Evaluation of techniques to detach particle-associated microorganisms from rumen contents

Effects of the most commonly used treatments to detach particle-associated microorganisms from rumen contents were investigated using rumen particles of different sizes. Particles were obtained before feeding from ruminally cannulated sheep. The extent of microorganism dissociation was determined using 15N as an external marker. The first experiment studied the effect of anaerobiosis on efficiency of 1 g/l methylcellulose, pH 8, and chilling (4 °C). Due to poor detachment, the anaerobic procedure was discarded. The following factors, separately or in combination, were then examined in aerobic conditions on two classes of particles obtained from whole ruminal contents (large: >400 μm; small: 100-400 μm) being: (a) stomacher pummelling (5 min); (b) Waring blender homogenisation (3×1 min); (c) chilling (4 °C); (d) pH 8; (e) 1 g/l methylcellulose; (f) 10 ml/l methanol and 10 ml/l tertiary butanol. Samples were incubated for 5 h, except for treatments (a) and (b), and washed after treatment for 2 min under running tap water in a 25 μm gauze. Blending proved to be the most effective treatment (from 50 to 57% removal). Combinations of treatments did not improve detachment. N losses from treated samples were linearly related to 15N removal. The percentage of particles removed was calculated using incubated and/or washed particles as the control, according to treatments. Results suggest that caution is needed when evaluating the effectiveness of treatments, because results are dependent on the type of particles chosen as the control.