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 salin e-washed ruminal digesta solids (SWRDS). Physical methods included chi lling and storage, homogenization, multiple extraction, and agitation with marbles. Chemical methods included use of low pH, Tween 80, forma ldehyde, methanol, tertiary butanol, and methylcellulose. Microbial re moval from SWRDS was determined directly by using epifluorescence micr oscopy 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 par ticle-associated protozoa (PAP), and 60 to 83% for PAB plus PAP. Two m ethods were considered most effective, based on microscopy; both remov ed 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 o f 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% methylcel lulose 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 homog enization 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.