A Method for Direct Viable Counts of the Facultatively Anaerobic Microflora in the Rumen of a Sheep maintained on a Hay Diet

Abstract

SUMMARY: By direct viable counts in deep cultures in a basal nutrient agar, consisting of buffered and very highly clarified nunen liquor, supplemented only with vitamin-free acid hydrolysate of casein, tryptophan, a fermentable carbohydrate and agar, the crude wet nunen contents of a hay-fed sheep has been shown to contain a population of c. lo8 viable and facultatively anaerobic bacteria per g., mostly Gram-positive cocci capable of fermenting'a wide range of soluble carbohydrates including starch and inulin but not mannitol. "he mannitol-fermenting population is of the order of lo6 organisms per g., consisting mostly of Gramnegative rods, including coliform bacteria. Addition of reducing substances such as sodium sulphide or memaptoacetate (thioglycollate) to the basal agar had little or no effect in increasing the counts. An extract of hay made With cleared and sterile rumen liquor did not yield the typical nunen streptococcal flora when counted by this method in rafFinose+rumen liquor nutrient agar.Although most workers with rumen micro-organisms seem to have assumed either that the important fermentative bacteria and protozoa in this very mixed culture must be obligate anaerobes, or that they will not act except under powerful reducing conditions (see, e.g. Hungate (1950) ; Huhtanen, Rogers & Gall (1952) and previous papers by Gall and associates on the methods for isolating nunen bacteria; Sijpesteijn & Elsden (1952) on methods of using washed suspensions of mixed rumen bacteria for fermentation studies), nevertheless there are good reasons for believing that the rumen normally contains a considerable population of facultative anaerobes which are active in fermenting carbohydrates. Thus, by a simple enrichment technique not carried on for more than 18 hr., Heald (1952a, b) isolated numerous pure cultures of coliform bacteria, capable rapidly of fermenting glucose and xylose, from the rumens of hay-fed sheep. MacPherson (1953), in a study of nunen amylolytic bacteria, without any difficulty isolated numerous facultatively anaerobic streptococci, which resembled Strep. bovis in fermentation reactions, from the rumens of both concentrate-fed and hay-fed sheep. These bacteria were absent from the fodder. Sugden & Oxford (1952) showed that an important and ubiquitous group of sheep's rumen ciliate protozoa, the holotrichs, which are very active in fermenting glucose (Heald & Oxford, 1958), are not a t all exacting in their demands for anaerobic conditions and will

Section A. Physiology of dairy cattle: The Biochemistry and Microbiology of the Rumen

“…The scheme put forward by Moir & Masson would seem to afford a method of quantitative evaluation of a bacterial population through differential counts, and might provide a useful control in many studies of a biochemical nature. Heald, Krogh, Mann, Appleby, Masson & Oxford (35) have devised a direct method for making viable counts of facultative anaerobes, and have reached the conclusion that facultative anaerobes play a more important role in rumen metabolism than had been previously believed, this early lack in recognizing their importance being due to a concentration of attention on the strictly anaerobic process of cellulose digestion. Mann, Masson & Oxford (36), in a further study of the facultative anaerobes of the previous paper, have shown that of 120 streptococcal isolates 82% showed fermentation and other reactions similar to those of Streptococcus bovis, 6 % resembled Strep, faecalis and the rest (12%) were unclassifiable.…”

Commensalism in Pathogenic Anaerobic Actinomyces Cultures

“…Anaerobiosis was obtained by flushing the container vessels with nitrogen in the manner described in that paper. Water/gel cultures were prepared in small screw-capped bottles according to the method of Heald, Krogh, Mann, Appleby, Masson & Oxford (1953), with the modification that the concentration of agar in the solid layer was only 0.5 yo. When this nutrient layer incorporating the inoculum had set, the bottle was carefully filled with freshly-boiled cold sterile water up to the rim and the cap screwed down.…”

Facultative Anaerobic Bacteria from the Sheep's Rumen

“…In a recent paper (Heald et al 1953) a method of counting viable saccharolytic bacteria from a sheep's rumen was described (hereinafter referred to as the 'bottle count ') in which the basal medium consisted of rumen liquor itself, suitably clarified and sterilized after withdrawal , from hay-fed sheep and supplemented by nothing more than a vitamin-free mixture of amino acids, a soluble fermentable carbohydrate and agar. In this way it was hoped that only bacteria genuinely active under rumen conditions would be counted.…”

“…By picking off colonies from the agar-shake cultures, some 300 isolates were eventually obtained in pure culture representing, for the most part, small cocci and Gram-negative rods present in numbers exceeding lOO,OOO/g. wet rumen contents (see Table 2 in Heald et al 1953). Since the methods used ensured that only the active carbohydrate fermenters would grow, these isolated bacteria were regarded as of actual or potential importance in rumen fermentations.…”