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just as the rise in pH became evident), were washed twice in 0.1 M potassium phosphate buffer (pH 6.4), and were suspended in various media at a concentration equivalent to that of the original culture. The more pertinent results are summarized in Table 2. Results comparable to the G medium control were obtained in a buffered medium containing L-glutamic acid, L-methionine, lactic acid, and a mixture of inorganic salts (CDS medium). Favorable results were also observed in a medium containing acetic acid together with smaller amounts of lactic acid. These acids, added in the concentration at which they are normally produced from glucose during growth, were shown to be rapidly utilized once sporulation begins (Nakata, J. Bacteriol. 86:577, 1963). The replacement of lactate with equimolar concentration of acetate, however, was not consistently successful. These observations, coupled with those demonstrating a rapid conversion of lactate to acetate and carbon dioxide during the prespore stage (unpublished data), suggest a requirement for a reducing potential, together with acetate, for sporogenesis. In this CDS medium, lactic acid is added in an amount nearly equivalent to the combined molar concentration of acetate and lactate normally produced from 0.2% glucose. The spores produced in CDGS or CDS media are microscopically indistinguishable from those produced in the G medium, and survive 20 min at 80 C without loss in viability. Only slight differences were noted in their dipicolinic acid (DPA) content, as determined by the method of Janssen et al. (Science 127:26, 1958). At pH 6.2, the CDGSand CDS-grown spores contained 7.41 and 7.40% DPA, respectively, as compared with 6.44% for spores produced in G medium buffered at the same pH. At pH 7.0, the DPA contents were 6.30, 6.67, and 6.33%, respectively, for CDGS, CDS, and G medium spores.

SELECTIVE MEDIUM FOR ISOLATION OF MIMA AND HERELLEA ORGANISMS