The rumen protozoa can be assigned to three different groups; one consisting ofthe large protozoa of the genus Diplodinium and related types which ingest quantities of fibrous plant material; a second group of smaller protozoa in the genus Entodinium which actively digest starch but seldom are found to contain the cell walls of plants; and a third group, the holotrichs, which seldom containplant materials. This latter group includes the species I8otricha pro8toma, I. inte8tinaU8 and the smaller Da8ytricha ruminantium.Previous investigators (Heald, Oxford & Sugden, 1952;Heald & Oxford, 1953) gave small amounts of glucose to strained sheep-rumen contents placed in separatory funnels. Metabolizing the carbohydrate very rapidly, the three species of holotrichs settled to the bottom of the funnel and could be obtained free of the other ruimen protozoa. Heald & Oxford (1953) analysed the fermentation products of the mixed holotrich protozoa and found carbon dioxide, hydrogen, lactic, acetic and butyric acids, and a trace of propionic acid as the products of glucose fermentation. Cellobiose was utilized by the mixture but at a rate much slower than glucose.This paper deals with attempts to separate the three species of holotrichs, to determine their individual characteristics, and to estimate their significance to the host. MATERIALS AND METHODSPreparation of buffer 8olutions and rumen fluid. The inorganic salt solution used in the culture media and manometric experiments contained 0-5 %The composition of this solution is similar to that of the inorganic salt solution described by Hungate (1942) for cultures of Diplodinium neglectum, except that he used 0-6% (w/v) NaCl. Anaerobic conditions were maintained with CO2 gas, and 0.5 % (w/v) NaHCO3 was added to give a suitable pH.Samples of rumen contents were removed by tube and suction bulb (Hungate, 1950) from two fistulated steers.When required for use in culture medium, the rumen fluid was placed in a refrigerator overnight to allow the protozoa and plant debris to settle. The supernatant fluid was found * Public Health Service Research Fellow of the National Microbiological Institute.by microscopic examination to be free of protozoa but to contain many bacteria. The upper portion was drawn off and stored in the refrigerator until required. Media containing either heated or Seitz-filtered rumen fluid did not support growth as well as those made up with untreated fluid. When the holotrich protozoa were to be used in Warburg experiments or fermentation product analyses, the rumen contents were filtered through bolting silk to remove the large debris and immediately incubated with glucose.Separation of protozoa. A portion of the rumen sample (usually about 300 ml.) was placed in a separatory funnel with 0.5 % (w/v) glucose and kept for 30-60 min. at 390 according to the method described by Heald et al. (1952). Many I8otricha and some Dasytricha collected in the bottom of the funnel and were drawn off for further purification.After the remaining rumen contents were drained o...

SYNOPSIS. Starvation of Isotricha prostoma for 72–96 hours decreased the cellular amylopectin granules and facilitated the microscopic search for bacterial feeding. I. prostoma selected and ingested only certain rods from among many types of rumen bacteria. In order to isolate the bacteria important as a food source for Isotricha, the starved protozoa were allowed to feed on mixed rumen bacteria, washed, and the crushed protozoan contents quickly cultured for bacteria. Several strains of bacteria were isolated in pure culture. Three of the rod strains isolated were rapidly ingested by I. prostoma when fed to the ciliate. In a monobacterial culture I. prostoma divided once before succumbing.

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SYNOPSIS. In cattle fed a high‐starch diet, species of Entodinium and Diplodinium ingested associated ruminal bacteria. Stained preparations of diluted rumen contents showed Entodinium caudatum, E. minimum, E. dubardi, (syn. E. simplex), E. longinucleatum, E. bursa, E. nanellum, E. exiguum, and E. vorax contained gram‐positive diplococci. Starch grains with adherent gram‐positive diplococci were observed within Entodinium spp. Diplodinium ecaudatum forma ecaudatum, D. ecaudatum forma caudatum, D. neglectum and an unidentified species of Diplodinium also ingested ruminal diplococci. Bacteria were isolated from mixed species of Entodinium by washing and culturing the protozoa in a starch feed‐extract agar medium. The strains isolated from the ciliates were gram‐positive diplococci, 0.8 times 1–1.5 μm, which attached themselves to starch granules and were able to digest the starch. Conclusive evidence of bacterial ingestion by the oligotrichs was obtained by providing the bacterial cultures to Entodinium species (E. dubardi and E. minimum) which had been starved 24 hr. Gram‐stained preparations showed the ciliates readily ingested the bacteria. The amylolytic cocci utilized by Entodinium spp. were identified as Streptococcus bovis.

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