Synchronous sporulation in Bacillus megaterium and some aspects of commitment to sporulation.

Most previous attempts to obtain synchronous sporulation involved Bacillus cereus and serial transfers of heavy inocula (H. 0. Halvorson, J. Appl. Bacteriol. 20:305, 1957). Synchronous sporulation in B. cereus could be inhibited by the addition of actinomycin D during certain stages of the process [P. C. Fitz-James, Colloque, Intern. Centre Natl. Rech. Sci. (Paris) 124:529, 1963. The use of serial transfers in B. megaterium had not been reported, although a new filtration technique (H. Imanaka, J. R. Gillis, and R. A. Slepecky, J. Bacteriol. 93:1624, 1967) was successful. Synchronous sporulation can be obtained by serial transfers of B. megaterium, and the inhibitory effects of actinomycin D on sporulation are similar to the effects on B. cereus. B. megaterium (obtained from C. F. Robinow) was maintained on nutrient agar at 4 C and was grown in 25 or 50 ml of the Slepecky and Foster medium (J. Bacteriol. 78:117, 1959), using glucose instead of sucrose. Salts, buffer, and glucose were autoclaved separately and combined aseptically in 500-ml nephelometer flasks, which were incubated on a wrist-action shaker (300 arcs of 2.0 cm/min) at 30 C. The relationship between optical density (OD) of the culture, disappearance of glucose, and appearance of heat-resistant spores is shown in Fig. 1. The exhaustion of glucose coincided with the initial peak in OD. A similar relationship exists in B. cereus grown in a richer medium (F. Buono, R. Testa, and D. G. Lundgren, J. Bacteriol. 91:2291, 1966). The peak in OD was followed by a decline of 4 to 5 hr before the OD rose again for 6 to 8 hr as heatresistant spores (75 C for 15 min) were produced (final count, 1.0 X 108/ml = 100%). After this final rise, the OD remained essentially unchanged. Microscopic observation showed that the final rise in OD was not due to growth but to the formation of spores which remained in chains up to 30 hr (Fig. 2). Blending for 3 min after heating (Waring Blendor, high speed)