68 CLARENCE F. SCHMIDT 



Informal Discussion 



Campbell: I would like to comment on the effect of L-alanine on germina- 

 tion and the relationship of this to colony counting as noted by Dr. Schmidt. 

 If spores of B. terminalis are placed in a medium which supports outgrowth, 

 the addition of alanine gives almost 100% germination in 5 to 10 minutes, 

 as evidenced by darkening under a dark phase contrast microscope. If you 

 follow the changes that occur over an 8 hour period, you find that only 20 

 to 30% of the germinated spores give rise to vegetative cells. The remaining 

 70 to 80% do not swell, elongate, or divide. However, in this 8 hour period 

 the vegetative cells have formed heat stable spores again. This complicates 

 the problem of counting colonies since the count will fluctuate as the heat 

 stable spores are formed. 



MuRTY: With regard to germination requirements, I wish to bring to your 

 notice what our experience has been with spores of B. terminalis. These 

 requirements can vary widely with the method of preparation and storage — 

 the so-called history of the spores — even when the same organism is used. 

 As a consequence, we do not call a suspension of spores clean and fresh 

 unless it satisfies the requirements that have been originally worked out for 

 this organism. Even freshly produced spore suspensions can differ in germi- 

 nation requirements if something went wrong with the washing or the medium. 

 We could not prevent germination by any of the so-called enzyme inhibitors 

 like arsenate, cyanide, etc. Furthermore, the spores would germinate even 

 under strictly anaerobic conditions. Heavy metal ions like copper, chromium, 

 or mercury, and the organic mercurials like phenylmercuric acetate and 

 phenylmercuric benzoate are the only ones we know of that can inhibit 

 germination. This inhibition could be reversed partially by washing and 

 adding -SH reagents. Church and Halvorson found that spores of B. termi- 

 nalis, which do not specifically respond to glucose, do so only under certain 

 conditions such as prior activation of the spores. One then wonders if at 

 least the first phase of germination, which cannot be inhibited by any of the 

 inhibitors of glycolysis or the TCA cycle or by anaerobiosis, is an energy 

 requiring process. We are inclined to believe that it is not an energy 

 requiring process. We found that, during germination, a large part of the 

 dipicolinic acid of the spores was released into the medium and that DPA 

 was also released on autoclaving the spores. We are therefore inclined to 

 believe that the first phase of germination is essentially some sort of a 

 depolymerization process, a view also shared by Powell and coworkers. 



Harlyn Halvorson: Spore germination has been described as an 

 exothermic reaction. The supporting evidence generally involves the ability 

 of spores to germinate either spontaneously or under conditions where energy 



