M. P. Bryant
1952 Journal of Bacteriology  
A small spiral organism has been observed repeatedly in the rumen contents of cattle. These observations have been made by direct microscopic examination (Hastings, 1944; Baker, 1943) and through the use of culture techniques (Hungate, 1947; Sijpesteijn, 1948) . During the course of the present investigation similar organisms were found in the rumen of all cattle examined. A close examination showed that it was actually a spirochete. The ease with which it could be cultivated made it
more » ... y suitable for further study, and the paucity of experiments on the physiology of spirochetes made it seem profitable to investigate the characteristics of this strain from the rumen. ISOLATION The spirochete was isolated by using the anaerobic technique and medium for the isolation of cellulolytic bacteria described by Hungate (1950). One ml of a fresh sample of bovine rumen contents was serially diluted to 109 dilution in rubber stoppered culture tubes containing 9 ml of rumen fluid-cellulose agar. The medium was of the following percentage composition: rumen fluid, 40; cellulose, 1.0; agar, 1.0; resazurin, 0.0001; cysteine hydrochloride, 0.02; NaCl, 0.09; (NH4)2S04, 0.09; K2HPO4, 0.045; KH2PO4, 0.045; CaCl2, 0.009; MgSOt, 0.009; and NaHCO3, 0.5. The cellulose was prepared from absorbent cotton according to the procedure of Farr and Eckerson (1934). A 5 per cent suspension was ground with distilled water in a pebble mill for 72 hours. Sodium bicarbonate solution was sterilized by filtration and added to the medium after autoclaving. Oxygen-free carbon dioxide was obtained by bubbling the gas through a chromous acid solution. The ingredients of the medium, minus sodium bicarbonate, were placed in a round bottom flask and boiled to dissolve the agar and to drive off oxygen. At the same time carbon dioxide was passed into the flask by means of a Pasteur pipette connected by rubber tubing with the gas source. The Pasteur pipette was removed and the flask was rubber stoppered so as to prevent the entrance of oxygen. The rubber stopper was wired in place before autoclaving the medium. After sterilization the medium was cooled to 48 C. The rubber stopper was
doi:10.1128/jb.64.3.325-335.1952 fatcat:peijwn2my5bvpi3yjrc6izxnsa