Requirements for Production and Germination of Spores of Anaerobic Bacteria

Abstract

The important role of amino acids in spore germination was established by Hills (1949) and Stewart and Halvorson (1953). Their studies, however, were limited to the aerobic forms because the anaerobes could not be made to produce sufficient quantities of spores in media within a specified time. Using an autoclaved 1 per cent solution of glucose phosphate, Wynne et al. (1954) germinated anaerobic spores in 1 hr, but rapid germination in a medium of well-defined nutrients has not been reported. The investigation reported here had three phases: (1) the development of media and methods for growing and harvesting anaerobic spores, (2) the determination of the optimum conditions and minimal nutritional requirements for their rapid germination, and (3) a study of the effect of inhibitors on germination. MATERIALS AND METHODS Organisms. Cultures of Clostridium butylicum strain 6014, Clostridium roseum strain 6012 (both of the American Type Culture Collection), and Clostridium acetobutylicum were tested for their ability to sporulate in various media and under different conditions. Stock cultures maintained on brain heart infusion medium (Difco) were used to inoculate 150 ml of clear sporulation medium in 6 oz bottles. The sporulation medium (TS-5) giving optimum spore production contained trypticase (BBL), 1.5 per cent; glucose, 0.2 per cent; sodium 1 This research was supported in part by Swift and Company, Office of Naval Research, and taken in part from a dissertation presented by the senior author in partial fulfillment of the requirements for the Ph.D.

Factors Affecting the Germination of Spores of Clostridium botulinum Type E

“…Although we used the same controlling methods, our results disagreed with those obtained by Knight and Fildes [10] who found the limiting potential of Eh +110 mV at pH 7.2 for the germination of spores of C. tetani. Moreover, it was reported that anaerobic conditions were essential for the germination of spores of some clostridial species [8]. The discrepancies existing between our results and those obtained by other investigators may be attributed to either of the following two causes:…”

“…The specific compounds responsible for the rapid germination of spores of C. botulinum type E were L-alanine, glucose, lactate, and bicarbonate (CO2). The requirement of L-alanine for the germination of spores of Clostridia has been reported by several investigators [2,3,8]. The involvement of lactate as a spore germinant has recently been reported with C. bifermentans [3] and C. tetani [15].…”

Low Temperature Growth Characteristics of Clostridia

“…sporogenes (Uehara & Frank, 1965;Mehl & Wynne, 1951), Cl. roseum (Hitzman, Halvorson & Ukita, 1957) and Cl. bifermentans (Gibbs, 1964).…”

“…Anaerobic conditions were essential for the germination of Cl. roseum spores (Hitzman et al, 1957) and germination could be stopped at any time by theintroduction of air.…”

“…(I) L-a-alanine (1-44 mg/ml) ; L-phenylalanine (0.285 mg/ml) ; L-arginine (0.285 mgiml); sodium thioglycollate (0.05%); pH 7.0 (Hitzman et al, 1957); glucose (2% w/v); 0.02 M phosphate buffer, pH 7.0 (Wynne, Mehl & Schmieding, 1954) ; ~-a-alanine (60 mM); sodium pyrophosphate (35 mix); pH 8.5; i.e. optimal levels of the two constituents at the optimal pH in the study of Uehara & Frank (1965); (IV) calcium chloride (35 mm) ; dipicolinic acid (35 mx); pH 7.0 (Riemann, 1963).…”