The effect of different centrifugation conditions for the isolation of mixed rumen bacteria, on their nitrogen and diaminopimelic acid content

Three cows were given two rations, a silage diet (3 animals) and a green forage diet (2 animals). Samples of rumen content were collected and aliquots of these were separated in a fraction of feed particles and protozoa (FP-fraction) and a fraction of mixed bacteria, varying the conditions of differential centrifugation. The low speed centrifugation was practised at 100 X g/5 min, 400 X g/10 min, 1000 X g/10 min, and 2000 X g/10 min. High speed conditions were 30,000 X g/30 min 4 degrees C. The lyophylisated sediments were used for determination of N and DAP. The content of N gave similar results for all fractions of mixed bacteria, the mean value being 7.43 +/- 0.48% (n = 20), while the N-content of the FP-fractions being 5.68 +/- 0.37% (n = 19). The N:DAP-ratio gave similar values for the cows fed the silage diet, the values were 29.45 +/- 1.56 (n = 12). The values for the cows receiving the green forage diet differed, the mean values were 23.08 +/- 0.88 and 42.01 +/- 5.81 (n = 5), respectively. In all five experiments highest ratios were found at 100 X g. Further investigations showed that storage at -20 degrees C rumen fluid after isolation of feed particles and protozoa decreased both the N- and DAP- content without affecting the N:DAP-ratio. Centrifugation at low speed with 100 X g resulted in a markedly decreased contamination with DAP in all the FP-fractions. Optimal conditions for separation of feed particles and protozoa from rumen fluid to get a fraction best reflecting the rumen bacterial populations are 100 X g/5 min.