An integrated investigation of the sequence of events during the germination of Bacillus megateriwn spores produced on three different media-Liver "B" (LB), synthetic, and Arret and Kirshbaum (A-K)-is reported. Heat-activated spores were germinated in a mixture of glucose and Lalanine. For studies of dipicolinic acid (DPA) release and increase in stainability and phase-darkening, germination levels were stabilized by the addition of 2 mm HgCl2. Heat resistance was measured by conventional plating techniques and by a new microscopic method. The sequence (50% completion time) of LB spore germination events was: loss of resistance to heat and to toxic chemicals (3.0 min); DPA loss (4.7 min); stainability and Klett-measured loss of turbidity (5.5 min); phasedarkening (7.0 min); and Beckman DU-measured loss of turbidity (7.2 min). The time difference between 50% completion of stainability and complete phase darkening was 1.5 min, in excellent agreement with the microgermination time of 1.49 min as determined by observation of spores darkening under phase optics. Alteration of the sporulation medium modified the 50% completion times of these germination events, and, in some cases, their sequence. In the A-K spores, the rates of loss of heat resistance and DPA were substantially higher than those of the other germination events, whereas in spores produced in the LB and synthetic media all germination events followed an approximately parallel time course. This is discussed from the point of view of spore population heterogeneity and germination mechanisms. We present here a less cumbersome and more 1811

Bacillus tuegateriuiiii spores, groN-n on variously, supplemented media, had varying concentrations of P, Ca, MIn, or dipicolinic acid. Supplementation with CaC12 yielded spores with increased heat resistance; addition of i-glutamate, L-proline, or increase of the phosplhate concentration yielded spores wvit,h leduced heat resistance. Germination characteristics depended on both the sporulation medium and the germinant (glucose, i-alanine, L-leuicine. or K-NO3); pronounced differences were demonstrable with glucose and L-alanine, which