Sampling
Approximately 250 ml rumen fluid was harvested by suction per fistula from a non-lactating Holstein dairy cow receiving a high forage diet. The fluid was strained twice through four layers of cheesecloth and transported to the laboratory in an insulated container to avoid cold shock. The fibrous residue remaining after sraining was likewise taken to the laboratory and was examined for fungal rhizobia.
Half of the fluid was gassed with carbon dioxide and incubated at 39 degrees for later examination. The remainder was diluted with 50% formalin and separate aliquots were stained with methylene blue, brilliant green, or iodine to emphasize structural components and aid identification (Dehority, 1993. Laboratory Manual for Classification and Morphology of Rumen Ciliate Protozoa. CRC Press, Inc., Boca Raton, FL.)