In a previous paper, El-Bisi and Ordal (1956a) reported that increased levels of phosphate, in the growth and sporulation medium, significantly reduced the thermal resistance of spores of Bacillus coagulans var. thermoacidurans. It was postulated that the phosphate anion lessened the availability of divalent cations to the sporulating cells and that spores so produced possessed a reduced thermal resistance. A relationship between the calcium content and the degree of thermal resistance of bacterial spores has been suggested by Curran (1952) and by Sugiyama (1951). Williams (1929) reported that the addition of magnesium to peptone containing media yielded spores of Bacillus subtilis of increased thermal resistance. In this investigation, the effect of the divalent cations, calcium, magnesium, and manganese, added to the sporulation medium, was studied in more detail for their effect on the thermal resistance of spores of B. coagulans var. thermoacidurans. MATERIALS AND METHODS Test culture. B. coagulans var. thermoacidurans, American Type Culture Collection no. 8038, was used in this investigation. Stock cultures were maintained on thermoacidurans agar (Difco) containing additional 1 ppm MnSO4. Media. Spore crops were produced either on agar surface cultures or in broth shake cultures. Basal media used were thermoacidurans agar (proteose-peptone, 0.5 per cent; yeast-extract, 0.5 per cent; glucose, 0.5 per cent; K2HPO4, 0.5 per cent; and agar, 2 per cent) and thermoacidurans broth (the same components omitting the agar).

SUMMARY 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 usin...

A survey of several strains of Bacillus coagulans var. thermoacidurans revealed considerable variation in sporulation when the organisms were cultured on a variety of peptone containing media. Our culture of strain 43P was especially lacking in sporulation ability, although it sporulated when cultured on a medium such as tomato juice agar.

It is well known that the heat resistance of bacterial spores is influenced markedly by the nature of the medium in which the spores are heated. This fact is an important problem relating to the practical sterilization in bacteriology and in the food industry. Hence voluminous data on the heat resistance of