EFFECTS OF CARBOHYDRATES, PROTEINS, AND BACTERIAL CELLS IN THE HEATING MEDIA ON THE HEAT RESISTANCE OF CLOSTRIDIUM SPOROGENES
It is well known that the heat resistance of bacterial spores is influenced markedly by the nature of the medium in which the spores are heated. This fact is an important problem relating to the practical sterilization in bacteriology and in the food industry. Hence voluminous data on the heat resistance of Clostridium botulinum and Clostridium porogenes in various kinds of foods or food juices have been published (Townsend et al., 1938; Reed et al., 1951; Reynolds et al., 1952). From these data, it can be easily pointed out that the resistance value of a strain in a food is larger than that in phosphate buffer, whereas in another food the resistance value of the same strain is smaller than that in neutral phosphate buffer. Although the pH of the heating media has a marked influence on the thermal death rate of bacteria, it appears that the cause of such a difference in resistance values of a strain cannot be attributed wholly to the variation of pH values of the foods (Sognefest et al., 1948). In fact, there are evidences that some of the ingredients of food might exert a protective effect or a death accelerating effect for the organism during the heating. Weiss (1921) reported that the longer survival time was obtained when the spores of C. botulinum were suspended and heated in the foods with the higher sugar concentration. The similar protective effect of sugars on the bacterial spores has been demonstrated by Anderson et al. (1949) and Sugiyama (1951). Murray and Headlee (1931) found that the presence of peptone in the heating medium had shown sometimes a protective effect for the spores of C. tetani. Recently, Kawai and Tanaka (1952) have shown that several kinds of higher molecular substances, such as gelatin, starch, gum arabic, and alginic acid, have the protective effects for the spores of Bacillus mesentericus and Bacillus brevis. The test organisms used by these workers are relatively less resistant than the most resistant food spoilage bacteria, and the kinds of the substances tested on each strain are rather limited. Thus it seemed important to study the effects of various substances on the most resistant strain. Using the spores of Cameron's putrefactive anaerobe 3679 (Clostridium sporogenes, ATCC 7955), the effects of various carbohydrates, proteins, and bacterial cells were studied. In the previous papers Amaha (1952a,b, 1953) has clearly indicated that the thermal death time of spores of certain strains is influenced markedly by the nature of the recovery media. In general the more favorable was the nature of a medium for the development of heated spores, the longer was the survival time. Hence, it should be considered that the substances added in the basal heating medium have the two stages in their action to the spores; the first, during the heating time, and the second, after the substances were transferred in the subculture medium. In this paper an attempt was made to distinguish the two effects, i.e., the first effect, a true protection, and the second effect, a false protection, of each of substances tested.
Physiological Studies on Spore Germination, with Special Reference to Clostridium botulinum
Unsaturated C18 fatty acids, particularly oleic, are known to inhibit the growth of a great variety of bacteria (see Humfeld, 1947, for literature summary). In contrast, virtually nothing is known of the effect of such agents on the germination of spores preparatory to vegetative development. Castor soap has been reported inhibitory to Clostridium botulinum (Gibbs et al., 1926), but it is not clear whether spores or vegetative cells or both were employed as inoculum. In a previous paper (Wynne and Foster, 1948a) we observed that incorporation of 0.1 per cent soluble starch in the spore-counting medium resulted in substantially higher counts on a pasteurized suspension of C. botulinum spores than nonstarch control media, and furthermore that the characteristic dormancy of botulinum spores was eliminated under these conditions. The starch evidently neutralizes some factor(s) inimical to germination of the spores. Ley and Mueller (1946) studied a similar situation in which a factor present in ordinary agar was inhibitory to gonococci and also was neutralized by small amounts of starch. The inhibitory material in agar had properties similar to oleic and stearic acids. We have found that small amounts of oleic, linoleic, and linolenic acids inhibit the germination of Clostridium spores, that starch neutralizes this action, and that the presence of these acids in complex natural materials may account for the depressed germination rate and counts in the absence of starch.
Physiological studies on spore germination with special reference to Clostridium botulinum; 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