Improved techniques for dissociating particle-associated mixed ruminal microorganisms from ruminal digesta solids

Combinations of physical and chemical methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from saline-washed ruminal digesta solids (SWRDS). Physical methods included chilling and storage, homogenization, multiple extraction, and agitation with marbles. Chemical methods included use of low pH, Tween 80, formaldehyde, methanol, tertiary butanol, and methylcellulose. Microbial removal from SWRDS was determined directly by using epifluorescence microscopy and indirectly by measuring removal of diaminopimelic acid and total purines. Different combinations of methods resulted in removals of 46 to 82% for particle-associated bacteria (PAB), 52 to 98% for particle-associated protozoa (PAP), and 60 to 83% for PAB plus PAP. Two methods were considered most effective, based on microscopy; both removed similar amounts of PAB (79 to 82%) and PAB plus PAP (80 to 83%). In one method, SWRDS were stored for 24 h at 4 degrees C in a solution of pH 2 saline, .1% Tween 80, 1.0% methanol, and 1.0% tertiary butanol. In the other method, SWRDS were incubated for 30 min in .1% methylcellulose before storage for 24 h at 4 degrees C in pH 2 saline, .1% Tween 80, and 1.0% methanol. Common to both treatments was subsequent homogenization of the suspensions for 15 s followed by washing the digesta solids seven times with the treatment solutions. Both methods resulted in values that exceeded those reported previously for removal of PAM from ruminal digesta solids.