a nalidixic acid-resistant Salmonella typhimurium strain to artificially contaminate carcasses. On milliliter of a saline suspension containing 107 cells/ml prepared from an 18-h culture was randomly pipetted over the surfaces of each visceral cavity. A 5-minute contact time was allowed before sampling. All experiments using the S. typhimurium marker organism were performed in the laboratory. Cultural methods. Rinse samples were stored in ice for transport to the laboratory. The delay between sampling and plating did not exceed 3 h. Total aerobic plate counts were determined by making appropriate dilutions in sterile 1% (wt/vol) sodium citrate followed by plating in Standard Methods agar (BBL). Plates were incubated 72 h at 20 C. The S. typhimurium artificial contaminant was grown on brain heart infusion agar (Difco) incubated Funnels. The size of the large end of the funnel cone was important relative to achieving the desired location and fit in the carcass neck opening. We found empirically that a cone diameter of 38 mm was suitable for the carcass size range normally encountered in this study. Since disposable funnels of this size were not commercially available, we cut larger funnels to size on a band saw. The funnels were then washed, heated for 5 min in 85 C water, and packaged in sterile plastic bags. RESULTS AND DISCUSSION Recovery of rinse solution. Success of the method depended mainly upon recovery of a high percentage of rinse solution and efficient removal of microbial contaminants. A total of six replications with 30 samples each was performed over a period of several months. Recoveries from a 10-sample segment of a typical experiment are shown in Table 1. The means, standard deviations, and coefficients of variability for all replicates are given in Table 2 Microbial counts. Total aerobic counts from a typical trial are given in Table 1. We selected the unit, microorganisms per carcass visceral cavity, (i) because it was adequate and convenient for our experiments and (ii) because of the difficulty of determining visceral cavity surface area. Comparison of our counts with those obtained by other visceral cavity sampling methods using gravimetric (2) or surface area (8) units is not possible. Although no data are available in the literature relating carcass weight to visceral cavity surface area, the methods described by Goresline and Haugh (3) and Simonsen (7) could be used to obtain this in-formation and thereby permit determination of counts per square centimeter. A comparison of total aerobic plate counts and recovery of an artificially applied S. typhimurium contaminant was made on a count per visceral cavity basis using the mean of 10 samples for both rinse and swab methods. The mean total aerobic plate counts (1.1 x 108/cavity) for the rinse method was 4.2 times greater than that obtained by the swab method (2.6 x 107/cavity). Recovery efficiency of S. typhimurium was 68% by rinsing and only 9% by swabbing. The differences between the mean log,, total aerobic plate counts per cavity obtained by the two methods, as well as the mean log10 S. typhimurium recovered per cavity, were statistically significant at the 5% level of confidence (see Table 3). Thus, the rinse method gives higher total counts and better recovery efficiency of artificial contaminants. Practical experiences. Mention should be made of some experiences encountered using the total internal rinse method. Occasionally a polystyrene funnel would break during insertion, necessitating discard of that carcass if the broken funnel could not be easily removed. Sometimes a loose piece of tissue would temporarily clog the funnel stem, preventing free drainage of rinse fluid. This problem usually could be overcome by inserting a sterile plugged pipette into the funnel cone and blowing out the blockage. An insufficiently tight seal, resulting in leakage outside the collection funnel, occurred in about one of 30 carcasses sampled. We found that with practice about 12 carcasses APPL. MICROBIOL. could be sampled per hour. Also a small area of the neck region was not included in the rinse because of the position of the funnel, but this area was very small relative to the total visceral cavity surface area. We think that our total internal rinse method provides more representative microbiological samples of the highly irregular visceral cavity surface of broiler carcasses than other cavity sampling methods.

v3-fos

2018-04-03T00:16:28.056Z

1975-09-01T00:00:00.000Z

10043337

s2

Metalloprotease from Bacillus thuringiensis. Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization... The nature of proteolytic enzymes of microbial origin is less well known than that of the proteolytic enzymes of animal or plant origin. In recent years, however, the discovery of applied uses for some of the microbial enzymes and their possible relationships to sporulation in bacilli (16) have served to stimulate research in this area. Morihara (12) has classified the microbial proteases into four groups. These are (i) the serine proteases formed by some Streptomyces and by Bacillus subtilis (the subtilisins), (ii) the thiol proteases formed by Clostridium histolyticum and Group A streptococci, (iii) the acid proteases formed by aspergilli and some other fungi, and (iv) the metal chelator-sensitive proteases formed by B. subtilis, Bacillus megaterium, Bacillus thermoproteolyticus, Bacillus polymyxa, and Bacillus cereus (4,6,12). The ability to synthesize serine and/or metal chelator-sensitive protease seems to be widespread among bacteria of the genus Bacillus. Whether or not these enzymes have a specific role in sporulation or some other aspect of secondary metabolism is an open question. One member of the genus, Bacillus thuringiensis, forms a proteinaceous paraspore or crystal at the time of sporulation. The paraspore is responsible for the toxicity of the microorganism for Lepidoptera larvae (15). With the exception of a report dealing with enzymes associated with mature endospore release (9), no information is available about the extracellular hydrolytic enzymes produced by B. thurin-giensis or about the possible relationship of extracellular protease to paraspore formation. In this report we present information about the first part of this question, i.e., the nature of extracellular protease produced by B. thuringiensis. We describe the appearance early in the sporulation sequence of an extracellular, metal chelator-sensitive protease, the conditions for its synthesis, and some of its properties. MATERIALS AND METHODS Organism and cultural conditions.B. thuringiensis var. kurstaki (strain HD-1) was maintained on slants of nutrient sporulation agar (NSM). NSM agar was slightly modified from the formulation of Fortnagel and Freese (5) and contained 23 g of nutrient agar (Difco) per liter, 7 x 10-4 M CaCl2, 5 x 10-5 M MnCl2, and 10-3 M MgCl2. Inocula for growth curve experiments were prepared by a modification of the active culture technique of Nakata and Halvorson (13). This consisisted of overnight static growth in nutrient broth (Difco) at 30 C, followed by the inoculation of 10 ml of nutrient broth in a 125-ml flask with 1.0 ml from the static flask. The 10 ml of broth were shaken (200 rpm, New Brunswick G76 shaker) for 3 h at 32 C, and 1.0 ml was then used to inoculate a second 10 ml of nutrient broth. This transfer was repeated with a third shaken flask and this was used as the source of young cells to make a 10% (vol/vol) inoculation of 50 ml of broth in a 500-ml flask for the growth curves. The 50 ml of broth for all growth curves were shaken at 220 rpm (32 C). Growth was followed by measuring absorbance at 660 nm in a Klett-Summerson colorimeter with 12.5mm cuvettes. 354 on March 21, 2020 by guest http://aem.asm.org/ Downloaded from Formation of heat-stable spores was determined by removing 1.0 ml of broth from a growth curve flask, sonicating the 1.0 ml for 1 min to declump cells, heating at 80 C for 12 min, diluting in water, and plating in nutrient agar. Declumping was carried out by placing 1.0 ml of cells and spores in a sterile plastic tube (12 by 75 mm), floating the tube in water in the chamber of a Ratheon 10-kc sonic oscillator, and treating at full power for 1 min. Culture supernatant from which the B. thuringiensis protease was purified was prepared by inoculating 200 ml of protease production medium in a 2liter flask with 20 ml of B. thuringiensis from the last flasks in an active culture sequence. Protease production medium contained 8 g of nutrient broth per liter, 7 x 10-3 M CaCl2, 5 x 10-M MnCl2, and 10-3 M MgCl2. The 2-liter flasks containing protease production medium were shaken (220 rpm, New Brunswick G25 shaker) for 12 h at 32 C and the cells were removed by centrifugation. Enzyme assays. Protease activity was determined by digestion of azo-albumin. Each reaction mixture was run in duplicate and contained 1.0 ml of 0.5% (wt/vol) azo-albumin which had been prepared in 0.1 M buffer at the appropriate pH (see below), 0.7 ml of water, and 0.3 ml of enzyme preparation. The mixture was incubated 1 h at 30 C, the reaction was stopped by addition of 2.0 ml of 8% trichloroacetic acid, and the precipitate was removed by centrifugation (27,000 x g for 15 min). Two milliliters of the supernatant was added to 2.0 ml of 0.5 N NaOH and the absorbance was measured in a 1.0-cm cuvette at 440 nm in a Hitachi Perkin-Elmer 139 spectrophotometer. The blank was prepared by adding trichloroacetic acid to the substrate before adding enzyme. One unit of protease is that amount of enzyme which produced an adsorbance increase of 0.01 per h under the assay conditions. Specific acitivty is units of protease per milligram of protein. When enzyme activity at various pH's was determined, substrate was prepared in the following 0.1 M buffers: pH 5.8 and 6.1 in 2 (N-morpholino) ethane sulfonic acid; pH 6.5, 7.0, 7.5, and 7.9 in morpholinopropane sulfonic acid (MOPS); pH 8.5 in tris(hydroxymethyl)aminomethane; and pH 9.10 and 9.75 in glycine-NaOH. Amylase was determined by the method of Bernfield (1) using 1% soluble starch (Difco) in 0.02 M potassium phosphate buffer, pH 6.9, as substrate. One unit of amylase liberated 1.0 mg of maltose in 3 min at 25 C. Esterase was determined by the method of Prestidge et al. (14) using benzoylarginine ethyl ester, acetyltyrosine ethyl ester, or p-nitrophenylacetate as substrate. Protein was measured by the method of Lowry et al. (10) using bovine serum albumin as a standard. Enzyme purification. Crude culture supernatant prepared as previously described was concentrated 18.7-fold by ultrafiltration with an Amicon stirred cell (model 52) using a Diaflo ultrafilter UM10 membrane under 55 lb/in2 of nitrogen at 5 C. The concentrated culture supernatant was clarified by centrifugation at 27,000 x g for 20 min and 2.0% (wt/vol) calcium acetate was added. Solid (NH4)2S04 was added slowly, with constant stirring to the concen-trate, and the mixture was allowed to stand 10 min at 4 C before centrifugal recovery of the precipitate at 45, 60, and 65% saturation. The precipitates were dissolved in 0.1 M MOPS-0.1% calcium acetate buffer, pH 7.0, and dialyzed against the same buffer at 4 C for 6 h. Amylase and pigments were removed by adding 10% (wt/vol) pulverized potato starch and 12% (vol/vol) ethanol to the dialyzed (NH4)2504 fraction. The enzyme preparation was stirred for 10 min at 4 C and the starch was removed by vaccum filtration. The procedure was repeated twice. Acrylamide gel disc electrophoresis. The number of proteins present at various stages of purification and the apparent molecular weight of the 92.3 x purified protease were determined (17) by electrophoresis at 24 C in tubes of 7.5% acrylamide-0.1% sodium dodecyl sulfate at pH 7.2 (0.1 M sodium phosphate buffer). Electrophoresis was performed from cathode to anode in a unit supplied by Hoefer Scientific Instruments (San Francisco, Calif.) with a constant current of 4 mA per tube. Samples (250 ILI) were prepared by adding 0.1 volume of 10% sodium dodecyl sulfate in 0.01 M sodium phosphate buffer, pH 7.2, and 0.1 volume of 10% mercaptoethanol to the enzyme. The mixture was boiled 1 min and cooled. Two drops of glycerol and two drops of dilute bromophenol blue were added and the sample was applied to the top of the gels. After electrophoresis for 10 to 12 h, the gels were removed, fixed in 20% sulfosalicylic acid for 8 to 12 h, stained for 14 h with 0.25% Coomassie brilliant blue, and destained with 7.0% acetic acid in a diffusion destainer (Hoefer Scientific Instruments, Inc.). For molecular weight determinations the gels were scanned at 540 nm in a Gilford spectrophotometer equipped with linear transport. The distance from the top of the gel to the peak of absorbance indicated on the paper was measured and plotted against the molecular weight of the protein to produce a standard curve. Standard proteins used and their molecular weights were bovine hemoglobin (16,000), pepsin (35,000), ovalbumin (43,-500), and bovine serum albumin (65,400). The single protein band found upon electrophoresis of the 92.3 x purified protease was proven to be protease by preparing and electrophoresing the sample under nondenaturing conditions. The gel was cut into 5-mm segments, each segment was crushed with a glass rod in the bottom of a culture tube, and 1.0 ml of 0.01 M MOPS-0. 1% calcium acetate (pH 7.2) and 1.0 ml of 5 mg of azo-albumin per ml were added. The segments were incubated 24 h at 30 C and the protease activity was determined as previously described. Sucrose density gradient centrifugation. The molecular weight of the 92.3 x purified protease was determined by centrifugation in the SW50.1 head of a Beckman model L3-50 ultracentrifuge using a 5 to 20% sucrose gradient. Calculation of the molecular weight and assumptions regarding the partial specific volume of the protease are described by Martin and Ames (11). The standard protein was bovine hemoglobin. Five-drop samples were collected by bottom puncture of the centrifuge tube. Hemoglobin was located by absorbance at 405 nm and protease by assay of each fraction. Maltose and soluble starch were obtained from Difco Laboratories, Detroit, Mich. Pulverized potato starch was obtained from Baker and Adamson Chemicals, Morristown, N. J. RESULTS Protease response to pH and inhibitors. B. thuringiensis was grown 12 h in NSM broth and the protease activity in the culture supernatant was assayed at several different pH's. Figure 1 illustrates the activity response to pH. The activity was maximum in the pH 6.5 to 7.5 range and declined rapidly below pH 6.5. The activity was maintained at a higher level in the alkaline range although it had declined markedly by pH 9.1. The loss of activity in the alkaline pH range suggested that most if not all of the activity was due to a metal chelator-sensitive protease (sometimes referred to as a neutral protease). This was tested by the addition of 1.0 mM o-phenanthroline to protease assay mixtures at pH 7.0 and 8.5. The activity was inhibited by 97 and 86% at pH 7.0 and 8.5, respectively (Fig. 1). Ethylenediaminetetraacetic acid (50 mM) inhibited protease activity 89% at pH 7.0 and 1.0 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, was not inhibitory at pH 7.0 or 8.5. Time of appearance of proteolytic activity. Extracellular proteases of bacilli have often been shown to appear at the beginning of the stationary phase of growth (16). Figure 2 reveals that B. thuringiensis produced only 25 U of activity through h 4, the time of completion of exponential growth. From the h 4 to 9, protease activity increased steadily, reaching a maximum of 180 U at h 9. The peak in protease activity occurred within 1 h of the appearance of heat-stable spores. Phase contrast microscopy examination showed little or no cell lysis during the period when protease activity appeared in the medium. Further indication that the protease activity was excreted rather than the result of cell lysis was the absence of esterase activity againstp-nitrophenylacetate in the supernatant. Cell extracts made by sonication contained very active esterase activity-against this substrate. Effect of medium on protease production. NSM broth was used for the experiments described above because preliminary experiments had shown it to allow greater protease production than glucose-yeast extract-salts broth (19), casein-casitone-glucose broth (3), or yeast extract broth (3). The role of the metal salts in NSM broth was investigated by growing B. thuringiensis in shake flasks of nutrient broth containing each of the metals, Mn2+, Mg2+, and Ca2+, individually. The data presented in Fig. 3 show that no more than 25 U of activity was produced in flasks containing nutrient broth alone or nutrient broth plus 10-3 M Mg2+. Although 5 x 10-5 M Mn2+ allowed the greatest production of protease of the single metals, the enzyme activity was lost after h 9. The combination of the three salts (NSM broth) allowed production and maintenance of a high level of activity. The three metals were then tested in pairs with the results given in Fig. 4. Although both the Mn2+-Mg2+ and the Mn2+-Ca2+ combinations allowed production of 110 to 120 U of activity in 11 h, the activity in the flask lacking Ca21 was lost after 11 h. The Mg2+-Ca2+ combination did not support the development of good APPL. MICROBIOL. activity. The addition of Mg2" to the Mn2+-Ca2" combination (NSM broth) allowed production of a high level of activity. The data in Fig. 3 and 4 demonstrated the importance of certain cations to the development of protease activity in culture supernatants. It was unclear if this was an effect upon enzyme synthesis or if it represented activation of a protein apoenzyme that was excreted into the medium in all of the growth situations tested. If the latter were the case, the supernatant of a nutrient broth culture which had little activity in the usual assay might gain activity if cations were added to the assay tube. This possibility was tested by adding Mg2", Mn2", and Ca2" to a nutrient broth supernatant at a final concentration equal to that in NSM broth and then assaying for protease activity. No increase in activity was observed. The Mg2+, Mn2+, and Ca2+ did not seem to function by activating an apoenzyme. Several metal chelator-sensitive proteases have been shown to be zinc-containing enzymes (12). The effect on protease production of the addition of various levels of ZnSO4 to NSM broth is presented in Table 1. The addition of as little as 10-6 M ZnSO4 was inhibitory. Up to 10-4 M Na2SO4 supplementation of NSM broth had no effect. Purification of B. thuringiensis protease. The sequence of steps used to achieve partial purification of the B. thuringiensis metal chelator-sensitive protease is listed in Table 2. Cells were removed from 2.4 liters ofprotease production medium broth and the culture supernatant was concentrated 18.7-fold by passage through a UM10 membrane in an Amicon ultrafiltration cell. About 57% of the total activity was retained and the specific activity was increased 1.4x. After the addition of 2% calcium acetate to increase enzyme stability, the concentrated supernatant was fractionated with (NH4)2SO4. Most of the protease activity was found in the 45 to 60% fraction. This step resulted in a total 58.1 x increase in specific activity in the 45 to 60% fraction. The amylase activity had also increased (7.2 x) in this fraction and brown pigment remained. For the first time more than one band appeared on sodium dodecyl sulfateacrylamide gels of the enzyme (Fig. 5). Both new bands were slower moving than the main band. Treatment with starch removed all of the brown pigment and 95% of the amylase. Both of the slower moving gel bands were removed by this treatment. The specific activity of the protease had been increased 92.3 x. The pH activity profile of the partially purified enzyme was very similar to that found with the crude culture supernatant. The partially purified enzyme was inhibited by ethylenediaminetetraacetic acid and o-phenanthroline to about the same extent as the culture supernatant. The single band found by electrophoresis of the 92.3 x purified enzyme was shown to be protease by elution from a nondenaturing gel and activity upon azo-albumin. Determination of the apparent molecular weight of the protease. A semilogarithmic plot of the molecular weight of reference proteins versus band migration distance is shown in Fig. 6. From this plot the apparent molecular weight of the protease was estimated to be 37,- 500. The molecular weight was also determined by sucrose density gradient centrifugation (11). By this method the apparent molecular weight was estimated to be 40,800. Esterase activity. No esterase activity against benzoylarginine ethyl ester, acetyltyrosine ethyl ester, or p-nitrophenylacetate was detected with either the crude culture supernatant or the 92.3 x partially purified protease. Thermal stability. The partially purified enzyme dissolved in 0.1 M MOPS-0.1% calcium acetate, pH 7.0, was heated for 10 min at the temperatures indicated in Fig. 7. The enzyme was stable for 10 min at 60 C. It retained about 78% of its activity after 10 min at 70 C but only about 8% of its activity after 10 min at 80 C. DISCUSSION B. thuringiensis var. kurstaki grown aerobically in nutrient broth supplemented with Mn2+, Mg2+, and Ca2+ excreted a proteolytic enzyme during the early stationary phase of growth. Since the appearance of enzyme activity in the culture supernatant was not accompanied by cell lysis or by the appearance of intra-cellular esterase activity in the supernatant, the protease was apparently excreted from the intact, sporulating cells. The appearance of extracellular proteolytic activity during the early stages of sporulation resembles the situation reported for B. licheniformis (2) and B. subtilis (14) but is unlike the exponential phase-related synthesis reported in B. polymyxa (6). The enzyme appeared in the culture supernatant when nutrient broth was supplemented with either Mn2" or Ca2" but it appeared at very low levels when nutrient broth was unsup- plemented or when supplemented with Mg2". Almost twice as much protease activity appeared in Mn2+-supplemented nutrient broth as in Ca2+-supplemented broth but the enzyme activity was not stable in the absence of Ca2 . The combinations of Mn2+-Ca2+ or Mn2+-Mg2+ allowed good enzyme production but again, in the absence of Ca2+ supplement, the activity was lost. These results indicate that Mn2+ is important to achieving a high level of enzyme Thermal stability of the partially purified B. thuringiensis protease dissolved in 0.1 M MOPS-0.1% calcium acetate, pH 7.0. The enzyme (03 ml) was heated for 10 min at the indicated temperature, rapidly cooled in an ice bath, and warmed to 30 C before addition of0.7 ml of water and 1.0 ml of azoalbumin to start the reaction. activity and that Ca2l is important to maintaining that activity in the supernatant. Mn2" is known to be important in the synthesis of various secondary metabolites (18) but its site of action is unknown. It did not appear to be involved in activating a protease apoenzyme. Ca2" is known to stabilize some proteolytic enzymes (12) and it seemed to fulfill that role in these experiments. Mg2" did not by itself support good protease development but, when combined with Mn2' and Ca2+, it produced a higher activity than the Mn2+-Ca2+ pair. Although Zn2+ is involved in the activity of several metal chelator-sensitive proteases (12), the addition of Zn2+ to the medium was inhibitory rather than stimulatory to enzyme activity. Apparently there was an adequate level of Zn2+ in the complex medium and excess was inhibitory. The information obtained in this study points to the synthesis of a single, metal chelatorsensitive protease, although the existence of very small amounts of other protease types cannot be completely ruled out. The pH profile of enzyme activity showed a peak at neutrality with little residual activity in the pH 9.5 to 10.0 range where serine protease is most active (7). The enzyme activity was largely but not completely inhibited by chelating agents and the serine protease inhibitor, phenylmethyl sulfenyl fluoride, produced no inhibition at pH 7.0 or 8.5. In view of the very broad pH activity range shown for serine proteases from bacilli (7), their presence should have been detectable by phenylmethyl sulfonyl fluoride inhibition even at pH 7.0 or 8.5. Serine proteases have frequently been shown to have esterase activity (12). The absence of esterase activity in the culture supernatant further argues against the presence of serine protease. Purifications of the protease free of pigment and much of the amylase provided a preparation giving a single band on disc electrophoresis. The apparent molecular weight of 37,500 determined by disc electrophoresis or 40,800 determined by sucrose density gradient centrifugation is close to the values of 35,100 and 40,500 reported by Keay and Wildi (8) for the metal chelator-sensitive proteases of B. subtilis NRRL B3411 and B. subtilis var. amylosacchariticus, respectively. The metal chelator-sensitive proteases appear to be somewhat larger molecules than the serine proteases whose molecular weights fall in the range of 20,000 to 30,000 (7,12). Although metal chelator-sensitive proteases are generally reported to be less stable than serine proteases, the B. thuringiensis partially purified protease at pH 7.0 in 0.1% calcium acetate retained most of its activity after 10 min at 70 C.

v3-fos

2020-12-10T09:04:20.847Z

1975-06-01T00:00:00.000Z

237230005

s2

Extracellular Polysaccharide from the Black Yeast NRRL Y-6272: Improved Methods for Preparing a High-Viscosity, Pigment-Free Product When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol or hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins or synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants, obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality. The black yeast strain NRRL Y-6272 (15) produces an acidic extracellular polysaccharide that contains both N-acetyl-D-glucosamine and N-acetyl-D-glucosaminuronic acid (15,27). This polysaccharide appears to have industrial potential, since it contains both acidic (carboxyl) and masked basic groups (N-acetyl) and since it disperses readily in water to give extremely viscous solutions. When the polysaccharide was isolated at maximum viscosity of the culture after 4 days of incubation by precipitation with either ethanol or hexadecyltrimethylammonium bromide (28), the product was colored gray to black by adhering pigment(s). When the polysaccharide. was isolated after 10 days of incubation, however, all the pigment was sedimented along with the cells when the culture was centrifuged (15). Incubation for 6 days beyond the time of maximum culture viscosity (5,000 to 6,000 centipoises) not only was impractically long, but also resulted in an irreversible decrease in viscosity to 2,000 to 3,000 centipoises. Just as with pullulan (35), pigment can be removed from polysaccharide Y-6272 by the Sevag fractionation technique (32). This technique, however, is impractical because it has to be applied numerous times for complete pigment removal and because yield of pigment-free polysaccharide is low (35) (up to 15% of the polysaccharide is lost with each application). We have found that the pigment contaminating polysaccharide Y-6272 is melanin-like, except for its apparent solubility in water. Reportedly, melanin binds many compounds (2) including chitin (4) and pullulan (35). In seeking a practical method for obtaining not only a pigment-free polysaccharide, but also a product of high viscosity and in an apparent narrow range of molecular weight distribution, we tried to find: (i) methods that separate and remove black pigment from the polysaccharide, (ii) methods that prevent formation of pigment, and (iii) mutants that would produce pigmentfree polysaccharide. Culture maintenance and propagation. Stock cultures of strain Y-6272 were maintained on yeastmalt agar slants (14) which, after being streaked, were held at 25 C for 2 days and then stored at 4 C. Stock cultures were transferred at 1-month intervals. For experimental polysaccharide production, 300-ml Erlenmeyer flasks containing 75 ml of media (plus inoculum) were incubated at 25 C on a rotary shaker (200 strokes/min, 2-inch eccentricity). To develop preinoculum, the stock culture was first transferred to fresh yeast-malt agar slants, incubated for 2 days, and then inoculated into yeast-malt broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 1.0% D-glucose). After 2 days of incubation, this preinoculum (7.5 ml or 10% by volume) was transferred to an inoculum flask containing 75 ml of test medium, and the culture was shaken for 1 day. Then this inoculum (3.75 ml or 5% by volume) was transferred to production flasks containing 75 ml of the test medium. With the native strain, the culture liquor generally reaches maximum viscosity (in media 1 and 3) after shaking for 4 days and is highly cohesive and ropy like egg white. Recovery of polysaccharide Y-6272. Four-day cultures, having high viscosity (4,000 to 6,000 centipoises; Brookfield viscometer, model LVT, spindle no. 4, 30 rpm), were diluted with 3 volumes of water to reduce the viscosity (-175 centipoises) and then centrifuged (35,000 x g, 30 min) to remove cells. By addition of KCl (1%) and ethanol (95%, 2 volumes), the total polysaccharide separated as a stringy precipitate that wound around the stirrer. After this precipitate was dissolved in water, hexadecyltrimethylammonium bromide (1%, Eastman Organic Chemical) (28) was added to remove the major fraction (acidic), again as a tough stringy precipitate that wound around the stirrer; the minor (neutral) fraction remained in solution. The acidic fraction was dissolved in 2 M KCl and, after dilution by addition of 1 volume of water, was reprecipitated by addition of 2 volumes of ethanol. The acidic polysaccharide (in K salt form) was then redissolved in deionized water and dialyzed until free of extraneous salt. The dialyzed solution was adjusted to pH 6.25 with dilute KOH and then freeze dried. RESULTS Characteristics of the soluble pigment. The absorption spectrum of the pigment in cell-free supernatants, which also contain the extracellular polysaccharide, has no unique features; the optical density at 400 nm was used as a measure of the amount of soluble pigment, as has been done for the black yeast Phialophora jeanselmei (11,12). Various physical, chemical, and enzymatic procedures were ineffective for separating pigment from polysaccharide. Physical treatments tried were: dialysis; addition of Celite, diethylaminoethyl-cellulose, or activated carbon followed by centrifugation; precipitation of the polysaccharide with ethanol from neutral, acidic, or basic solutions; precipitation with hexadecyltrimethylammonium bromide; and extraction with several organic solvents. Chemical treatments included the following. Addition of ferric salts, although expected to precipitate melanin (16), formed a waterinsoluble polysaccharide-pigment complex; acidification with HCl precipitated pigment along with polysaccharide; NaOCl not only bleached the pigment to straw color, but also degraded the polysaccharide. Neither Pronase nor papain was effective in freeing polysaccharide from pigment. In its behavior underthese various treatments, the pigment contaminant of polysaccharide Y-6272 appears to be melanin-like (13,16,30) except for its apparent water solubility. Several compounds that were previously reported to inhibit tyrosinase or melanin formation in other organisms (7,23,24), when added to the culture medium of Y-6272, adversely affected either growth of the organism or poly-saccharide biosynthesis. Attempts were also unsuccessful to inhibit pigment formation by complexing suspected phenolic precursors (18,19) with such synthetic polymers as PVP. Factors involved in pigment and polysaccharide formation. A chemically defined medium (no. 2) (11, 12) was selected, and the effect of systematic addition of other ingredients was observed. Growth of Y-6272 on this defined medium was slow; the yeastlike cells were brown; cell-free culture supernatants were straw colored and contained little polysaccharide, as indicated by both the low viscosity of culture liquors and the low yield of isolated material. Adding L-asparagine to the medium caused black pigment to occur in both the cells and cell-free culture supernatants and increased viscosity. Whereas addition of filter (Millipore Corp., 0.22-Am pore size)-sterilized BSA (11,12), with L-asparagine either present or absent, resulted in highly pigmented cell-free culture supernatants of low viscosity, addition of heat-sterilized BSA plus asparagine resulted in moderate viscosity with less pigment than with asparagine alone. Addition of L-asparagine evidently stimulates polysaccharide Y-6272 biosynthesis, and addition of BSA, when autoclaved, diminishes soluble extracellular pigment. The stimulative effect of L-asparagine on polysaccharide production noted with defined medium (no. 2) was an important factor in the development of the more practical medium, no. 3. Among the various media compositions we have tested, medium no. 3 was best for production of polysaccharide Y-6272 from D-glucose at 25 C. The viscosity of cultures grown on medium no. 3 is 5,000 to 6,000 centipoises in contrast to 2,000 to 3,000 centipoises obtained with medium no. 1 (15). When grown on either medium no. 1 or 3, cultures reach maximum viscosity in 4 days with essentially no change in pH (6.8 to 7.2). When shaking was continued to 10 days, cultures on medium no. 3 retained their viscosity and soluble pigment, whereas for those on medium no. 1 viscosity decreased and pigment became insoluble. To prepare both pigment-free and high-quality polysaccharide, further information on the combined roles of L-asparagine and BSA seemed necessary. Effect of BSA on soluble pigment. After being heated under sterilizing conditions, BSA lowers the amount of pigment in cell-free supernatants of Y-6272 grown on medium no. 3, as well as on medium no. 2. The BSA may either be autoclaved with part A of the medium or heat sterilized separately and then added aseptically to the other sterile ingredients. In either case, however, the BSA is more effective when present in the medium at the time of inoculation rather than when autoclaved and added aseptically to the culture 2 or 4 days after inoculation. This effect is shown by optical density measurement of soluble pigment in cell-free supernatants when fermentation is terminated at 4 days (Fig. 1A). Heat-sterilized BSA, whether present at inoculation or added later to the growing culture, does not affect viscosity (Fig. 1B). In contrast to the effect of heat-sterilized BSA, filter-sterilized BSA stimulates formation of soluble pigment (Fig. 1A) and lowers viscosity (Fig. 1B) throughout the course of fermentation. Thus the polysaccharide having the most favorable quality, as compared with the control, is obtained under the conditions of points la and lb in Fig. 1A and 1B, respectively. The optimum concentration of BSA and length of autoclaving (121 C) are 0.2% (wt/vol) and 5 to 15 min, respectively. After culture fluids were centrifuged, the cell pad of cultures of Y-6272 to which heat-sterilized BSA had been added before inoculation had two visible layers: a large one at the bottom, which is a mixture of black yeast cells and insoluble BSA (white chunks), and a small gray-black one on top of the cell layer. This gray-black layer does not contain yeast cells and appears to be a pigment-BSA material. Control samples without added BSA do not have the top gray-black layer. We have found that filtration (Millipore Corp., 0.65-,um pore size) is also effective in reducing the level of pigment in culture supernatants, primarily by removal of the last traces of suspended pigmented cells and debris. Filters of pore size less than 0.45 um remove the polysaccharide and readily become clogged. Filtration of whole-culture broth is not practical, because filters rapidly become clogged with cells. Effect of various proteins and synthetic polyamides on soluble pigment. To test their applicability as agents for removing soluble pigment, proteins other than BSA were added to part A of culture medium no. 3 before autoclaving (15 min) ( Table 1). Of the materials tested, only EA was as effective as BSA, whereas almost all the materials tested actually increased the amounts of pigment in culture supernatants. After autoclaving, BSA and EA became insoluble. The amount of insoluble protein, as observed visually, was related in- versely to the amount of soluble pigment in polysaccharide preparations; i.e., test media with the greatest amounts of insoluble protein gave the lowest amounts of pigment in cell-free supernatants. The optimum length of autoclaving (15 min) for both BSA and EA was established experimentally; however, for the other proteins optimum conditions were not sought. The yield of polysaccharide was not affected by the addition of either BSA or EA; when Promine-D, gelatine, or Plasdone was added, the apparently high yield probably was due to large amounts of contaminating pigment ( Table 1). The synthetic polyamide PVP, when added in an insoluble form (Polyclar AT), remained insoluble in the medium and appeared to be inert. Addition of PVP in a soluble form (Plasdone-C) resulted in low viscosity and high pigmentation of cell-free supernatants ( Table 1). Pink mutant strains. After irradiation of the black parent strain Y-6272 with ultraviolet light, two mutants were selected that are pink initially when streaked on yeast-malt agar slants (14). When these mutants were stored on slants at 4 C for 14 days, mutant Y-6272 pink (colony 10-2) remained pink, whereas Y-6272 brown (colony 10-1) gradually turned brownblack. After growth in medium no. 3, cell-free supernatants of both mutant strains were straw colored, and the extracellular polysaccharide when isolated was colorless. All the pink coloration remains with the cells. To test whether L-aspartic acid, L-glutamic acid, or L-glutamine is a better nitrogen source than L-asparagine for polysaccharide production, the parent and two mutant strains were compared after 4 days of culture growth on medium no. 3 supplemented by each of these amino acids (Fig. 2). The samples fall into two distinct categories: (i) those from both mutants grown on L-asparagine or L-glutamine, additives which apparently provide unfavorable nutritive conditions (Fig. 2, lower right); and (ii) all the others, which fall on the same viscosity-yield line and for which nutritive conditions apparently were more favorable. After isolation, all samples of group (ii) gave nearly the same viscosity on a dry-weight basis and so are similar in nature but differ in yield. The addition of L-asparagine, as compared with the other nitrogen additives tried, doubles the yield of polysaccharide with both the native and mutant strains (Fig. 2); however, the product from the two mutant strains appears to be degraded since the viscosity of these samples (10 and 85 centipoises) is much lower than that from the native strain (6,500 centipoises). Analysis of the isolated polysaccharides by the NaOCl-amylose-KI method (26) for amino sugars, by the phenol-sulfuric acid assay for neutral sugars (9), and by paper chromatography of acid hydrolysates indicates that all three strains produce the N-acetyl-D-glucosamine-N-acetyl-D-glucosaminuronic acid polysaccharide as the major product regardless of nitrogen additive; however, the mutant strains produce more neutral polysaccharide(s) composed mainly of mannose and glucose. DISCUSSION Production of both black pigment and extracellular polysaccharide by the unidentified black yeast NRRL Y-6272 appears to be closely interrelated. The addition of known inhibitors of tyrosinase or melanin formation, culture conditions that preclude black pigment formation by the parent strain, either diminished or eliminated polysaccharide production. Mutant strains induced by ultraviolet irradiation, which contain pink but not black pigment, apparently produce a main (acidic) polysaccharide product that is compositionally the same as that from the black parent strain but varies in viscosity with the nitrogen source in the growth medium. Except for its water solubility, the black pigment appears to be melanin-like, as indicated by its chemical and physical behavior. Failure to remove the pigment by dialysis indicates either that the molecular weight is so large that the pigment cannot pass through the membrane, or the pigment itself is small but interacts with the polysaccharide so strongly that the pigment-polysaccharide will not dialyze out. Removal of pigment from polysaccharide Y-6272 by the Sevag technique (32), generally used to separate noncovalently bound protein from polysaccharides, suggests that contaminating pigment is physically associated and has some characteristics of a protein. The low recovery of polysaccharide Y-6272 with the Sevag technique appears to be due mainly to its loss into the emulsion layer from which a considerable amount of polysaccharide can be removed. Characterization of fractions of this recovered polysaccharide indicates that most of it is equivalent to the main product. Loss of some polysaccharide would be expected if a small percentage of the polysaccharide molecules are firmly complexed with pigment and so are removed by the Sevag technique. Further indication that the pigment has some proteinaceous character is the stimulation of soluble pigment formation by addition of soluble proteins, such as BSA, to culture media. With the black yeast P. jeanselmei (11,12), addition of soluble BSA solubilized the black pigment, normally found in mycelia, by attachment to the BSA molecule. These same studies also showed that the size of the BSA-pigment complex increased with incubation time until it was so large that the complex precipitated from solution. Similar behavior may take place when Y-6272 is grown on medium no. 1 since the soluble pigment seen at 4 days is removed by centrifugation when incubation is extended to 10 days. In this instance, soluble proteins in the medium may act like BSA. Failure to remove pigment from polysaccharide Y-6272 by treatment with Pronase or papain before isolation by alcohol precipitation may indicate either that the pigment is inhibitory to the enzymes or the proteinaceous portion of the pigment has been modified sufficiently to resist enzymatic cleavage. The mechanism of action of BSA and EA is not established by our observations, but apparently autoclaving medium no. 3 for 15 min places BSA and EA in an optimum physical state for acting as an insoluble anchor to which pigment can be attached preferentially. There does not seem to be any correlation between the amount of tyrosine in the various proteins tried and pigment removal since gelatine and Protamine-D, which have no tyrosine (31), stimulate extracellular pigment formation (see Table 1). Proteins, such as BSA, may be involved in the regulation of melanin biosynthesis (20). The correlation between low viscosity and high pigmentation of polysaccharide supernatants is not due to the precipitation of the polysaccharide by the added protein, since we found that pH of culture fluids remains near neutrality (6.8 to 7.2) throughout the 4-day culturing period and that pH must reach pH 4.5 or below for BSA to carry a positive charge and thus be able to precipitate acidic polysaccharide. This behavior is similar to that reported for an a-1,4-linked poly-N-acetylgalactosaminuronic acid (Vi antigen) polymer (33) and is the basis for a turbidimetric assay of the Vi antigen and other acid polysaccharides (8,25). The lowering of viscosity by added protein may be either due to the stimulation of biosynthesis of pigment, which binds the polysaccharide, or to the stimulation of an enzyme, such as the Vi antigen-degrading enzyme (1), which depolymerizes the polysaccharide. The stimulation of polysaccharide Y-6272 production by addition of L-asparagine is not completely understood, especially in view of our finding that L-glutamine, not L-asparagine, is involved in the enzymatic biosynthesis of hexosamine; i.e., L-glutamine, but not L-asparagine, is a substrate in the assay of fructose 6-phosphate amidotransferase (EC 2.6.1.16) in washed-cell homogenates. Two hypotheses have been proposed to account for the effect of L-asparagine: (i) L-asparagine may relate to the biosynthesis of a glycoprotein that is vitally related to polysaccharide synthesis, possibly as a bridge between protein and carbohydrate (17,29); (ii) L-asparagine may induce in the microorganism a morphological change (5) that must take place before polysaccharide production can begin. The inability to classify Y-6272 taxonomically is due mainly to our failure to induce sporulation. By an alternate approach of seeking taxonomic relationship through comparison of composition of polysaccharide products, we isolated extracellular polysaccharide from strains of a number of classified black yeasts. None of these produced extracellular polysaccharide comparable to that of Y-6272. The majority of strains were Aureobasidium pullulans, which produced glucans rather than amino sugar polysaccharides.

v3-fos

2020-12-10T09:04:20.886Z

1975-03-01T00:00:00.000Z

237230039

s2

Bacteriological Survey of Raw Beef Patties Produced at Establishments Under Federal Inspection At the time of manufacture, 76% of 74 sets of raw beef patties collected in 42 federally inspected establishments had aerobic plate counts of 1,000,000 or fewer/g; 84% contained 100 or fewer coliforms/g; 92% contained 100 or fewer Escherichia coli/g; and 85% contained 100 or fewer Staphylococcus aureus/g (geometric means of 10 patties/set). Salmonellae were isolated from only three (0.4%) of 735 beef patties. A survey was conducted to determine the bacterial levels of raw beef patties during preparation and as packaged for shipment from establishments under federal inspection in the United States. Beef patties are fabricated from chilled lean beef cuts (labeled as boneless beef, cow meat, or bull meat) mixed with chilled, higher fat content, beef trimmings. The meats, weighed in proportions not to exceed the fat-to-lean ratio specified by the purchaser, are blended in a chopper or mixer (frozen meats are first passed through a flaker) and then ground. The ground meat is transferred to a patty-forming machine. The patties emerging from the machine are separated by squares of paper and are packed manually into cartons. The product is placed in either frozen or refrigerated storage. Generally, nonfrozen patties are shipped to outlets within 24 h after fabrication. Bacterial growth is unlikely during the production of raw beef patties, because the meat trimmings and cuts are chilled or frozen, the process is rapid, and the production area is maintained at or below 10 C. Measurable contamination from food contact surfaces appeared unlikely because raw trimmings and cuts have a substantial initial bacterial content, and the observed conditions of sanitation in the firms were acceptable. MATERIALS AND METHODS Sampling. From August 1971 to June 1973, samples were collected from 42 federally inspected establishments producing raw beef patties. Nine of the firms were located in the Northeast, 11 were in the South and Southeast, 20 were in the West and Midwest, and two were on the West Coast. Twentytwo establishments froze the patties before packing; 'Statistical Services Staff, Washington, D.C. the other firms froze a portion of the production to maintain a small inventory. A total of 690 production line samples and 735 finished patty units were collected and analyzed. A group of samples from each plant included samples of the beef trimmings and cuts utilized for the patties, samples at each stage of processing, units of the finished patties related to the production line samples, and, from 31 plants, units of finished patties fabricated prior to the date of the plant visit. Nearly always, a group included 10 samples of the meat utilized for the patties and a set of 10 finished patty units per production date. A total of 74 sets of finished patties units was collected. The samples were frozen promptly and were shipped under dry ice to the laboratory. Generally, analysis was begun 3 to 4 weeks after collection. Of the 42 firms, 34 added nothing to the ground meat, two added small amounts of salt and pepper, and six added 12 to 20% seasoned soy protein in water. Laboratory methods. Methods used for aerobic plate counts (APC), coliforms, Escherichia coli, Staphylococcus aureus, and salmonellae have been described (9). Figure 1 shows the distribution of bacteria (APC at 35 C) in the individual samples of trimmings and patties and demonstrates that the grinding and mixing resulted in averaging the highest and lowest count trimmings to yield patties with intermediate bacterial levels. A similar effect was noted for coliforms, E. coli, and S. aureus. RESULTS This study deals with a non-negative random variable (bacterial counts) having distributions skewed to the right. Figure 1 shows that the skewness is much more pronounced in the trimmings than in the patties. Hence, the difference between the arithmetic average and SURKIEWICZ ET AL. the median would be more pronounced in the trimmings than in the patties. Plots of the geometric means of APCs versus the medians of APCs of the trimmings and of the patties (not presented in this paper) show that the geometric means are approximately equal to the medians. Since the geometric mean indicates the location of the median, a plot of the geometric means of the APCs of paired sets of trimmings and patties (Fig. 2) shows that most of the points are above the diagonal line, because the medians of the trimmings and patties are significantly different. Thus, Fig. 2 reflects the difference in the medians of the bacterial distribution in the trimmings and the patties, not a difference in the numbers of bacteria. Figure 3 shows that the geometric means of APCs and the arithmetic averages of APCs of patties. Also, the scattered pattern of the points indicates that the processing of trimmings into patties contributed little, if any, bacteria to the finished product. Collectively, Fig. 1 through 4 refute the belief that the physical process of grinding meat causes an increase in bacterial counts. The incidence of salmonellae was very low. Only one of the 690 production line samples and only three of the 735 beef patty samples were salmonellae positive. APCs of 491 samples of the trimmings were determined at both 35 C (48-h incubation) and 20 C (4-day incubation). Of the 491 samples, 48 (9.8%) had APCs at least 10 times greater at the 20 C incubation temperature. These findings suggest that some of the meat from which the patties were fabricated had been under prolonged refrigerated storage. The bacterial content of the raw beef patties is shown in Table 1. At the time of manufacture and by the laboratory methods employed, 76% of 74 sets of raw beef patties had APCs of 1,000,000 or fewer/g; 84% contained 100 or fewer coliforms/g; 92% contained 100 or fewer E. coli/g; and 85% contained 100 or fewer S. aureus/g (geometric means of 10 patties per set). DISCUSSION Stringer et al. (8) measured the microbial contamination of fresh beef from the time of slaughter to retail display. They reported that the number of bacteria on carcasses before shipment from the packing plant increased as time increased, that the number on carcasses increased during transportation from the packing plant to the retail store, and that an appreciable amount of contamination was transferred to steaks through the cutting procedures at the retail store. A reasonable projection of Stringer's findings is that bacterial counts increase further on the trimmings and cuts accumulated and stored for subsequent grinding. Most beef patty processors use purchased trimmings and cuts; only two of the 42 establishments used beef from animals slaughtered on the premises. Because this survey shows that the bacterial content of patties depends primarily on the bacteriological quality of the trimmings, the examination of patties at plant level measures the accumulative bacterial increase of meat from time of slaughter to packaged trimmings and cuts at time of use, rather than conditions of sanitation during fabrication of patties from the trimmings. The examination of patties collected at retail outlets would measure, in addition, any subsequent deleterious effects of transportation, further handling, and time and temperature of storage. Reports by other investigators indicate that raw ground beef collected at retail levels may have high bacterial levels. Duitschaever et al. (1) found that 64% of 213 ground beef samples had APCs in excess of 107/g and some in excess of 108/g; Kirsch et al. (3) found seven of 20 samples over 107/g; Rogers and McClesky (6) found ground beef in excess of 107/g from 16 of 24 retail markets and, upon examination of 59 samples collected by a local inspector, found 20 in excess of 107/g and 33 over 108/g; Weinzirl and Newton (8) reported that 36% of ground beef samples from 50 retail outlets had APCs over 5 x 106/g. Geer et al. (2) reported that 13 of 20 samples of unfrozen hamburg steak had counts in excess of 107/g, and that frozen storage caused a material reduction in the numbers of bacteria; however, 10 samples stored at 0 F (-17.8 C) for 1 month had counts in excess of 106/g. As aptly put by Mercure (5): "One must realize that meat comes from an animal which was skinned, eviscerated, cooled, then cut into pieces and lastly carried from one establishment to another. These manipulations are made in a non-aseptic milieu. Even in taking the best S. aureus 32 31 9 1 1 a All sets but one consisted of 10 patties. VOL. 29,1975 SURKIEW hygienic precautions, the presence of numerous microorganisms on the surface of the meat is unavoidable. Since ground meat is, more often than not, made with trimmings, and since it offers a wider surface to contamination, one can conclude that the microbial flora of that product would be rather high." (Translated from French) We agree with Mercure, but we also believe, as suggested by Duitschaever (1) and Rogers (6), that the bacteriological quality of ground meat can be improved with improved practices in the handling and storage of meat from the time of slaughter to the consumer.

v3-fos

2020-12-10T09:04:20.627Z

1975-06-01T00:00:00.000Z

237233119

s2

Microflora and Invert Sugars in Juice from Healthy Tissue of Stored Sugarbeets Bacterial populations increased in juice of healthy tissue of sugarbeet roots stored at 5 C. Average counts showed a sixfold increase after 150 days of storage. Invert sugar levels increased over threefold in “American 4 Hybrid A” and remained fairly constant in “Mono-Hy D-2.” The former cultivar also had significantly higher bacterial colony counts than the latter before 90 days of storage. Of 36 isolates identified, 16 were Pseudomonas spp. including P. chlororaphis; 6 Bacillus spp. including B. subtilis; 5 Arthrobacter spp. including A. globiformis; 4 yeasts; 2 Erwinia spp.; 2 Flavobacterium spp. including F. aquatile; and Streptomyces longisporus. Isolates of all genera except S. longisporus were able to hydrolyze sucrose in vitro. The survival of bacteria in healthy storage tissue of potato, red beet, turnip, carrot, and kohlrabi has been established (4). MacDonald et al. (3) aseptically excised sugarbeet root tissue, and plate counts of tissue homogenates averaged 104 bacteria/g of tissue before incubation. Counts increased to 108 to 109/g after 4 days of incubation in aerated washing at room temperature. Sugarbeets are harvested and stored outdoors for 60 to 120 days in regions where freezing temperatures could prevent winter harvest. Extractable sucrose decreases during this storage period. The metabolism of microbial inhabitants, particularly the production of invertase, might contribute to this decrease in sucrose content and juice quality. The objectives of these studies were to (i) measure bacterial populations of healthy sugarbeet roots during storage at 5 C, (ii) identify the most prevalent microorganisms, and (iii) study the relationship of bacterial populations with invert sugar levels. MATERIALS AND METHODS Bacterial populations were measured in two cultivars, American 4 Hybrid A (4A) and Mono-Hy D2 (D2). Roots were stored at 5 C in perforated plastic bags (45 by 80 cm). Humidity in the storeroom was 90 to 95%. Cores 18 mm in diameter were removed with a cork borer from each of 50 roots for each cultivar. The same roots were sampled at 30-day intervals during a 150-day storage period. The epidermal ends of the cores were removed (2 to 5 mm), and the cores were trimmed to 10 g. Each core was disinfected by suspension in 1% sodium hypochlorite for 15 s and then rinsed once in sterile distilled water. Juice was extracted from each core with a vegetable juicer fitted with a paper filter. The juicer was flushed with 70% ethanol after each extraction. Sterile water was used instead of a core after 25 extractions to determine the effectiveness of the 70% ethanol wash. Filters were replaced after three extractions. Juice samples were diluted and plated in triplicate by the pour plate technique with nutrient agar (Difco) as the medium. Incubation was at 26 C for 4 to 5 days. Identification was based on Bergey (2). Invert sugar levels in the extracted juice were measured by the 3,5-dinitrosalicylic acid reagent (1). RESULTS AND DISCUSSION During this experiment, 42% of sterile water checks through the juicer remained sterile. The average colony count was 1.4/plate on the remaining checks. This count was considered tolerable because of the amount of plant material sampled and the number of plates poured at each sample date. Colony counts of the checks were not used to adjust test counts. The bacterial counts at harvest were 0.72 x 105/ml of juice for 4A and 0.5 x 105/ml for D2. This difference was statistically significant (Fig. 1). The bacterial numbers increased up to a maximum between 90 and 120 days of storage. Maximum average counts were 3.7 x 105/ml of juice for D2 and 4.5 x 105/ml for 4A. The cultivar 4A had significantly more bacteria per milliliter of raw juice than D2 up to 60 days storage. Thereafter, 4A continued higher than D2, but considerable variability in both cultivars removed statistical significance. The invert sugar levels varied less than the bacterial counts. Cultivar 4A had significantly more invert sugar than D2 at harvest and throughout storage (Fig. 2). The invert levels in 4A increased sharply after 90 days, whereas those in D2 remained constant. The higher bacterial count in 4A, plus the higher invest level, suggests that the microflora could contribute to a storage loss of sucrose by inversion and use. Further, it is not known why 4A supported a greater population of bacteria than D2. The sucrose contents of both cultivars was 11.2% after storage for 150 days. Roots of both cultivars used in these experiments were produced in the same field plot. The following genera (with numbers of cultures) were represented in the 36 colonies that were isolated and purified: Pseudomonas, 16; Bacillus, 6; Arthrobacteri, 5; Erwinia, 2; Flavobacterium, 2; and Streptomyces, 1. Of special interest because of the potential economic importance was the ability of 20 of the 36 cultures to hydrolyze sucrose in vitro. All genera except S. longisporus showed this ability. Experiments are needed to determine if microbial hydrolysis of sucrose in vivo contributes toward sucrose loss and invert sugar accumulation.

v3-fos

2018-04-03T03:39:50.514Z

1975-07-01T00:00:00.000Z

7399027

s2

Incidence of Listeria monocytogenes in Nature During a research project on the occurrence of Listeria monocytogenes 194 strains were isolated in southern West Germany during the years 1972 to 1974: 154 from soil and plant samples (20.3%), 16 from feces of deer and stag (15.7%), 9 from old moldy fodder and wildlife feeding grounds (27.2%), and 8 from birds (17.3%). The highest number of isolates was obtained from uncultivated fields. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the. first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the. first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources. In spite of the growing literature on Listeria monocytogenes the question whether this organism is primarily soil born or originates from animals excreting L. monocytogenes with feces (3,4,6,8,13) remains unresolved. Therefore, this study has focused on the occurrence of L. monocytogenes in plants, soil, and feces of wild animals and birds. MATERIALS AND METHODS Samples of plants and soil from various parts of southern West Germany to be tested for L. monocytogenes were collected from: (i) cultivated fields (maize, wheat, oats, barley, potatoes, etc. and pastures and meadows), (ii) uncultivated fields (fields and meadows that had lain fallow for years); (iii) forests, wildlife feeding grounds, and mud from creeks, rivers, and ponds); (iv) feces and residues of fodder from wildlife feeding grounds. Each sample of plant or fodder specimen from wildlife feeding grounds was collected with sterile rubber or disposable plastic gloves or with sterile scissors and was packed in plastic bags. The soil samples were obtained from the surface of the ground, as well as from a depth of 10 cm, using small sterile shovels. It was taken into consideration that soil samples from the surface are regularly contaminated with plant residues and that contamination could not be avoided when soil samples were collected from the depth. Shredded plants, soil samples (15 to 20 g), or feces (1 to 10 g) were suspended in 50 to 400 ml of enrichment broth and grown in 500-ml glass bottles. The following enrichment media were employed: (i) potassium thiocyanate medium (according to Lehnert [19]) and potassium thiocyanate medium substituted with 10 ug/ml of Acriflavin (Serva) per ml (K. Hbhne, Ph.D. thesis, Justus-Liebig University, Giessen, West Germany, 1972); (ii) tryptose broth (Difco); and (iii) brain heart infusion broth (Difco). For medium (i) the seeded containers were maintained at 22 C for 7 days. After 2 and 7 days, samples were plated on four tryptose agar plates (Difco) with 40 #g of nalidixic acid (Serva, Heidelberg; subsequently called TN medium) added per ml. This TN medium was incubated at 37 C for 24 h and for an additional 24 h at 22 C. Then the growth was checked for typical colonies with a microscope by use of the Henry illumination technique (1,2). Using enrichment media (ii) and (iii), cultures were maintained for 12 months at 4 C. After 1, 3, 5, and 12 months, one loopful of the material was plated on four TN agar plates. In addition, a sample from each culture was transferred into Stuart's liquid with the aid of an alginate tampon and incubated at 22 C for 1 week. Subsequently, this tampon was suspended in 10 ml of Ringer solution and one loopful was plated on four TN agar plates (5). Additionally, brain heat infusion (5.0 ml) was inoculated with 0.1 ml of material from the brain heart infusion enrichment broth, incubated at 22 C for 2 weeks in the dark, and streaked on four TN agar plates (15). All colonies suspected of being Listeria were examined for catalase activity and motility and tested further as described by Seeliger (11). The virulence of the isolated Listeria strains was tested in an outbred strain of mice (NMRI/Han) weighing 18 to 20 g. Four mice each were injected with 0.5 ml of an 18-h glucose broth culture intraperitoneally. A strain was considered to be virulent if the mice died within 3 weeks and Listeria could be isolated from the organs at necropsy. The determination of serovars was done according to the procedures outlined by Seeliger (11). RESULTS Cultivation in the enrichment broth of Lehnert (9) was found to be an excellent method for 30 WEIS AND SEELIGER the isolation of Listeria. The same medium substituted with acriflavin yielded even better results, but the time of its use was too short for a definite evaluation. L. monocytogenes occurs in a high proportion of plants, soil samples, and feces of deer and stags as well as of birds ( Table 1). The highest positive results were obtained from the surface of soil specimens and plants, particularly in fields that had lain fallow for years and were overgrown with grass and small shrubs ( Table 2). Only a few strains of L. monocytogenes could APPL. MIcaomOL be isolated from the depth of soil from uncultivated fields. Faded and decayed grass was a direct indicator of the presence of Listeria. The same Listeria serovar was found repeatedly at the same place at half-year intervals. In some instances two different serovars were detected in the same sample. Positive findings were irregular at the same site. From a meadow near Kaiserstuhl that had not been used as pasture land for some years two strains were isolated in the autumn of 1971, none in the spmmer of I I I I 1 2 a New antigen combinations, not yet designated (see Table 3). 1972, and again two in the autumn of 1972. Somewhat unexpected was the isolation of L. monocytogenes from the leaves of shrubs 50 cm above ground, in addition to those on the ground. L. monocytogenes could be isolated from samples of mud in relatively great numbers. It appears to survive and to multiply particularly well in a moist environment. The lowest number of positive results was obtained from fields and meadows used for agricultural purposes. An entirely different distribution of L. monocytogenes was noted in the upper Black Forest, where it was isolated exclusively from the surface of the soil of wildlife feeding grounds. On the other hand, the incidence of Listeria in the deciduous forests of the foothills and in the valleys, as well as in the plain, was rather scattered. The low incidence of L. monocytogenes in upper Black Forest was associated with low pH values of the local soil (sometimes below 3.5). A high percentage of samples collected in a small forest north of Salem, near the Lake of Constance, yielded Listeria. This forest is surrounded by cultivated fields, and the pH value of the soil samples was found to be between 4.8 to 7.6. In this area Listeria was cultured from 22 out of 46 specimens in a single wildlife feeding ground, in its vicinity as well as at a distance of 200 m. A great number of Listeria isolates were obtained from feces and old moldy specimens of fodder collected from wildlife feeding grounds; 17.3% of the birds examined were Listeria positive. In a pheasant and a partridge, septicemia was noticed, whereas two blackbirds, a sparrow, a cnaffinch, a hawk, and another songbird harbored the organism only in the intestinal tract. Among the isolated strains, serovars 1/2b and 4b were found to be predominant ( Table 1). The serovars of 20 of the Listeria strains (Table 3) belonged to antigen combinations not listed on the extended scheme of Seeliger (12). Six of these strains, including two serovars carrying the 0 antigen XV, showed no hemolysis on sheep blood agar. The colonies of the other isolated serovars exhibited varying patterns of hemolysis, i.e., from very pronounced betahemolysis to hemolysis that was hardly visible or even completely lacking. Sometimes hemolysis first became visible in subcultures. Only 37 out of 103 strains tested were virulent for mice, and they were found practically in all areas. Most isolates of serovar 4b, but only two of serovar 1/2b, were virulent (Table 4). (16) indicating that L. monocytogenes has a saprophytic life. The present study confirms that Listeria can be isolated frequently from old, faded, or moldy plants. Although it was recovered during all seasons, there was a slight increase during autumn. In this context it may be mentioned that the isolation of L. monocytogenes from the above sources was independent of the incidence of listeriosis among domestic animals in the same areas. The existence of the various serovars with and without hemolysis does not allow any definitive conclusion at this time as to whether or not certain nonhemolytic bioor serovars are merely saprophytes and perhaps belong to subspecies different from L. monocytogenes. The occurrence of Listeria in wildlife feeding grounds and in the feces of wild animals raises the question whether L. monocytogenes is an inhabitant of the normal intestine and whether there is a cycle between animals and the soilplant environment (14), as suggested by Kampelmacher and van Noorle Jansen (7), who stated that L. monocytogenes might have a cycle similar to that of Salmonella. It is rather difficult to evaluate the role of birds with respect to the spread of Listeria in nature. Birds carry L. monocytogenes in the intestinal tract and are thus obviously able to spread it, as is suggested from its isolation from shrub leaves. One may also speculate whether L. monocytogenes survives the winter in a manner similar to that of insect-spread enterococci (10). It seems unlikely that birds and other wild animals are the essential or only source responsible for the distribution of Listeria in nature. The high incidence of Listeria in plant and soil samples would indicate that, according to the present concept, it is primarily saprophytic. A on March 18, 2020 by guest http://aem.asm.org/ Table 3). "Number of strains. subdivision of L. monocytogenes in a hemolytic and a nonhemolytic nonvirulent subspecies may perhaps help to clarify in the future the still obscure pattern of distribution. Although many questions remain unresolved, at this time the following tentative conclusions are drawn. (i) L. monocytogenes and a nonvirulent variety apparently exist as a saprophytic organism in soil and on plants. (ii) L. monocytogenes can also be present as an intestinal inhabitant of animals and thus can be spread to the environment. (iii) Humans and animals are exposed to this organism frequently in the natural environment.

v3-fos

2020-12-10T09:04:20.442Z

1975-03-01T00:00:00.000Z

237231251

s2

Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C1-C7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO4 as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 μmol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of taxonomic significance. microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 ,mol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of taxonomic significance. An important feature in the identification of anaerobic bacteria is an analysis of the acid fermentation products formed in culture media (9). These volatile (C1-C,) and nonvolatile (lactic, fumaric, and succinic) acids have been analyzed by various gas-liquid chromatographic methods. Problems arising from direct chromatography of the volatile components include free acids interacting with metal columns and adsorbing to polar supports and stationary phases, resulting in poor quantitation due to sample loss, peak tailing, and ghosting (10,13). Moreover, many stationary phases have low thermal stabilities and tend to desorb ("bleed") from supports with repeated use. In a report by Lambert and Moss (11) a procedure was described for the preparation and analysis of the butyl esters of short-chain volatile and nonvolatile acids on a Chromosorb-W support coated with a new, highly stable stationary phase, Dexsil 300 GC. This method is useful since it eliminates those problems associated with direct analysis of free acids, and both volatile and nonvolatile acids can be separated on a single column. The procedure described by Lambert and Moss, however, includes solvent extraction and evaporation steps which could lead to considerable loss of free acids from samples (5,8). In this publication we report a modified and simplified Lambert and Moss method for butylation of the sodium salts of carboxylic acids which provides for chromatography of multiple samples of fermentation media. When acids were esterified from samples and compared with authentic butyl esters, the apparent recoveries were 80 to 90% for most acids. Furthermore, in these studies the addition of an internal standard (heptanoic acid) to samples facilitated quantitation of the volatile and nonvolatile acids. MATERIALS AND METHODS Gas chromatograph and auxiliary equipment. The gas chromatograph used was a Hewlett-Packard model 5754B equipped with a hydrogen flame ionization detector. Additional components used with this instrument were also Hewlett-Packard equipment and consisted of the following: (i) an autosampler (model 7670A) with operating controls and injection units set for single analysis per sample and minimum wash cycle; (ii) a strip-chart recorder (model 7123A) of 100 mV full-scale deflection set for a chart speed of 0.5 inch (1.3 cm)/min, and (iii) a digital integrator (model 3373B) set for minimum input sensitivity (with a resolution of 1 jsV/s) and capable of printing retention time (midpoint of the peak area). Column and chromatographic conditions. Fatty (7). The coated packing was loaded into columns by gentle tapping with a vibrator. Silanized glass wool was used to plug the column ends. The column was conditioned prior to use by heating to 250 C for 24 h and under a flow of nitrogen gas. Sample application was made by on-column injection. The following additional chromatographic conditions were employed throughout this study: (i) gas flow rates of 84, 260, and 50 ml/min for hydrogen, air, and nitrogen, respectively; (ii) injection port temperature, 230 C; (iii) detector temperature, 270 C; and (iv) temperature program, initial column bath was set at 50 C for 3 min followed by 10 C/min temperature increase to 250 C. The total time for chromatographic separation and integration of each sample was 24 min. A 5-min column bath cooling period followed after the last ester (dibutyl fumarate) was detected and integrated. Sample preparation, butylation procedure, and recovery of esters. A standard mixture containing 40 gmol each of the following acids per ml was prepared in distilled water, and the pH was adjusted to 9 to 10 with 10 N NaOH: formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, caproic, heptanoic, lactic, fumaric, and succinic. Dilutions of the standard mixture were made to obtain additional samples containing 5, 10, 15, 20, and 30 Mmolml. (All samples were made alkaline to convert the free acids to the ionized species and thus prevent their loss during subsequent lyophilization.) Samples (1 ml) of each fatty acid mixture (or individual fatty acids) were placed in culture tubes (13 by 100 mm) (Kimble Products), frozen in an alcohol-dry ice bath, and dried overnight on the shelf unit of a continuous freeze dryer (New Brunswick, model V-13). To the dry salts of the acids were added 0.8 ml of chloroform (or hexane) and 0.2 ml of 1-butanol saturated with anhydrous HCl. (Butanol was saturated with anhydrous HC1 by bubbling until a pH of 1 or less was achieved. The butanol-HCl mixture will remain saturated in a stoppered bottle for about 1 month.) Butanol (0.2 ml)-HSO. (0.05 ml) and 0.2 ml of boron trifluoridebutanol (14% wt/vol; Applied Science) were also compared as butylating agents. After mixing on a Vortex spinner, the tubes were tightly capped (Teflon-lined, screw cap), and the mixture was heated at 80 C in a temperature block (Lab-Line Instruments, Melrose Park, Ill.) for 2 h. Tubes were then cooled to room temperature and 0.2 ml of trifluoroacetic anhydride (TFA; Sigma Chemical Co.) was added to each; the solution was mixed and allowed to react for 1 h. The TFA was used to (i) react with: hydroxy acids (e.g., lactic) forming the trifluoroacetyl esters and (ii) react with any excess butanol in the reaction mixture. Samples were then washed twice with 1-ml aliquots of distilled water to remove excess TFA reagent, and the water layer was discarded. The chloroform layer (adjusted to 1 ml) which contained the butyl esters was placed into 1-ml vials with Teflon-lined aluminum seals (Wheaton Glass Co., Millville, N.J.). Two microliters of sample was injected and analyzed by gas chromatography. Cultures of rumen anaerobic bacteria were centrifuged to remove cells, and the supernatant (1 ml) was treated in the same way as the standard acid mixture. Authentic butyl esters of the various acids were made up in chloroform (5 to 40 umol/ml concentration) and analyzed either singly or as a mixture. Internal standardization and calculations. Calibration with an internal standard was performed by the addition of a known amount of heptanoic acid to standard mixtures of fatty acids or fermentation media samples before freeze drying and esterification. The formula used for calculating the quantity (micromoles per milliliter) of a sample unknown, containing an internal standard, is given by: (A.) (C,)/(RRF)-(A,), where A. and A, are areas of the unknown component in a sample and internal standard, respectively; C, is the concentration of internal standard in micromoles per milliliter; and RRF (relative response factor) is the ratio of AC,-AC,, where A, and C, are the area and concentration of prepared fatty acid butyl esters prepared from a standard calibration mixture containing 5 to 40 gmol each of C1-C7, lactic, fumaric, and succinic acids per ml. The percent recoveries of prepared butyl esters were calculated by comparing peak areas (in microvolts per second integrator units) with those of authentic esters. Mean peak areas and standard deviation of samples run in triplicate were also determined for each butyl ester. Monocarboxylic (C1-C7, iso C,, iso C., and lactic) and dicarboxylic (fumaric and succinic) acids were obtained as the free acid from Eastman Kodak Co. Cultures. Rumen anaerobic bacterial strains used in this study were obtained from M. P. Bryant (University of Illinois). Bacteroides ruminicola 23, Bacteroides melaninogenicus ATCC 25845, Butyrivibrio fibrisolvens D1, Megasphaera elsdenii B159, Ruminococcus albus 7, Eubacterium ruminantium GA-195, and Lactobacillus vitulinus T-185 were grown for 7 days at 37 C in a rumen fluid-glucose VOL. 29, 1975 SALANITRO AND MUIRHEAD medium (4) under a 10% C02-90% N2 gas phase. Cultures were centrifuged to remove cells, and the fermentation medium was processed as described above for preparation and recovery of butyl esters. Uninoculated media samples served as controls. RESULTS AND DISCUSSION Effects of butylating agents on esterification of acids. Data on esterification and apparent recovery of various volatile and nonvolatile acids using different butylating agents are shown in Table 1. With butanol-HCl, recovery of C l-C, and lactic acids (relative to authentic butyl esters) varied from 80 to 95% (see also Table 2); recoveries for dibutyl succinate and dibutyl fumarate, however, were 78 and 68%, respectively. Using butanol-H 2SO as the butylating agent, recoveries of acids were comparable to those with butanol-HCl, except that only 30% of the formic acid was recovered as the butyl ester. The low recovery of butyl formate with butanol-H2SO4 may be due to oxidation of the formic acid to CO2 and water by the concentrated sulfuric acid. In contrast, esterification of similar acid mixtures with BF,- butanol resulted in butyl ester recoveries varying from 25 to 95%; poorest recoveries were obtained for succinic and fumaric acids. Several aspects of the butylation reaction with butanol-HCl or butanol-HSO have been considered. Fatty acids can be reacted and extracted in hexane as well as in chloroform with no appreciable difference in recovery or alteration of the chromatographic profile. Diethyl ether, however, is not a desirable reaction solvent because of its high volatility (boiling point, 35 C). Maximum butylation of fatty acid mixtures at concentrations of 5 to 40 umol/ml each was achieved (particularly for formic and acetic acids) when the reaction was carried out for 2 h at 80 C. We have observed that recoveries of formic, acetic, and fumaric butyl esters were at least 5 to 10% lower when the butylation reaction was carried out for 30 to 60 min. Extending the heat step reaction beyond 3 h resulted in reduced recoveries (30 to 40% less) of the C1-C7 acids. It is necessary to allow the mixture to react with TFA to remove excess butanol, since butanol adsorbs tenaciously to the Dexsil column and interferes with elution of the butyl esters. TFA also reacts rapidly with the hydroxyl group of lactic acid forming the trifluoroacetyl ester of butyl lactate. In this respect, TFA esterification of lactic acid, presumably as the TFA and butyl derivative, was 10 to 15% (based on recovery) lower when the TFA was allowed to react for 15 to 30 min. However, authentic trifluoroacetyl butyl lactate (synthesized from TFA and butyl lactate, and structure confirmed by nuclear magnetic resonance spectroscopy and elemental analysis) co-chromatographs with butyl lactate under the conditions used in this study. In addition to having the same retention time, butyl lactate and trifluoroacetyl butyl lactate have similar response factors. These esters, therefore, were chromatographically indistinguishable. It is also likely that a mixture of butyl lactate and trifluoroacetyl butyl lactate is formed during the esterification reactions. For purposes of lactate quantitation, however, it is not necessary to know which derivative is formed, since the response factors for these esters are similar. Although pyridine has been used as a catalyst to enhance trifluoroacetylation of hydroxyl groups on acids (2), our studies indicate that pyridine is not necessary for this reaction. In fact, addition of pyridine (10 to 50 ,ul) to the reaction mixture reduced recoveries of butyl lactate (15 to 70% less) and most other butylated short-chain acids (10 to 30% less), depending upon the butylating agent used. It is common practice to extract the watersoluble, short-chain, volatile and nonvolatile acids into organic solvents (diethyl ether or chloroform) from acidified aqueous media. However, poor solvent extraction and recovery of volatile (e.g., formic and acetic) and nonvolatile (lactic and succinic) acids is a frequently reported observation (6,8). Hankinson et al. (8) noted that only continuous shaking of aqueous samples with a mixture of diethyl ether and petroleum ether resulted in recovery of 57% of the formic acid and 80 to 100% of the other volatile acids present in milk. Doelle and Manderson (6) determined that it was necessary to extract these acids from acidified microbial fermentation media for 2 to 3 h with diethyl ether to recover 90 to 100% of the volatile acids. In this latter study, losses of 10 to 20% occurred due to evaporation of the mixture, whereas distillation resulted in losses of 80, 60, and 20% for acetic, propionic, and isobutyric acids, respectively. Our experience with rapid solvent extraction procedures of acids from aqueous media confirms observations of others (1); only 10 to 30% of the formic and acetic acids and 60 to 80% of the C,-C. acids and lactic, succinic, and fumaric acids are recovered with diethyl ether or chloroform. Neither the use of hot solvents nor saturation of samples with salts significantly improves the extraction and recovery of most acids. If the extracted sample is evaporated to a smaller volume (e.g., 0.1 to 0.2 ml), we lose approximately 60% of the formic acid and 20 to 30% of the other volatile acids. Our method of lyophilizing media yields a greater recovery of fatty acids and, therefore, has an advantage over extractions and distillation techniques. Chromatographic profiles and calibration curves of acid butyl esters. Typical gas chromatographic separations of a mixture of authentic butyl esters and those prepared from the salts of acids are shown in Fig. 1. The chromatographic profile of authentic and prepared esters of volatile (C -C7) and nonvolatile (lactic, succinic, and fumaric) acids are superimposable, and individual acids were baseline separated on the Dexsil-Chromosorb W column (Fig. la and c). Only lactic and isovaleric did not separate as well, and thus integration and quantitation of the lactic and isovaleric acid peaks were affected. The butyl ester of 2-methyl butyric acid could not be separated from that of isovaleric acid. Retention times of the prepared esters are given in Table 2. A comparison of elution times between authentic and prepared butyl esters indicated they were highly reproducible and usually did not vary by more than 0.5 to 1.0% at the concentrations of acid tested. The profile of acids shown in Fig. 1 has been maintained for the last 6 months on the same preparation of column material (over 1,000 injections), demonstrating the remarkable stability of the Dexsil 300 GC stationary phase with repetitive column temperature programming. Baseline shifting or peak broadening, which may be indicative of desorption or breakdown of the liquid phase, was not observed. The reagent blank chromatogram in Fig. lb shows that reagents or by-products of the butylation reaction contribute a minor peak (between 5 and 6 min) to the butyl formate peak; another peak elutes at about 9.5 min but does not interfere with the separation of any other butyl ester. Unknown peaks (U1, U2, and U,) in the chromatogram of the prepared butyl esters of acids (Fig. lc) were due to trace components contaminating the commercially available succinic and fumaric acids. Similarly, separation of the butyl esters of valeric and caproic acids individually produced minor peaks (trace amounts) which chromatographed under butyl propionate and butyl butyrate, respectively. All other purchased acids and authentic butyl esters were chromatographically pure and free of detectable contaminating components at the concentrations employed. Standard curves relating peak areas and concentration of fatty acid are given in Fig. 2 problems associated with the analysis of fatty acids in a sample arise from the fact that usually an external standard (mixture of fatty acids) and a sample are run as separate injections at different times. An internal standard compensates for variations between runs in column temperatures, gas flow rates, detector sensitivity, sample preparation, and injection of different amounts of sample, since both sample and the internal standard are co-chromatographed and thus are influenced by these factors to the same relative extent. Heptanoic acid (as the butyl ester) fulfills several criteria for use as an internal standard for determining those fermentation acids considered here; it is (i) chemically similar to the sample species but normally not present in such samples, (ii) available as a pure and stable compound which may be accurately added to the sample, (iii) nonreactive with other acid components in a sample, and (iv) resolved from other acid peaks but similar in its retention time. Table 3 gives a list of the calculated RRFs of various prepared acid butyl esters relative to that of the internal standard, butyl heptanoate (added to each concentration mixture as heptanoic acid). These values show that the RRFs vary with concentration by 0.4 to 9% for most esterified acids. Butyl lactate, however, varied as much as i 30% (standard deviation, 0.122). The apparently low RRF for butyl lactate at sample concentrations of 5 and 10 gmol/ml is partly explained by the fact that the butyl lactate peak is not completely resolved from butyl isovalerate, and consequently there is interference with integration of this peak. In most fermentation media samples, isovaleric acid is a minor component and would not interfere with quantitation of butyl lactate. In our experience with microbial fermentation media, we have made use of an average RRF (see Table 3) and observed that most acids in a sample can be accurately quantitated to within 5 to 10% when they are present in concentrations ranging from 5 to 40 ,mol/ml. Application of the methods described in this paper to samples of microbial fermentation media are illustrated in Fig. 3 and Table 4. Chromatograms of fermentation products produced by rumen bacterial species in a rumen fluid-glucose medium are shown in Fig. 3, and quantitation of acids as the butyl esters using an internal standard is given in Table 4. Similar fermentation patterns (major and minor products) for these bacterial strains have been described by Bryant (3) and illustrated in the VPI Anaerobe Laboratory Manual (9). These data indicate that acids (C1-C , lactate, and succinic) as the butyl esters can be detected and quantitated with little interference from other components of the medium. The methods described in this paper are suitable, therefore, for analysis of acid fermentation products produced by anaerobic bacteria for purposes of identification. Preparation and analysis of the butyl esters of monocarboxylic and dicarboxylic acids have also been useful to determine milk fatty acids (14) and to identify species of Pseudomonas (12) a Products formed were determined from duplicate cultures of each strain. Calculations are based on the internal standard, heptanoic acid, and are corrected for acids in uninoculated (control) media. Abbreviations: F, formic; A, acetic; P, propionic; B, butyric; L, lactic; iV, isovaleric; V, valeric; C, caproic; S, succinic; Fu, fumaric. b Uppercase letters refer to acids formed in amounts of 10 Mmoles/ml of medium or greater, whereas lowercase letters refer to amounts less than 10 jimol/ml. Products formed in less than 0.5 jsmollml amounts are not given. LITERATURE CITED

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2020-12-10T09:04:20.569Z

1975-06-01T00:00:00.000Z

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Microflora of Maize Prepared as Tortillas Very little is known of the microflora in tortillas, the major component in the diet of many Guatemalans and other Central Americans. Based in a Guatemalan highland Indian village, this study examined the types and amounts of bacteria, yeasts, and molds in tortillas and in their maize precursors. Coliforms. Bacillus cereus, two species of Staphylococcus, and many types of yeast were the main contaminants, but low concentrations of alpha-hemolytic Streptococcus, facultative Clostridium, and other bacterial types were also found. When tortillas were first cooked, the bacterial counts dropped to 1,000 or fewer organisms per g, a safe level for consumption. Under village conditions, bacterial counts regained precooking levels (about 108 organisms/g) within 24 h and rose even higher after 48 h. Reheating caused very little change; hence, bacterial levels remained very high in old tortillas kept for later consumption. A search for the sources of contamination showed that most came from water used in preparation and from the soiled hands of women preparing the tortillas. As an attempt to correct certain nutritional needs of the population, the Institute of Nutrition for Central America and Panama initiated a tortilla fortification project in the Guatemalan village. The bacterial counts in fortified tortillas did not differ significantly from those in ordinary tortillas. Furthermore, the level of contamination was constant among tortillas of different sizes and among tortillas made from different types of maize. The importance of tortillas as a food product, the high incidence of bacterial gastrointestinal diseases among Guatemalan Indians (5), and the lack of information concerning this food's microbiological content led to this study. The microbiological implications of fortification were examined by comparing differences in bacterial growth between fortified and unfortified tortillas. Studies were made for tortillas of different sizes and thicknesses and for those made with different strains of maize. The work was done in the Mayan Indian During the period of the study environmental temperatures ranged from 10 to 28 C, with an average rainfall of 3.5 mm/day (range, 0 to 28 mm/day; this rainy season extends from June to October). Indian women prepared all the tortillas in their houses; many village households supplied the samples, which were collected only up to 48 h after preparation because tortillas are not kept longer under ordinary circumstances. Tortillas are usually consumed within several hours after preparation; older tortillas are eaten only on trips. In an attempt to find the sources of bacteria in tortillas, we examined the maize precursors: dry maize, nixtamal (maize that has been cooked with limestone for 50 min and then soaked in water for 14 h [2 ]), masa (a wet, pasty flour that results from the grinding of nixtamal at the mill), and the water used in preparation. The masa is rolled and patted into a flat pancake and then cooked for 4 or 5 min on a comal, a large, flat, hardened clay plate placed over an open fire. Reheating is also done on the comal, but for a shorter time interval (1 to 2 min). Although data were obtained in Santa Maria Cauque, conditions in other highland villages are almost identical, and results can be safely generalized to cover all such highland villages. MATERIALS AND METHODS Samples. Samples, made in individual homes, were randomly selected from among the tortillas and maize sources being prepared for the family's daily consumption. The tortillas were left towel-wrapped in a basket in the same house where they were prepared, until time of culturing (24 or 48 h after preparation). The same women who had made the tortillas also reheated them for later stages of study. Samples of masa and the water used to moisten it were obtained during tortilla preparation. Maize was obtained the previous night so that it would be from the same batch used in the other samples; nixtamal was obtained at the mill just before being ground into masa. All samples were cultured 15 to 30 min after receipt, with special care taken to process fresh and reheated tortillas immediately. Dilutions. Tortillas were brought directly to the laboratory at the village health clinic. A 10-g portion of tortilla pieces was ground in a mortar with sterilized sand and placed in a bottle containing 90 ml of charcoal water (tap water that had previously been adsorbed with charcoal, filtered, and sterilized) (14). This 10-dilution was mixed and then agitated for 5 min; progressive dilutions to 10-9 were prepared in charcoal water as needed (5). Samples for maize, nixtamal, and masa were prepared as above; kernels were ground in a Waring blender when necessary. The original sample of water was used as 10°d ilution, with further dilutions to 10-' prepared. Media used. Table 1 shows the media and types of incubation used. Total bacterial count was determined by using Trypticase soy agar (supplied by Baltimore Biological Labs, Cockeysville, Md., as were GasPak generators and all other media, except for cereus-selective agar, which came from Merck & Co., Inc., Rahway, N.J.), pour-plate method; Tergitol-7 agar with 0.004% triphenyl-tetrazolium chloride, Salmonella-Shigella agar, and brilliant green agar were used to select for coliforms. One gram of minced tortilla was also placed in tetrathionate Selenite-F Enrichment broths and restreaked on Salmonella-Shigella and brilliant green agars after 24 h of incubation to select for pathogenic coliforms. Mannitol salt agar was used to select for staphylococci; cereus-selective agar with 10% egg yolk emulsion in physiological saline (1:1) solution was the medium used to select for Bacillus. Schaedler base medium (14) was modified by adding 1% Trypticase soy broth instead of Trypticase and was used in anaerobic culturing (10). Sabouraud dextrose agar and Mycosel agar were used for yeasts and molds. Media inoculation and incubation. In inoculating plates from various dilutions, we used a 0.01-ml calibrated platinum loop and placed four dilutions on each plate. The plates for anaerobic culture were inoculated with 1.0 and 0.1 ml of the 101-dilution by the spread-plate method. Anaerobic plating was done both before and after boiling the 10-dilution for 30 min. Anaerobiosis was achieved by means of GasPak disposable generators (3). Table 1 lists the incubation periods and temperatures. Enumeration and identification of bacteria. Enumeration was made directly from the plates; representative colonies from each plate were Gram stained (Kopeloff method) and further identified through biochemical tests (1,6,7,9,12,13). All colonies found in anaerobic culture were subcultured aerobically at 37 C for 24 h; almost all were found to be facultative. Microflora of tortillas. Coliform levels of 103 to 107 organisms/g were obtained in 24to 48-h-old tortillas; Alcaligenes faecalis, Klebsiella sp., and Escherichia coli were the most common of those encountered (Fig. 1), indicating a high level of fecal contamination during tortilla preparation. These same species were encountered in the water at levels of 104 to 10l organisms/ml. Cooking the masa killed most coliforms, but a sufficient number survived to return the concentrations to precooking levels within 24 to 48 h of storage at room temperature (Fig. 2). Neither Shigella nor Salmonella were observed, even with tetrathionate and Selenite-F Enrichment; however, this finding does not rule out tortillas as a possible vector for pathogenic coliforms. Such bacteria would probably be much overgrown by other coliforms. The high fecal contamination rates make tortillas a prime candidate for further study in this area. Furthermore, the high E. coli counts suggest the probable presence of enteropathogenic E. coli. Staphylococci. Staphylococci levels in tortillas reached 107 to 108 organisms/g (Fig. 2), with equal numbers of Staphylococcus aureus and S. epidermidis found both in tortillas and in their maize sources. The high counts of S. aureus in the older tortillas could have significant health implications, because certain strains may produce a heat-stable enterotoxin in foods and also because of the possibility of staphylococcal infection. Bacilli. Bacillus cereus, B. macerans, B. megaterium, B. polymyxa, and B. subtilis were found in the maize precursors, with no one species predominating. Most species of Bacillus were killed during cooking, leaving almost exclusively B. cereus. Certain strains of B. cereus, in high concentrations, can cause food poisoning by producing an enterotoxin (8). Hence, the high B. cereus levels observed in tortillas after 24 or 48 h of storage (up to 109 organisms/g) may make their consumption hazardous (Fig. 2). B. subtilis and B. megaterium are also occasionally observed in tortillas. Clostridia. All clostridia encountered in anaerobic culture were found to be facultative. In tortillas, they appeared in low concentrations (101 to 102 organisms/g). Further identification was not pursued because facultative clostridia are nonpathogenic. Streptococci. Streptococci were encountered only in anaerobic culture and never in concentrations greater than 102 organisms/g. The streptococci present were almost exclusively alpha-hemolytic, so further identification was not pursued. Other anaerobes. A variety of other species were encountered, including Sarcina, Lactobacillus, Coccobacillus, and Micrococcus, always in very low dilutions and almost always facultative. Because these species are not pathogenic to man, they were not further identified. Yeasts. Both tortillas and maize sources supported a great quantity and variety of yeasts. Because yeast identification is both difficult and complex, only precursory examination was done, although further work in this area would be of great interest. Examinations revealed species of a red-pigmented Rhodotorula, Candida, Trichosporon, Geotrichum, Torulopsis, Saccharomyces, Pytirosporum, and others. Molds. Molds were found in very high concentrations in tortilla precursors, but were almost completely absent in tortillas, even up to 4 days after preparation. Colony morphology showed species of Penicillium, Aspergillus, Neurospora, and Rhizopus in the maize precursors. No further identification was carried out because these species were not present in any of the tortillas. However, the possibility of mycotoxin production by Aspergillus and Penicillium during the growth of maize should be investigated. Some mycotoxins are thought to be carcinogenic after repeated consumption (4). Unless they are inactivated by the limestone treatment of maize kernels (2,15), their presence could have serious health implications. Microfloral population of tortilla precursors during and after preparation. During early stages of tortilla preparation, bacterial levels rose rapidly, with most initial bacterial contamination resulting from the water used in preparation (Fig. 3) and probably from the hands of the women preparing the tortillas. Most bacteria were killed by cooking (Fig. 2). Enough survived, however, to return bacterial concentrations to precooking levels after 24 h and to even higher levels after 48 h. Yeast contamination was found in maize and throughout preparation. Molds did not reappear after preparation of tortillas, indicating that cooking effectively killed them. After 7 to 10 days, molds grew again on tortillas because of air contamination during storage. Fortified tortillas supported slightly greater aerobic growth than unfortified tortillas, both after 24 and 48 h, but the high levels present make small variations relatively insignificant. A 2-logarithm range in B. cereus levels between fortified and unfortified tortillas was the most important disparity; the several-logarithm difference in nonpathogenic yeasts had little effect on the safety of tortillas. Therefore, the microbiological difference between fortified and unfortified tortillas is minimal with respect to the safe consumption of either kind. Fresh tortillas are safe for consumption, but stored tortillas have sufficiently high bacterial concentrations within 24 h to make them dangerous to health. Most significant in tortillas more than several hours old are the extremely high levels of S. aureus, B. cereus, and coliform bacteria. Tortillas of various sizes and from several different types of maize showed no significant difference in bacterial counts or species isolated. Effects of reheating tortillas. In 24-h-old tortillas, reheating dropped the bacterial count 2 to 3 logarithms, but the counts nevertheless remained very high (Fig. 4). After 48 h, the reheating decrease was slightly less, leaving extremely high bacterial counts (105 to 107 organisms/g). This indicated that the usual 1to 2-min reheating time for the older tortillas did not render the tortillas safe for consumption. Possible existence of enterotoxins from B. cereus and S. aureus further increased the danger. Because such enterotoxins are often heat stable and pathogenic, a study of their existence in tortillas 48 h old or more would be worthwhile. Toasting or longer periods of reheating would possibly lower bacterial concentrations to safe levels. This is currently unfeasible, however, because additional heating would alter taste and texture and also increase fuel consumption. DISCUSSION Tortillas offer a very rich medium for bacterial growth. With the Indians' relative disregard for sanitary procedures, plus the high levels of bacterial and fecal contamination found in the water used during tortilla preparation, a great amount of contamination occurs during the preparation process. Although cooking destroys many of these bacteria, a sufficient number survive to bring bacterial concentrations up to precooking levels within 24 to 48 h. Because of the possible production of enterotoxins and because of the possible contamination by other pathogens, the Indians should avoid consuming older tortillas that have been stored unrefrigerated. The results indicate that sanitation and the use of comparatively clean water should be stressed to those living in such villages. Some of the water samples from tortilla preparation arrived in our laboratory clouded with masa and containing dead flies. Water used in tortilla preparation should be changed daily instead of being used for an extended period. Adopting such simple sanitation procedures would do much to lower contamination in tortillas that are not consumed quickly after preparation, especially in tortillas prepared for trips. This study involved 46 women in one highland village where conditions were similar to those of other indigenous villages. At lower elevations in Central America, ambient temperatures are much higher, and the bacterial growth can be assumed to be even more rapid. Hence, the need for better sanitary methods in the preparation of tortillas must affect populations throughout Central America.

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2020-12-10T09:04:20.627Z

1975-06-01T00:00:00.000Z

237229681

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Laboratory Method for Fermentation of Meat and Poultry Sausages in Fibrous Casings The construction and operation of a relatively inexpensive cabinet for sausage fermentation studies is described. Temperature can be controlled to ±1 C with a relative humidity of approximately 95%. Laboratory-scale fermented meat studies have generally employed beakers rather than conventional sausage casings to hold the meat (3), is economical but has the disadvantages that (i) the ratio of air to meat surface is considerably different and restricted from that ) controlled via warm water bath heated by found with casings, and (ii) the fermented material cannot readily be smoked, cooked out, or dried for possible additional study. Small commercial scale air-conditioned smokehouses, providing temperature, humid-ity, and air-flow control, are quite expensive and not always available. Therefore, we designed a simple, relatively inexpensive cabinetheater combination that will permit fermentation studies in typical fibrous or natural casings. The cabinet (Fig. 1) was a conventional vat with lid, both made of a reinforced plastic material and of the type previously used for icing down poultry (Model GE795, Goodyear Aerospace Corp., Jackson, Ohio). The vat is available through most meat supply houses. Holes were drilled as shown to accommodate eight stainless-steel rods from which sausage chubs or links were suspended. Temperature control within the cabinet was achieved with a 16-liter external water bath equipped with a 1,000 W heater-circulator pump. This pump was used to circulate heated water from the external bath to the cabinet via tygon tubing, 9.5-mm inside diameter by 1.6-mm wall (0 by %A6 inch). A coil of copper tubing, 6.1 m by 9.5-mm inside diameter (20 feet by % inch), in the bottom of the cabinet was connected to the tygon tubing and served as a heat exchanger by transferring heat from the external water bath to approximately 25 liters of water covering the coil in the bottom of the cabinet. Cabinet air temperatures (dry bulb) above the coil-heated water of 30 and 38 C were achieved with external water bath settings of approximately 38 and 52 C, respectively. Sausage material with an initial internal temperature after stuffing of 10 to 12 C rose to 30 C in about 2.5 to 3 h and to 38 C in a total of about 4.5 h. The preceding rates were found for sausage stuffed in 52 mm diameter dry sausage fibrous casings (Union Carbide). Use of smaller or larger diameter casing would change the heat penetration rate. Humidity within the chamber averages approximately 95%, based on wet-bulb and drybulb readings. In industrial practice, a humidity control between 90 to 98% is obtained by introducing steam as the only heat source. It may be possible to achieve a lower humidity, near 90%, through use of various salt solutions although none have been tested in our laboratory. The rates of pH reduction for replicate sausage fermentations using the above cabinet and an air-conditioned industrial scale smokehouse have shown no significant differences. The rates of pH reduction in these units for a summer sausage mix inoculated with a frozen concentrate of Pediococcus cerevisiae (Lactacel, Merck and Co.) and held at 38 C are shown in Fig. 2. Lactic acid bacteria, as enumerated on the V-8 medium of Fabian et al. (4), were initially (postinoculation) at 8.6 x 106 cells/g and increased to 6.3 x 108 cells/g within 24 h. Studies of microbiological, meat chemistry, physical, and processing parameters involving fermentation with the above system have been reported elsewhere (5-7).

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