70 CLARENCE F. SCHMIDT 



that, in the so-called resting state, are killed by some of these time-tempera- 

 ture relationships, no matter how carefully they are selected. 



To define germination in terms of loss of heat resistance implies that heat 

 resistance is a constant characteristic which can be defined by some given 

 time-temperature relationship — but it isn't. 



Brown: Getting back to the germination process, I wonder if any of you 

 have considered the possibility that this could be the removal of an inhibitor 

 that permits the germination process to proceed. We have recently completed 

 some work that seems to indicate this and with your permission I would 

 like to briefly outline it on the board. 



We used Dr. Lund's mutant strain of Putrefactive Anaerobe Number 3679 

 that was described yesterday. The spores were grown in the spent medium, 

 treated with lysozvme to remove the vegetative cells, and washed 10 to 14 

 times with sterile distilled water using centrifugation to separate the spores 

 from the wa h water. 



Four methods were used to measure spore germination. The methods 

 used were: optical densitv changes as measured with a spectrophotometer; 

 refractivity changes as measured under the phase microscope; differences in 

 staining properties; and the loss of heat resistance. The loss of heat resist- 

 ance due to germination was determined by exposing the treated spores to a 

 temperature of 97 °C for 10 minutes. The number of surviving spores was 

 determined by a dilution count procedure using eugon agar as the recovery 

 medium. The count was then determined from the tables of Halvorson and 

 Ziegler. Statistical methods were employed to study the relation -hip between 

 the 4 measurements of spore germination. The correlation coefficients were 

 highly significant at the 1% level but the relationship was not high enough 

 to permit the easier measurements to be used in lieu of the viable count. 

 Consequently, the viable count was used in all of the experiments as a 

 measure of spore germination. 



A wide variety of organic compounds were tested for their ability to 

 germinate the mutant strain spores. Of the compounds tested, none were 

 able to change the refractivity of the spores during a 30-minute interval. It 

 was considered possible that a metal inhibitor could be blocking the germi- 

 nation process. Germination of the mutant PA 3679 spores was accomplished 

 by treatment with ethylene diamine tetraacetic acid (EDTA) at an optimum 

 concentration (M/180) when the clean spore suspension was adjusted to a 

 constant turbidity with the colorimeter. When the concentration of EDTA 

 was too high (M/10) no germination occurred. Other chelating agents were 

 tested but only the EDTA type compounds were effective. 



Tt was hypothesized that treatment of a spore suspension with stronger 

 solutions of EDTA (M/10) resulted in the removal of both an inhibitor and a 



