BACTERIAL SPORE GERMINATION 39 



have become heat labile. The total heating time at 75°C in the control tubes 

 without incubation is equalized with that of incubated tubes. Spore levels 

 are determined by plating into a counting medium such as Yesair's pork 

 infusion agar with 0.1% soluble starch and 0.5% BBL thioglycolate sup- 

 plement, or the yeast extract starch bicarbonate agar recently described 

 (Wynne, Schmieding and Daye, 1955), with an anaerobic overlay of 3-4 

 ml of the counting medium of 1.5% agar containing BBL thioglycolate 

 supplement. Counts are considered to represent ungerniinated spores. The 

 number of ungerniinated spores remaining after incubation is subtracted 

 from the level of spores prior to incubation, and the difference is considered 

 to be spores which have germinated. Percentage germination is then calcu- 

 lated by dividing the number of germinated spores by the spore level prior 

 to incubation, or after incubation in water or buffer. Generally, duplicate 

 or triplicate platings are made from triplicate tubes, with statistical analy- 

 sis of the data for significance. 



In testing a given agent or condition for possible effects on germination, 

 it has been emphasized that the length of the incubation period is impor- 

 tant (Wynne and Foster, 1948a; Wynne. 1952 L For stimulatory effects it 

 is desirable to use an incubation period sufficiently brief to insure relatively 

 little germination in the controls. On the contrary, inhibitory effects are 

 best shown with an incubation period of such length that germination in 

 the controls is essentially complete. However, the incubation period must 

 not be long enough for appreciable resporulation. Furthermore, it is essen- 

 tial that the test conditions be such that dormancy does not occur to any 

 significant degree (Foster and Wynne, 1948; Wynne and Foster, 1948a; 

 Wynne, 1952). 



In testing a given agent or condition for possible effects on spore germi- 

 nation, it has been emphasized in previous publications (Wynne and Har- 

 rell, 1951; Wynne et al, 1952) that a change in level of recoverable spores 

 may result from (1) an effect on germination, (2) a sporicidal effect, (3) 

 inhibition of colony development in the counting medium, or (4) any com- 

 bination of these. A sporicidal effect may be tested for by exposure to the 

 agent or condition under consideration in an environment in which germi- 

 nation does not occur, e.g., by incubation in water or buffer. Inhibition of 

 colony development in the counting medium may be detected by adding the 

 agent tested directly to the counting medium. It is possible to test for spori- 

 cidal effect and/or inhibition of colony development by a single control 

 consisting of spores in water or buffer containing the agent studied and 

 inci>bated for the same time as parallel systems containing spores in (1) 

 water or buffer, (2) germination medium, and (3) germination medium 

 plus agent (Wynne and Harrell, 1951; Wynne et al, 1952). 



