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I. THE ISOLATION TECHNIQUE Gases. Oxygen-free carbon dioxide, hydrogen, or nitrogen is obtained by bubbling the cylinder gases with low oxygen content through a chromous acid solution similar to that ' These investigations have received financial support from The Society of the Sigma Xi

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An average of 11 (range, 2 to 47) μmoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k , times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K m , for formate conversion to CH 4 was estimated at 30 nmoles/g. Also, the dissolved H 2 was measured in relation to methane production, and a K m of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K m of 1 nmole of H 2 /g, but the K m for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO 2 and H 2 are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.

Direct microscopic and c~~l t u r e coi~nts were made on samples of rumen contents obtained by stomach tube from each of 25 cows a t monthly intervals during 3 months. The ration \\.as timothy hay plus various amounts of cottonseecl 111ea1, rolled barley, and salt.No correlation between different rations and differences in the microbiota c o~~l d be detected. Slreplococczls boois was identified by its rapid growth in the feed nledii~nl and sho\\red counts ranging between 2 X 10j and 1 .4X108. Most animals showed a S. bovis count between 1 and 20 million with an average of about 10 million per milliliter. The characteristics of some rumen strains are described. The cellulose-digesting bacteria were referable to four chief groups and the numbers of each were recorded. The cellulolytic cocci varied in alrnost every character studied. Two types were distinguished as representing the greatest divergence. R~~~n i~z o c o c c l~s albz~s n. sp. differs from R. juvefacims in being Gram-negative, forming little or no yellow pigment, and producing no succinic acid. A cellulose-digesting sporeformer, Clostridiz~nt Ioclzlzeadii n. sp., was found in many of the sanlples. I t was estre~nely active in digesting cellulose, exceeding in this respect the species of anaerobic ccll~~lose digesters previoirsly isolated. Spores were fornled in abundance but rapidly disintegrated and many strains were lost before subculture.