. Internet address: leland@lenti.med.umn.edu.Brevinema for the single species that has been identified so far, Brevinema andersonii.
MATERIALS AND METHODSBacterial strains and culture conditions. The strains of Brevinema andersonii which we used are shown in Table 1. The spirochetes were cultured in a modified BSK medium (2) which is referred to below as shrew-mouse spirochete medium. Shrew-mouse spirochete medium was prepared as follows. To 900 ml of distilled water we added 3 g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Sigma Chemical Co., St. Louis, Mo.), 5 g of dextrose, 3 g of neopeptone (Difco Laboratories, Detroit, Mich.), 1 g of tryptone (Difco), 3 g of TC yeastolate (Difco), 0.8 g of sodium citrate, 2.2 g of sodium bicarbonate, 0.2 g of catalase (Sigma), and 0.4 g of cysteine (Sigma). After these components were fully dissolved, 100 ml of 10 X 1066 CMRL tissue culture medium (Gibco, Grand Island, N.Y.) was added, and the pH was adjusted to 7.6. The medium was then filter sterilized, and 35 ml of sterile fetal calf serum (Gibco) and 35 ml of bovine embryonic fluid (Sigma) were added. Finally, 200 ml of sterile 7.5% gelatin (Difco) was added before dispensing. The final pH was 7.4. This medium was dispensed into 7-ml tubes; each tube was filled to 95% of its capacity and tightly capped to provide a microaerophilic environment. Cultures were incubated at 30°C. The numbers of cells were determined by using a Petroff-Hausser counting chamber.Electron microscopy. For transmission microscopy, spirochetes were harvested by centrifugation at 10,000 X g, washed twice in phosphate-buffered saline (PBS) (pH 7.4) at 4"C, and resuspended in a small volume of PBS. Specimens were placed on Formvar-coated grids and stained for 1 min with 1% phosphotungstic acid (pH 7.4). Specimens for scanning microscopy were prepared by pretreating 10-mm glass coverslips with 1% bovine serum albumin and coating the preparations with cells for 20 min at 37°C. The cell preparations on coverslips were then immersed in 0.2% glutaraldehyde, washed in Hanks balanced salt solution, immersed in 1% glutaraldehyde, washed, and finally treated with 1% osmium tetroxide; in each fixation step the preparation was incubated at 4°C for 1 h. The cells were then dehydrated by using 15-min incubations in serially increasing concentrations of ethanol from 70% to absolute. The specimens were removed from ethanol and immediately subjected to critical point drying. Samples were then sputter coated and examined by conventional methods.Gas chromatographic analysis of cellular fatty acids. Cells were harvested at the mid-exponential growth phase by centrifugation at 10,000 X g and were washed once in PBS (pH 7.4). Fatty acids were extracted, separated, and analyzed as described previously (19) by workers at Microbial ID, Newark, Del.