ALBERT

Notes: Efforts were continued in an attempt to assess quantitatively the behavior of certain cellular components while the spores were being exposed to moist heat, and to correlate such behavior with the thermal death reaction.Spores of Bacillus subtilis were preheated over prolonged intervals in an attempt to induce the release of cellular dipicolinic acid (DPA) without exercising serious loss of viability, and then to evaluate the subsequent effect of DPA release upon the thermal death rate of the preheated spores. At 45°C in 2.5mM PO4 buffer at pH 7, the net DPA released amounted only to 1.6 and 2.7% at the end of 20 and 30 hr. The treated spores exhibited no loss in viability and when heated at 98.5°C in 25mM PO4 buffer at pH 7, exhibited no differenee in their thermal death rates. Prolonging the heat treatment up to 9 days did not materially change the final results. To induce faster and greater DPA release, the above experiment was repeated at 80 and 90°C. At 80°C the net DPA released was 3.6 and 4.1% at the end of 3 and 8.5 hr, without loss in viability. However, the treated spores exhibited some reduction in their thermoresistance at 98.5°C. At 90°C greater amounts of DPA were released, but were associated with substantial loss in viability, and the surviving spores exhibited marked reduction in their thermoresistance at 98.5°C. These observations suggest the existence of DPA in more than one structural form; free or loosely bound to the spore structure and easily exuded upon mild heating or even standing under refrigeration; and a second more strongly bound form which required severe heating for its release. It is this latter form which might be associated with the mechanism of thermoresistance. Along with DPA, following the same pattern but at much higher rates, ninhydrin-positive material was exuded during sublethal heating at 80, 85, and 90°C. It was suggested that such material might be of similar nature to that exuded during germination of Bacillus species as described by Powell (1957).Spore suspensions of Bacillus coagulans (thermoacidurans) of varied thermostability were heated in 25mM phosphate buffer at pH 7, at 95°C and both the rate of death and DPA release were established. Results showed a difference in kinetics between the two reactions. Death progressed at higher rates than DPA release. Higher rates of death were associated with higher rates of DPA release. Spores of same strain were heated in both water and 10mM glyeylglycine, a death accelerating agent, at 100°C, sampled at intervals and analyzed for survival, dry weight, calcium, manganese, magnesium and DPA. Results showed the death of spores and their concurrent exudation of DPA and divalent cations were both markedly accelerated in the presence of glycylglyeine. The 25-min survival and exudate levels in glycylglyeine were near equivalent to those at the 65-min levels in water.The kinetics of death and the concurrent release of DPA and calcium were further assessed in more detail in 5mM phosphate buffer at pH 7 at 96 and 99°C using spores of B. coagulans (thermoacidurans). Death again progressed at a higher rate than that of either DPA or calcium release. Based on the molar ratio of Ca/DPA released in the supernataut, the initial ratio was always greater than 1, then rapidly dropped. and eventually plateaued at a value of less than 1. Similar patterns of death and calcium and DPA release were obtained for B. cereus when heated at 86°C.All of the above observations demonstrate the association of the thermal death reaction with the exudation of ninhydrin-positive material, DPA and divalent cations into the heating menstruum. The exact relationship between such cellular components and the spore mechanism of thermoresistance remains inconclusive.