The dioecious nematode Pelodera strongyloides has been established in axenic culture. It has been maintained by serial subculture in films of nutrient media on agar and by continuous culture in media on glass fibers. Cultures cannot be maintained in deep media in test tubes, the adults being unable to mate without a suitable physical substrate. In axenic culture the optimal incubation temperature was 23 to 25 C; reproduction ceased at 33 C, and only dauer larvae survived 37 C. Axenic culture of Pelodera strongyloides Schneider, 1866 (Dougherty, 1953) in liquid medium was undertaken with the purpose of establishing cultures at 37 C, so that nutritional requirements at this temperature could be investigated. The possibility of culturing this nematode at 37 C was suggested by its xenic environment, bovine skin pustules (Levine et al., 1950). MATERIALS AND METHODS Pelodera strongyloides was obtained as a culture with mixed flora on blood agar from Dr. Norman Levine who had isolated it from bovine dermatitis. Culture media used were those developed for nutritional studies of Caenorhabditis briggsae: A. A basal medium of soy peptone and yeast extract, each at 3 g/100 ml, supplemented with heated liver extract at 20% by volume (Cryan et al., 1963), referred to hereafter as liver-peptone-yeast medium. B. A chemically basal medium supplemented with growth factor at 250 gug/ml and containing 8.2% Ficoll (Buecher et al., 1966), referred to hereafter as defined medium. C. A chemically basal medium supplemented with heated liver extract at 20% by volume. In initial tests, as indicated below, lower levels of supplement were used, i.e., 10% heated liver extract or 100 gug/ml growth factor. The media were used in 0.25-mi aliquots in 10by 75-mm test tubes, as a film on nutrient agar (Difco) slants in 16by 150-mm test tubes, or as a film on glass fibers in continuous culture vessels (Hansen and Cryan, 1966). Cultures were also carried on nutrient agar slants holding pieces of sterile rabbit kidney (Stoll, 1959). Transfers of individual eggs, larvae or adults were made by micropipette in a sterile chamber with the aid of a dissecting microscope. Axenization was initially carried out with a preparation of 10,000-unit penicillin and 1% streptomycin. AntiReceived for publication 11 October 1967. * Supported by NIH Grant AI 07359, formerly Al 00926. biotics were not added to culture media used subsequently. As a step toward axenization, a monoxenic culture with Escherichia coli was established. Loop transfers were made from the original xenic culture to nutrient agar slants preincubated for 2 hr with a film of inoculum from a broth culture of E. coli. After 12 days at room temperature, these cultures were transferred by loop to sterile nutrient agar slants and subcultured again 3 days later. Scrapings from the second subculture at 5 days were placed in antibiotic mixture in a BPI dish; adults and eggs were then removed to a second dish of antibiotics. The next day eggs and newly hatched larvae were removed to 0.066 Mi potassium phosphate buffer, pH 7, allowed to settle, and transferred by pipette to a nutrient agar slant inoculated with E. coli and preincubated at 37 C for 30 min. The culture was kept in a room where the temperature ranged 15.5 to 18 C. New larvae appeared on the 8th day. At 3 weeks a broth culture inoculated from this slant and streaked on agar showed no growth other than E. coli. This monoxenic culture has been maintained for 2 years by loop transfers at 3to 4-week intervals. It provided the source of worms for successful axenization.
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Open Access
Oct 1, 1970
James S Murphy 
