BACTERIAL Sl'OKK GERMI^'ATIO^ 35 



tigators have employed a 470-530 lUfi band filter (Levinson and Sevag, 

 1953), a 420 mfi filter (Church et al, 1954) or a 540-590 m;;. filter (Man- 

 dels et al., 1956) to follow the changes in optical density. This method 

 measures the loss in refractilitv of spores and has been correlated with the 

 appearance of translucent areas in the spores when observed by electron 

 microscopy (Hachisuka et al, 1954, 1955). A high degree of correlation 

 also exists between this technique and those which measure dye uptake, 

 heat lability, and darkening under a phase contrast microscope. The major 

 advantage of this method is the speed with which quantitative germination 

 data can be obtained. 



Manometric methods. Manometric methods, in conjunction with other meth- 

 ods, have been used to measure the effect of substances which stimulate the 

 basal respiration of spores. The marked increase in respiratory activity of 

 germinating spores has been shown to compare well with other methods 

 of determining germination (Levinson and Sevag, 1953; Levinson and 

 Hyatt, 1955, 1956; Hachisuka et al, 1956; Mandels et al, 1956). An excel- 

 lent discussion of this technique has been presented recently by Levinson 

 and Hyatt (1956). 



Definitions of Germination 



In early studies on spore germination, most workers defined germination 

 as the entire transition from the resting spore to an actively dividing vege- 

 tative cell. This view is still held by some modern investigators. Thus, 

 Knaysi (1951) defined germination of the endospore as "a process involv- 

 ing growth and [which] takes place under conditions that favor vegetative 

 growth." Fitz-James (1954) defined germination as the period "extending 

 from the resting stage of the 'mononculeate' spore to the vegetative or 'bin- 

 ucleate' cell." More recently Fitz-James (1956) has divided the germination 

 process into two parts: "The initial activation of the resting spores into a 

 respiring cell and the period of growth leading up to the formation of a 

 young vegetative bacillus." Preuner (1951) suggested the term "prevege- 

 tative" for the developmental sequence leading up to the vegetative form. 



Most investigators have recently defined germination in terms of some 

 convenient and easily demonstrable stage. Thus, germination has been de- 

 fined to include loss in heat resistance, stainability, decrease in optical den- 

 sity, darkening under a phase contrast microscope, marked increase in re- 

 spiratory activity, emergence from the spore coat, or the beginning of cell 

 division. Using stainability and increase in respiratory activity as criteria 

 of germination, Levinson and Sevag (1953) divided germination into two 

 phases: (a) "pregermination" or the process occurring when the spore 

 "becomes stainable and has started to consume oxygen, but before it has 

 elongated and become typically bacillary in shape," and (b) "germination" 



