Culture of Serum-induced Spheroplasts from Vibrio cholerae

Spheroplasts of Vibrio choleraetypes Inaba, Hikojima, and Ogawa were produced in serum. An excess of lysozyme was added to expedite transformation, and CaCl2was used as the stabilizing agent. At the optimal time for each strain, when less than 1% classical rod forms were observed microscopically, samples were plated on both conventional and modified L-form media. No growth occurred on conventional media; L-form type colonies appeared on L-form media after 2 to 6 days of incubation. L-form colonies could be subcultured on conventional media and required from four to six passages for complete reversion to classical parent forms. Reaction mixtures of Hikojima and Inaba types were passed through membrane filters (0.45 μ); L-form colonies were grown from both strains after spheroplast transformation. Appropriate controls were negative. It is suggested that the replicative particle may be filtrable.