Physiological Studies on Spore Germination with Special Reference to Clostridium botulinum: I. Development of a Quantitative Method.

By far the majority of studies hitherto made on germination of bacterial spores have employed the appearance of visible turbidity as the criterion of germination. Obviously this technique can reveal no quantitative characteristics of the germination process and is therefore of value only in establishing that some germination does or does not take place. Even here its value may be questioned, since it has been clearly demonstrated that germination of spores of various organisms may occur without significant subsequent vegetative proliferation (Itano and Neill, 1919; Knight and Fildes, 1930; Knaysi, 1945; Knaysi and Baker, 1947). Furthermore, various environmental conditions imposed upon germinating spores may have no influence on the germination time but yet may alter appreciably the rate of subsequent vegetative development (Evans and Curran, 1943). Our own experiments confirm this finding. Direct microscopic counts have been used for quantitative studies of the germination of aerobic spores (Eckelmann, 1918; Curran, 1931), but such a procedure is unduly wearisome and not readily adapted to use with anaerobes. Also, with certain species, for example, Bacillus anthracis, it may be very difficult to establish microscopically a criterion of germination, as noted by Fischoeder (1909), Swann (1927), and Cook (1932). The outstanding physiological difference between spores and vegetative cells of any one organism, namely, heat lability of the latter at a temperature innocuous to the former, has long been employed in quantitative approaches to spore germination, since it is assumed traditionally that when a spore cell is so changed that it becomes heat-labile, germination has taken place (Weil, 1901; Fischoeder, 1909; Evans and Curran, 1943). Although Curran and Evans (1937, 1945b) have indicated that the heat-labile state may actually precede rupture of the spore wall and that some morphological changes characteristic of germination may occur prior to the loss of thermal resistance of the spore, heat differentiation of the germinated vs. the ungerminated spore appears to be the most practicable approach. The fact that a definite reproducible standard end point may be selected, viz., survival at a definite temperature for a definite period of time, even though somewhat arbitrary, outweighs the overlapping