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activity. Media containing protein were used so that the proteolytic activity could be measured at stages in the isolation procedure, but to obtain adequate growth additions of non-protein nitrogen had to be made. Some rumen bac- teria were found to need rumen fluid as a nitrogen source or to provide growth factors, but many bacteria which were initially isolated in rumen fluid media were later found to grow in other media, so yeast extract was used in most of the media described here to give a more uniform medium for determination of proteolysis. METHODS Determination of proteolytic activity. The quantitative biuret method used was described in Blackburn & Hobson (1960). For qualitative determinations of proteolysis on solid media clearing of the cloudy medium around colonies was looked for either immediately, or after flooding with acid mercuric chloride. Semi-quantitative tests on liquid media were initially made by centrifuging off the organisms and adding trichloroacetic acid solution (8 vol. 0.7211) to a portion of the culture; the turbidity produced being compared against an uninoculated culture. As initial standards the turbidity produced by different concentrations of casein was measured under the same conditions. Later it was found unnecessary to centrifuge off the organisms. Reagents were described in Blackburn & Hobson (1960). Media Medium 1. The mineral solutions used were based on those of Hungate (1950) and contained: (a) 0.3% (w/v) K,HPO,; (b) (yo w/v). 0.3, KH2P04; 0.6, (NH,),so4; 0.6, NaCl; 0-6, MgSO,*7H2O; 0.6, CaCl,; (c) A-cysteine hydro- chloride, 0.2 g.; NaHCO,, 1.92 g.; in water 82 ml. A mixture was made usually containing, per 100 ml. : solution (a), 15 d.; solution (b) 15 d.; 0.1 yoresazurin solution, 0-1 ml.; protein 0.5 g. (based on N content of the material); yeast

Isolation of Proteolytic Bacteria from the Sheep Rumen