114 



MOLECULES, VIRUSES, AND BACTERL«l 



germinated spores. We found that in such cultures sporulation set in 

 very early; in fact, the process was complete in about seven hours. By 

 staining an hour or so before any dipicolinic acid was synthesized, we 

 obtained what appeared to be normal spores. Furthermore, the synthe- 

 sis of dipicolinic acid was complete, or nearly so, before any of the 

 spores had developed heat resistance. Heat resistance developed ap- 

 proximately an hour after the synthesis of DPA. The development of 

 a spore-like structure ( this study would indicate ) occurs independently 

 of the synthesis of DPA, and this precedes the development of heat 

 resistance. This is shown graphically in Figure 1. 



When we attempted to repeat this with the aerobe B. cereus, our 

 cultures would lyse about the time they should be producing spores. 

 Investigation showed this was due to a lack of oxygen ( Nakata, 1959 ) . 

 By using a very heavy inoculum we developed a condition in which 

 the demand for oxygen exceeded our ability to dissolve oxygen in the 

 water. This prompted us to make a study of the oxygen demand of 

 cultures during various stages of growth and sporulation. The results 

 of this study are shown in Figure 2. This shows that the oxygen de- 

 mand curve is bimodal. The first peak in this curve occurs about the 



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DPA IN Clostridium roseum 37 ° C 



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Figure 1. The relationship of heat resistance— total viable count— spores by stain— syn- 

 thesis of DPA in Clostridium roseum 37° C. 



