40 L. LEON CAMPBELL, JR. 



The general method of study of spore germination just described has been 

 illustrated in tables in a number of publications (Foster and Wynne, 1948; 

 Wynne and Foster, 194Sa, 1948b; Wynne and Harrell, 1951; Wynne, 1952; 

 Wynne et al, 1952; Wynne et al, 1954). In germination of five strains of 

 Clostridia in buffered glucose at 37 °C, a post-incubation heating period to 

 eliminate vegetative cells has proved unnecessary (Wynne et al, 1954). 

 Germinated spores apparently become non-viable from nutritional inade- 

 quacy of the medium (Powell, 1951) or during dilution and plating pro- 

 cedures. 



Recently in our laboratory considerable effort has been expended in the 

 study of an unexpected phenomenon. When spores of mesophilic Clostri- 

 dium are incubated at 75°C in glucose carmelized by overautoclaving, the 

 level of recoverable spores falls rapidly (Wynne, Galyen and Mehl, 1955; 

 Wynne and Galyen, 1956), with essentially no decrease in incubation in 

 brain-heart infusion broth. This rapid decrease in heat resistant units is 

 generally essentially complete in from 20 to 120 minutes, depending on 

 the strain. Furthermore, the process appears to be logarithmic in 3 to 5 

 test organisms, with a definite lag period of from 2 to 15 minutes. Turbid 

 spore suspensions incubated at 75°C in caramelized glucose have shown no 

 detectable loss in optical density. The vegetative cells of the five test species 

 do not grow at 50°C, and two of them do not grow at 48°C. 



We have been unable to explain the rapid loss in heat-stable units occur- 

 ring in overautoclaved glucose at 75 °C on any basis other than the occur- 

 rence of germination, and considerable evidence has been secured that ger- 

 mination does in fact occur. These findings have been published in abstract 

 form (Wynne, Galyen and Mehl, 1955; Wynne and Galyen, 1956) only, 

 but detailed publication is contemplated. 



References 



Evans, F. R. and H. R. Curran. 1943. The accelerating effect of sublethal 

 heat on spore germination of mesophilic aerobic bacteria. J. Bact. 46: 

 513-523. 



Fischoeder, F. 1909. Beitrage zur Kenntnis des Milzbrandes. Zentr. Bakt. 

 Parasitenk. Abt. I, Orig. 51: 320-417. 



Foster, J. W. and E. S. Wynne. 1948. Physiological studies on spore germi- 

 nation with special reference to Clostridium hotulinum. IV. Inhibition 

 of germination by unsaturated Ci.s fatty acids. J. Bact. 55: 491-501. 



Hills, G. M. 1949. Chemical factors in the germination of spore-bearing 

 aerobes. The effect of yeast extract on the germination of Bacillus an- 

 thracis and its replacement by adenosine. Biochem. J. 45: 353-362. 



Knaysi, G. 1945. Investigation of the existence and nature of reserve ma- 

 terial in the endospore of a strain of Bacillus mycoides by an indirect 

 method. .1. Bact. 49: 617-622. 



