Distribution and correlation of events during thermal inactivation of Bacillus megaterium spores.

Aqueous suspensions of Bacillus megaterium QM B1551 spores were heated at temperatures from 75 to 85 C. The rapid initial viability loss, followed by a more gradual, almost exponential decline, was not due to mixed populations with discrete heat resistances. The slight "tailing" below 0.01% survival was not the result of heat adaptation. Loss of viability was more rapid than loss of dipicolinic acid (DPA) and germinability and, although these events could not be correlated by use of simple kinetic plots, they had similar activation energies (80 to 90 kcal/mole). Probability (probit) plots of per cent survival as a function of logarithmic time yielded not the single line expected, if the heat resistances of individuals in the population were log-normally distributed, but two straight lines intersecting at a survival level of 1 to 6%. Probit-intersects occurred at times ranging from 8 min for spores heated at 85 C, to 310 min at 75 C. Probit-intersects for DPA release and loss in germinability occurred at the same time as for survival, but at much higher levels of retention. There appeared to be two subpopulations, both log-normally distributed but with different mechanisms of kill. Ninety-four to 99% of the spores died via injury to the cell-division process but retained germinability; the remaining smaller subpopulation (1 to 6%) was nonviable because of loss of the ability to germinate.

Activation energy for glucose-induced germination of Bacillus megaterium spores.

The maximum germination rate of Bacillus megaterium QM B1551 spores in glucose increased, and the lag before its attainment decreased, with increasing germination temperature. The activation energy for germination (mu = approximately 20 kcal/mole), based on rate or on lag, was consistent with an enzymatic mechanism.

Effects of temperature on activation, germination, and outgrowth of Bacillus megaterium spores.

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.

Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination.

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.

Sequence of events during Bacillus megaterim spore germination.

Levinson, Hillel S. (U.S. Army Natick Laboratories, Natick, Mass.), and Mildred T. Hyatt. Sequence of events during Bacillus megaterium spore germination. J. Bacteriol. 91:1811-1818. 1966.-An integrated investigation of the sequence of events during the germination of Bacillus megaterium 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 l-alanine. For studies of dipicolinic acid (DPA) release and increase in stainability and phase-darkening, germination levels were stabilized by the addition of 2 mm HgCl(2). 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); phase-darkening (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.