38 L. LEON CAMPBELL, JR. 



Wynne, E. S. 1952. Some physiological aspects of bacterial spore forma- 

 tion and spore germination. Bact. Rev. 16: 101-110. 



Discussion 



E. S. Wynne 



We agree with Dr. Campbell that development of turbidity is not a ser- 

 viceable criterion of spore germination. As pointed out earlier (Wynne, 

 1952), a number of workers have reported germination without appreciable 

 subsequent vegetative multiplication under a variety of experimental con- 

 ditions, including unfavorable oxidation-reduction potential (Knight and 

 Fildes, 1930) inadequate nutrients (Knaysi, 1945; Powell, 1951), and the 

 presence of appropriate concentrations of penicillin (Wynne and Harrell, 

 1951) or streptomycin (Wynne et al, 1952). On the other hand, salts of 

 Cis unsaturated fatty acids have been found to be potent inhibitors of ger- 

 mination, but much less effective against vegetative cell multiplication (Fos- 

 ter and Wynne, 1948). Finally, with putrefactive anaeiobe No. 3679, the 

 amount of germination occurring by the time of appearance of turbidity 

 has been found to vary with incubation temperature (Mehl and Wynne, 

 1951). 



In our laboratory, change in heat lability has been considered the most 

 workable criterion of germination (Wynne and Foster, 1948a; Wynne, 

 1952). The first investigator to employ loss of heat stability as a criterion 

 of germination appears to have been Fischoeder (1909), whose method was 

 a modification of a technique devised by Weil (1901) but was subsequently 

 discarded. Using spore suspensions of B. anthracis, Fischoeder removed ali- 

 quots at intervals during incubation and heated for 3 minutes at 80°C to 

 kill any vegetative cells resulting from germination. Plating then yielded 

 colonies representing only the heat-resistant ungerminated spores. Decrease 

 in the number of spores from the level present before incubation was used 

 to measure germination. Similar methods of study were employed by Evans 

 and Curran (1943) in their classical work showing the importance of pre- 

 heating spores before incubation for germination, and by Hills (1949). In 

 our studies, percent germination rather than numbers of ungerminated spores 

 has been used as a basis for quantitative measurement of germination, since 

 with this standard it is felt there is less likelihood of attaching a false sig- 

 nificance to small deviations (Wynne and Foster, 1948a; Wynne, 1952). 



As detailed elsewhere, (Wynne and Foster, 1948a; Wynne, 1952), the 

 method employed in our laboratory for studying germination with Clostri- 

 dia has generally involved a pre-heating period of 5-20 minutes at 75° C 

 for expelling dissolved oxygen and effecting any possible heat activation of 

 spores. After incubation in appropriate media under anaerobic conditions, 

 a second heating period of 20 minutes is employed to destroy units which 



