Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Enumeration and Isolation of Lactate-Utilizing Bacteria from the Rumen of Sheep

A highly specific medium was developed for the enumeration of lactate-utilizing bacteria in the rumen of sheep. This medium, which contained 2.0% lactate, 2.0% Trypticase, 0.2% yeast extract, and volatile fatty acids, hemin, and trace elements in place of rumen fluid, enabled high counts (42 × 10⁷ to 190 × 10⁷/g of ingesta) of lactate-utilizing bacteria to be made with a high degree of specificity (96%). The medium also supported the growth of all species of predominant lactate-utilizing bacteria reported to occur in the rumen and thus is of importance for ecological studies where the incidence and influence of the different species on lactate metabolism under changing conditions in the rumen cannot be predicted. The survival rate of isolates was increased from 60 to 96% by addition to the modified maintenance medium of 40% rumen fluid in place of the volatile fatty acids, hemin, and trace elements used in the counting medium. These results, together with the slow growth of colonies in roll bottles, showed that, although highly selective, the counting medium was not optimal for the types selected.     

Morphological and physiological characteristics of Gemmiger formicilis isolated from chicken ceca.

Morphological and physiological studies were made on chicken cecal isolates of the strictly anaerobic bacterial species Gemmiger formicilis. Structural features (phase-contrast and electron microscopy) of these microorganisms indicate they (i) are highly pleomorphic, (ii) possess a trilaminar cell wall like gram-negative bacteria, (iii) exhibit an unusual growth process characterized by polar swelling (resembling budding bacteria), and (iv) grow into elongated cells when exposed to a subinhibitory concentration of penicillin. The morphological data presented suggest that this species has a rod-shaped structure. These bacteria ferment a variety of sugars to produce formic, butyric, and lactic acids. There appear to be two groups of Gemmiger, one producing primarily lactate and the other producing formate as major fermentation metabolites. Growth of six strains in a basal medium, consisting of Trypticase, minerals, carbohydrate, Na2CO3 buffer, and cysteine as reducing agent, was stimulated by rumen fluid and yeast extract. Volatile fatty acids partially replaced the requirement for rumen fluid with some strains. Single deletions of vitamins (from a defined vitamin mixture) indicated that pantothenate, riboflavin, and thiamine were highly stimulatory to growth of the organism in a medium containing rumen fluid and Trypticase as source of vitamins. Other vitamin requirements were not studied.     

Isolation and some characteristics of anaerobic oxalate-degrading bacteria from the rumen.

Obligately anaerobic oxalate-degrading bacteria were isolated from an enriched population of rumen bacteria in an oxalate-containing medium that had been depleted of other readily metabolized substrates. These organisms, which are the first reported anaerobic oxalate degraders isolated from the rumen, were gram negative, nonmotile rods. They grew in a medium containing sodium oxalate, yeast extract, cysteine, and minerals. The only substrate that supported growth was oxalate. Growth was directly related to the concentration of oxalate in the medium (1 to 111 mM), and cell yields were approximately 1.1 g (dry weight)/mol of oxalate degraded. Oxalate was stoichiometrically degraded to CO2 and formate. These anaerobes occupy a unique ecological niche and are distinct from any previously described oxalate-degrading bacteria.     

Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars

A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.     

Cellulolytic cocci isolated from the cecum of guinea pigs (Cavia porcellus).

Five strains of anaerobic, gram-variable cellulolytic cocci, belonging to the genus Ruminococcus, were isolated from the cecum of a guinea pig. They differed from most previously described strains of cellulolytic ruminococci as follows. (i) Lactate was the major fermentation product; lesser amounts of formate and ethanol and a trace of succinate were also produced, along with an uptake of acetate. (ii) No growth occurred at 30 degrees C; however, good growth was observed at 38 and 45 degrees C, (iii) Glucose, cellobiose, cellulose, xylose, arabinose, xylan, sucrose, and lactose were fermented by all strains. Rumen fluid was required for growth in a complete medium containing all nutrients previously found to be required by species in this genus. Limited growth occurred when rumen fluid was replaced by yeast extract, and maximum, but delayed, growth occurred when a water extract of alfalfa was added to the complete medium. No qualitative differences were found in the cell wall amino acids and sugar composition of these strains as compared to Ruminococcus flavefaciens and Ruminococcus albus; however, cell walls of the guinea pig strains appeared to contain a higher proportion of glucose.     

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate.

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.     

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.     

Studies on the Cecal Microflora of Commercial Broiler Chickens

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.     

Effect of titanium (III) citrate as reducing agent on growth of rumen bacteria.

We compared the growth of 10 strains of rumen bacteria in an anaerobic medium reduced with cysteine hydrochloride, dithiothreitol, or titanium (III) citrate. The redox potential of medium reduced with cysteine hydrochloride was -167.8 mV; with dithiothreitol it was -175.8 mV; and with titanium(III) citrate it was -302.4 mV at a concentration of 5 X 10(-4) M titanium and -403.9 mV at 2 X 10(-3) M titanium. Maximum growth of the strains was generally lower with dithiothreitol or titanium(III) citrate than with cysteine hydrochloride, although growth was greater than in medium lacking an added reducing agent. Strains for which cysteine was required or markedly stimulatory grew only poorly with titanium(III) citrate. No strain grew in medium with sodium citrate as the energy source. Titanium(III) citrate could be used to reduce anaerobic media for some rumen bacteria if the exclusion of a sulfur-containing reducing agent is required.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Medium Without Rumen Fluid for Nonselective Enumeration and Isolation of Rumen Bacteria

Colony counts which approximated those in a habitat-simulating, rumen fluid-agar medium (RFM) were obtained in medium 10, a medium identical to the RFM except for the replacement of rumen fluid with 1.5 x 10(-6)m hemin, 0.2% Trypticase, 0.05% yeast extract, and a 6.6 x 10(-2)m volatile fatty acid mixture qualitatively and quantitatively similar to that in rumen fluid. Single deletion of Trypticase, yeast extract, or the volatile fatty acid mixture from medium 10 significantly reduced colony counts. Colony counts were also reduced when medium 10 was modified to contain higher concentrations of Trypticase or volatile fatty acids. Significant differences were found between colony counts obtained from diluted rumen contents of animals fed a cracked corn-urea diet, and the colony counts obtained from animals fed either a cracked corn-soyean oil meal or an alfalfa hay-grain diet. Qualitative differences were found between the predominant bacterial strains isolated from rumen contents of animals fed cracked corn diets and strains isolated from animals fed alfalfa hay-grain. Regardless of differences in the predominant flora associated with diet, medium 10 and the RFM supported growth of similar bacterial populations. The results show that medium 10 is suitable for enumeration and isolation of many predominant rumen bacteria.     

Enrichment and isolation of Acetitomaculum ruminis,gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen

Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H₂:CO₂ (80:20) or N₂:CO₂ (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H₂:CO₂. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H₂:CO₂ to form acetate in quantities 2 to 8-fold higher than when grown under N₂:CO₂. All presumptive acetogenic isolates were derived from either the 10^-7 or 10^-8 dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species. Portions of this work were presented previously (Greening RC, Leedle JAZ (1987) Abstr Annu Meet Am Soc Microbiol I 131, pp 194)     

Growth Factor Requirements of Ruminococcus flavefaciens Isolated from the Rumen of Cattle Fed Purified Diets

Eight strains of cellulolytic cocci were isolated from a 10^-8 dilution of rumen ingesta and were presumptively identified as Ruminococcus flavefaciens. Four strains were isolated from a steer fed a purified diet which contained isolated soy protein, and four strains were isolated from a steer fed a purified diet which contained urea. Certain growth factor requirements of these bacteria were determined. All strains grew with clarified rumen fluid added to the medium. However, fatty acids could substitute for rumen fluid in four strains. Two strains isolated from each steer either required or their growth was stimulated by isobutyric and/or isovaleric and/or 2-methyl-butyric acid. These results indicate that, even when a diet was fed which contained no branched-chain amino acids, the carbon skeleton precursors of branched-chain fatty acids, the cattle were still able to maintain a large population of cellulolytic bacteria that require fatty acids for growth. Therefore, the fatty acids appear to be provided by other bacteria, by protozoa, or by the host animal.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Production of alkaline phosphatase by epithelial cells and adherent bacteria of the bovine rumen and abomasum

Three distinct bacterial populations have been defined in the bovine rumen: the rumen fluid population; the population associated with food particles; and the population adherent to the rumen epithelium. Alkaline phosphatase activity has been reported in cells of the first two populations and here we report that assays of rumen epithelial samples containing both tissue and bacteria also contain the enzyme. The reaction product technique has localized the enzyme both in adherent bacteria and in cell of the stratified squamous rumen epithelium. The epithelium of the abomasum shows much lower levels of alkaline phosphatase activity.     

Anaerobic Roll Tube Media for Nonselective Enumeration and Isolation of Bacteria in Human Feces

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.