Acholeplasma Bactoclasticum Sp. N., an Anaerobic Mycoplasma from the Bovine Rumen JOHN P

INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 23, No. 2 April 1973, p. 171-181 Printed in U.S.A. Copyright 0 1973 International Association of Microbiological Societies Acholeplasma bactoclasticum sp. n., an Anaerobic Mycoplasma from the Bovine Rumen JOHN P. ROBINSON' and R. E. HUNGATE Department of Bacteriology, University of California, Davis, California 9561 6 A strain of a strictly anaerobic, filterable, bacteriolytic microorganism has been isolated from the bovine rumen. It has the microscopic and colonial morphology characteristic of mycoplasmas and is resistant to penicillin G. Sterols are not required for growth, and physiological properties show it to be distinct from Acholeplasma laidlawii, A. granularum, and A. axanthum. It is proposed that the organism be named Acholeplasma bactoclasticum sp. n. The type strain is ATCC 271 12. Microorganisms causing a partial digestion of A mineral salts basal medium was prepared by rumen bacterial cells included in an agar culture adding four volumes of deionized water to one volume medium were initially thought to meet their each of mineral solutions A (0.3% KH,PO,, 0.6% energy needs by a fermentation of protein (6, NaC1, 0.3% (NH, ), SO,, 0.06% MgSO, -7H, 0, 0.06% p. 79). A rapid digestion of the casein in skim CaC1, *2H, 0) and B (0.3% K, HPO, 1, all w/v. milk was observed, and addition of glucose to Minfav medium. Minerals salts-volatile fatty acid- the medium did not greatly increase the size of vitamin (Minfav) medium was prepared by adding vitamins and volatile fatty acids to the mineral salts the colonies. medium. The vitamin solution contained the follow- The work reported here describes the results ing, dissolved in 500 ml of water: biotin, 1 mg; of experiments and observations identifying a calcium pantothenate, 200 mg; folic acid, 1 mg; similar microorganism as a new species of inositol, 1,000 mg; niacin, 200 mg; riboflavine, 100 AchoZeplasma establishing some of its charac- mg; and thiamine hydrochloride, 200 mg. Each 10 ml t erist ics. of medium contained 10 pliters of this solution. The volatile acid mixture contained 17 ml of acetic acid, 6 MATERIALS AND METHODS ml of propionic acid, 4 ml of butyric acid, 1 ml of n-valeric acid, 1 ml of isovaleric acid, and 1 ml of Bacterial strain. The strain reported here was DL-methyl butyric acid; 0.03 ml of the mixture was isolated from rumen fluid siphoned from a fistulated neutralized with NaOH and added to each 10 ml of Jersey heifer. It was deposited in the American Type culture. Culture Collection, Rockville, Md. as ATCC 27 112. CRF medium. Clarified rumen fluid (CRF) medium Preparation of media and supplements. The orga- was similar to the mineral salts medium except that nism was cultured anaerobically under 100% CO, by two volumes of rumen fluid freed of cells by the roll-tube technique (7). Cysteine (0.03%) and centrifugation were substituted for two volumes of NaHCO, (0.05%) (both wt/vol in final concentration) deionized water. Rumen fluid was centrifuged at were added anaerobically with a syringe before 25,000 X g for 15 min, and the supernatant fluid was autoclaving. The 3% Na, S.9H2 0 solution routinely added to the cooled mineral salts solution. Rumen added to provide a low redox potential was prepared fluid medium (RF medium) was prepared by using by dissolving the crystals in boiled deionized water uncentrifuged rumen fluid freed of protozoa and plant cooled under 0, -free N, , tubed anaerobically under material. N,, and autoclaved. Final concentration in the Skim milk medium. This medium, used for viable medium was 0.03% Na, S-9H20. Carbohydrate solu- counts, contained 0.2 ml of sterile skim milk added to tions were autoclaved under CO, to minimize alkaline 4.25 ml of rumen fluid-agar medium. destruction during sterilization. Sulfide and carbohy- CC medium. Concentrated cells medium (CC me- drate solutions were injected aseptically and anaer- dium), relatively free of protozoa and plant material, obically into the melted medium held at 48 C. The was prepared by allowing the rumen liquor, as osmotic pressure of the media used was about 7 collected, to incubate at 38 C. Fermentation gases atmospheres, and the pH was 6.8. carried plant matter to the surface, the protozoa sank Skim milk was autoclaved in 10-ml volumes under to the bottom, and the rumen fluid containing N, in butyl-stoppered tubes. bacteria was drawn off from the middle part of the container with a 50-ml pipette. ' Present address: Department of Plant Biology and This rumen fluid was centrifuged at 25,000 X g for Microbiology, Queen Mary College, Mile End Road, 15 min. The pellet was suspended with CRF medium London El, England. to one-tenth the original volume, and one part of this 171 172 ROBINSON AND HUNGATE INT. J. SYST. BACTERIOL. 1OX concentrated bacterial suspension was added to gas was helium at a flow rate of 13 mllmin. The one part of mineral salt solution and one part of column temperature was 160 C. deionized water. Determination of fermentation products with uni- Tryptone (176, Difco), 3% tryptone-soy broth formly labeled C-galactose. The ’ C-galactose was (Oxoid), 1.3% nutrient broth (Oxoid), and 3.7% brain supplied in 20% ethanol solution. A 12-pliter amount heart infusion (Oxoid) were prepared under 100% was transferred with a micro-syringe to each of two CO, and buffered with 0.5% NaHCO,. culture tubes. The ethanol was evaporated in a stream Isolation procedures. Samples of rumen fluid were of air, and 0.6 ml of 9% galactose was added to each siphoned from a fistulated Jersey heifer and poured tube under CO,. Sterile, anaerobic RF medium was into a flask until it was almost full. The flask was prepared, and 9.4 ml of the medium was added to the stoppered, insulated, and quickly transferred to the galactose which had been sterilized under CO,. laboratory. Oxygen-free CO, was bubbled through the Control tubes lacked the Cgalactose. Initial 1-ml sample to displace the air above it, and 0.5 ml of the samples were removed after inoculation with 0.1 ml of stirred fluid was diluted with 1-ml syringes through a CRF/milk culture, and the cultures were incubated tubes of anaerobic medium. Lower dilutions were in at 43 C for 1 week. sterile 33% RF medium and higher ones were in the A 1-ml amount of 10 N sulfuric acid was injected CC medium containing 1.5% agar, which was melted through the stopper of the culture tube, and the tube and held at 48 C. The viable count was not detectably was shaken. The released CO, was collected in a changed by holding as long as 2 h at this temperature water-lubricated 10-ml syringe while the tube and before rolling the tubes in ice water. Tubes were syringe were shaken to insure equilibrium of CO, always rolled within 30 min after inoculation. The between gas and liquid. The volume of gas in the cultures were incubated at 39 C or 45 C and examined syringe as well as the pressure were noted at room periodically for clear zones due to partial digestion of temperature, and the gas was injected into a stoppered the rumen bacteria in the medium. flask containing 10 ml of 2,2,2-nitrilotriethanolamine, Pure cultures were obtained by picking individual a sample of which was assayed with the scintillation colonies with a Pasteur pipette drawn out to a thin counter . capillary and subculturing in agar medium until, in Samples of the acidified culture (0.5 or 1.0 ml) two successive dilution series from a colony in high were chromatographed on a Celite column by the dilution, only the clearing colonies appeared and their method of Wiseman and Irvin (13). Samples of the numbers in successive tubes decreased in approximate eluant were assayed for 4C in a liquid scintillation agreement with the dilution. There were no problems counter (Nuclear-Chicago Mk 11). with contamination. The C in trichloroacetic acid-insoluble material Hydrogen measurement. Hydrogen was measured was assayed by collecting the 5% cold trichloroacetic on a Perkin-Elmer thermal conductivity vapor frac- acid precipitate from a sample of culture on a 0.45-pm tometer 154B with a silica gel column and N, carrier membrane filter (Millipore Corp.). The filter was gas. The instrument detected lo3 pmol of H, in a washed with 5% trichloroacetic acid, dried, and 0.5-ml sample. dissolved in the counting fluid, 0.4 g of 1,4 bis[2-(5 The volume of excess gas in the culture and control phenyloxazolyl)] -benzene (POPOP, Packard Corp.) tubes was measured at atmospheric pressure with a 5- and 4 g of 2,5 diphenyloxazole in 1 liter of toluene. or 10-ml syringe and injected back into the tube after Preparation of samples for electron microscopy. each measurement. A sample in excess of 0.5 ml was Colonies in CRF-agar were prefixed for 1 h in a then drawn into a water-lubricated, sterile 1-ml glass Kellenberger buffer containing 0.2% glu tar aldehyde, syringe through a 2lgauge needle. The tip of the 0.1 g of OsO,/lO ml, and sufficient sucrose to make needle was drawn into the rubber of the stopper to the buffer and the medium isotonic. The buffer allow the gas to come to atmospheric pressure. The contained 5 ml of Verona1 acetate (2.94 g of sodium volume was noted, the needle was withdrawn, the barbiturate, 1.94 g of hydrated sodium acetate, and volume was reduced to 0.5 ml, and the gas was 3.4 g of NaCl in 300 mI), 13 ml of water, 0.25 mi of injected into the fractometer.

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