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.

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.

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 (C-1-C-7) 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 mumol 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 toxonomic significance.

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.