Experiments on the culture and physiology of holotrichs from the bovine rumen

Abstract

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...

Factors Affecting the Uptake and Metabolism of Soluble Carbohydrates by the Rumen Ciliate Dasytricha ruminantium Isolated from Ovine Rumen Contents by Filtration

“…Suspensions of Dasytricha with negligible contamination by bacteria or other protozoan species can be prepared in high yields, whereas diffe1 ential sedimentation often results in incomplete separation of the holotrich species. Methods relying on the adhesion of Dasytricha to glass surfaces (Guttierez, 1955) are unreliable and produce poor yields. Using the filtration technique to isolate Dasytricha, partial defaunation of the host animal is unnecessary (Eadie & Oxford, 1957), although in combination the two techniques should permit the establishment and recovery of pre-determined rumen protozoan SPY' cI ies.…”

“…Viable preparations were maintained in a supplemented buffer solution for long periods (24 to Isotricha spp. were separated from the accompanying Polyplastron remaining on the filter by repeated washing, using a separating funnel and allowing the Isotricha to adhere to the inner walls of the glass vessel in a manner analogous to that described for D. ruminantiurn by Guttierez (1955).…”

“…The rate of acid production was directly proportional to the amount of cell protein present. The average proportions of metabolic end-products from glucose present at 5 mg ml-l in 10 incubations were estimated to be: acetic acid, 2-3 % (1.2 %); butyric acid, 3-7 % (2-5 %); lactic acid, 19.8 % (18.8 %); carbon dioxide, 6.0 % (4.5 %), assuming C 0 2 and H, are produced in I : I proportion (Heald & Oxford, 1953;Guttierez, 1955); and storage polysaccharide, 68.2 % (73 %). The figures in parentheses are the proportions formed after corrections for endogenous metabolism have been made.…”

“…has been examined in vitro using cultural studies , and washed preparations of either single genera (Guttierez, 1955 ; or mixed holotrich species (Masson & Oxford, 1951;Heald & Oxford, 1953). Protozoa were separated from rumen liquor by sedimenting strained rumen contents (Heald, Oxford & Sugden, 1952) or by repeatedly washing the residue remaining after freshly-sampled rumen liquor had been filtered through surgical gauze (Oxford,195 1).…”

“…Protozoa were separated from rumen liquor by sedimenting strained rumen contents (Heald, Oxford & Sugden, 1952) or by repeatedly washing the residue remaining after freshly-sampled rumen liquor had been filtered through surgical gauze (Oxford,195 1). Dasytricha ruminantiurn was subsequently recovered by differential sedimentation of the mixed holotrich preparation, or by makkg use of the tendency of the protozoa to adhere to the inner walls of the glassware used in the initial sedimentation (Guttierez, 1955). Partial defamation of the host animal permits the introduction of specific protozoan species into the rumen simplifying the recovery process (Eadie & Oxford, 1957 ; Howard, I 957).…”