Isolation and growth of Leptospira on artificial media.

Studies on virulence of leptospires would probably be most fruitful if the leptospires could be grown in chemically defined media. Nonpathogenic leptospires found inwater, Leptospira biflexa, have been described in Bergey's Manual, and in the 1962 minutes of the taxonomic subcommittee on Leptospira of the International Committee on Bacteriological Nomenclature, as being saprophytic and able to be grown in simple and serum-free media. Viable leptospires were described for the first time by S. B. Wolbach and C. A. L. Binger (J. Med. Res. 30:23,1914) from pond water; however, these authors were unable to cultivate them. Although J. E. Walker (J. Infect. Diseases 41:164, 1927), V. T. Dimitroff (J. Infect. Diseases 40:508, 1927), and H. Noguchi (N. Y. State J. Med. 22:426, 1932) cultivated leptospires from water in this country, none of their cultures is now available. Nutritional studies have been performed in the absence of serum by B. Babudieri and 0. Zardi (Z. Vitamin-, Hormon-, Fermentforsch. 11:299, 1961) on leptospires initially cultivated from water on egg yolk and rabbit serum (B. Babudieri and I. Archetti, Rend. Ist. Super. Sanita 10:962, 1947). Growth of parasitic leptospires in the absence of serum has been studied by H. Vogel and S. H. Hutner (J. Gen. Microbiol. 26:223, 1961) and 0. H. V. Stalheim and J. B. Wilson (J. Bacteriol. 88:48, 55, 1964); however, growth was less than one usually sees in serum, and was considered by the latter authors to result from mutation. The CDC (origin unknown) and Patoc (B. Babudieri and I. Archetti, Rend. Ist. Super. Sanita 10:962, 1947) strains of L. biflexa have been found to require serum in our laboratory, and, to our knowledge, all cultures of leptospires available have been initially grown and transferred many times in serum. To isolate leptospires which would grow to good yields in a defined medium, surface waters were plated on three enrichment media. Serum agar contained (per 100 ml): pooled rabbit serum (Pel-Freez Biologicals, Rogers, Ark.), 10.0 ml; tryptose-phosphate (Difco), 0.2 g; and agar (Difco), 1.0 g; the pH was 7.6. The composition of SM-1 and SM-2 agar is given in Table 1. Water samples were held an average of 3 days at room temperature (23 C) before being filtered sequentially through Millipore filters of decreasing porosity. Pore sizes depended on gross appearance of the water, and the smallest pore size was always 0.45 ,. Filtrates were incubated at room temperature for an average of 21 days. A small drop of each filtrate was placed in the center of each plate of medium, and plates were incubated at 30 C. Of 106 water samples plated on serum agar and SM-1, leptospires were grown from 15 of the samples on serum agar, and from the same 15 plus 2 additional samples on SM-1. Growth appeared in 7 to 9 days as thin veils which moved away from other bacterial colonies where the drops of water had been placed. Growth was transferred from these veils to other plates and streaked for isolation. Cultures were purified by restreaking isolated colonies several times (C. D. Cox and A. D. Larson, J. Bacteriol. 73: 587, 1957; A. D. Larson et al., J. Bactkriol. 77:361, 1959). After the above filtrates had been permitted to stand at room temperature for 240 days, five still gave leptospiral growth on serum