Leptospiral Selection, Growth, and Virulence in Synthetic Medium

Stalheim, O. H. V. (National Animal Disease Laboratory, Ames, Iowa). Leptospiral selection, growth, and virulence in synthetic medium. J. Bacteriol.92:946–951. 1966.—The need for protein in leptospiral cultural medium may be circumvented by the use of strains which tolerate the lytic activity of polyoxyethylene sorbitan monooleate (Tween 80), a relatively nonlytic source of essential fatty acids. In an otherwise adequate medium, the primary function of a serum protein (bovine albumin fraction V) in the cultivation ofLeptospira pomonawas detoxification of fatty acids. Treatment to destroy or block end groups (amino, sulfhydryl, or hydroxyl) did not impair this function, but, after treatment with trypsin, albumin was inactive. Synthetic and derived peptides or polyvinylpyrrolidone did not substitute for albumin.L. pomonagrew in medium with surface tension values of 44 to 58 dynes/cm2; after growth, the values were increased slightly (5 to 8). The growth responses did not correlate with the surface tension of the medium, but they were in proportion to the concentration of Tween 80. Of six strains ofL. pomona, five were transferred from medium containing rabbit serum and were subcultured in Tween synthetic medium (TSM) containing low, nonlytic concentrations (0.002%) of Tween 80. The poor antigenicity ofL. pomonain carbon-limited TSM was associated with a deficiency of those carbonaceous cellular components which were extractable with 50% ethyl alcohol. After as few as four subcultures in TSM,L. pomonatolerated higher concentrations of Tween 80 (0.06% was optimal; MTSM). If grown on a shaker, the rate and amount of growth and the antigenicity ofL. pomonain MTSM equaled that in medium supplemented with rabbit serum. After cultivation in MTSM, all of the five strains were avirulent when administered to hamsters, guinea pigs, and swine. They were still avirulent after three subcultures in complex media or after two serial passages in hamsters.