34 L. LEON CAMPBELL, JR. 



Direct microscopic counts. Direct microscopic counts have been employed 

 for quantitative studies of spore germination. Such procedures are tedious 

 and are not readily adapted to studies with anaerobes. Furthermore, they 

 lack precision since with some species it is extremely difficult to establish 

 a microscopic criterion of germination I Fischoeder, 1909; Swann, 1924; 

 and Cook, 1932). 



Heat lability. One of the most accurate methods for determining spore 

 germination utilizes the loss in heat resistance as a quantitative measure 

 of the number of germinated spores. Briefly, the method consists of heat- 

 ing a spore suspension at a time-temperature relationship which is lethal 

 to the germinating spores but not to ungerminated spores and plating the 

 survivors. A control suspension of ungerminated spores is treated similarly 

 and the percentage germination calculated. Another technique often em- 

 ployed is to heat the spores at the beginning of the germination experiment, 

 followed by a second heating after incubation in a suitable medium. The 

 difference in spore counts before and after incubation is used for calculat- 

 ing the percentage germination. Several precautions must be taken when 

 using this method. These have been summarized by Wynne (1952) and 

 need not be enumerated here. Some investigators have criticized this method 

 on the basis that it is detailed and time consuming. However, the fact that 

 a definite time-temperature relationship can be established as a reproduc- 

 ible end point of germination outweighs these disadvantages. 

 Stainahility. Uptake of stains such as methylene blue has been used as a 

 measurement of spore germination (Powell, 1950; Levinson and Sevag, 

 1953). In this method air-dried smears of spores are stained with an aque- 

 ous solution of methylene blue. Ungerminated, heat-resistant spores appear 

 as unstained, refractile bodies, while germinated, heat-labile spores stain 

 blue. The total number of spores and the number staining blue are counted 

 and the percentage germination calculated. 



Darkening under a phase contrast microscope. When observed under a dark 

 phase contrast microscope, ungerminated spores appear as refractile bodies 

 while germinated spores appear homogeneously black or very dark blue 

 (Pulvertaft and Haynes, 1951). This technique of studying germination 

 correlates well with stainahility and heat lability methods. This method is 

 also excellent for observing the changes which take place in the transition 

 of germinated spores to actively dividing vegetative cells. It should be 

 pointed out that spores which do not darken never develop into bacteria. 

 Decrease in optical density of spore suspensions. A very rapid quantitative 

 method of measuring spore germination is that of following the decrease 

 in optical density of spore suspensions at some specified wave length, such 

 as 610 mfx (Powell, 1950, 1951; Hachisuka et al, 1954, 1955). Some inves- 



