Mildred T. Hyatt, Hillel S. Levinson
1961 Journal of Bacteriology  
Spores of Bacillus megateriumn germinate in the presence of glucose or L-alanine and such germination is stimulated when spores are heated before incubation with these substrates (Hills, 1950; Powell, 1951; Levinson and Sevag, 1953; Church, Halvorson, and Halvorson, 1954). Furthermore, the addition of small amounts of L-alanine increases the germination rate of spores in glucose (Levinson and Hyatt, 1955) . The present report is concerned with previously unpublished details of the influence of
more » ... f the influence of varying combinations and concentrations of glucose and L-alanine on the extent and rate of germination, and on the modification of these spore germination requirements by heat. The addition of KNO3 to spores in L-alanine or glucose also stimulates initial germination rate, and in some cases increases total germination. Only heated spores germinate in KNO3 by itself. Glucose supports postgerminative development of spores, but L-alanine does not. However, spores germinated in L-alanine are viable, and capable of subsequent growth, if glucose is added. MATERIALS AND METHODS Spores of B. megaterium (QMI B1551) were grown on charcoal-treated liver broth, harvested by centrifugation, washed at 4 C, and dried from the frozen state (Levinson and Sevag, 1953) . A pool of lyophilized spores, sufficient for all the experiments described here, was prepared in September, 1958. These spores were stored at 4 C, with no change in their germination requirements attributable to "aging" noted. Spores were suspended in a buffer containing KH2PO4 and NH4H2PO4 (25 mm each) adjusted to pH 7.2 with KOH. When indicated, spore suspensions were heated for 10 min in thermostaticallv controlled water baths. Unless otherwise stated, spores were used at a concentration of 1 mg (5 X 108 spores) per ml. All concentrations given are final, and are expressed as millimolar (mM). For the study of rate and extent of germination, 2.4 ml of unheated or heated spore suspensions (pH 7.2) and 0.6 ml of varying concentrations of substrates were shaken in 50-ml flasks in a water bath at 30 C. K2SO4 (0.4 mM) was added to the phosphate buffer when a medium suitable for postgerminative development was required (Hyatt and Levinson, 1957 Levinson, , 1959 . At intervals, aliquots of spores were stained with dilute methylene blue and examined microscopically. Since essentially no spores stained in the absence of added substrate, stainability was used as an index of the percentage germination (Levinson and Hyatt, 1960) . Glucose utilization during germination of spore suspensions (30 ml in 500-ml flasks) was estimated by assay of the glucose remaining in supernatant fluids, according to the dinitrosalicylic acid method of Sumner (1925) or by Miller's (1959) modification of this method for microquantities of glucose. RESULTS M11odification of germination requirements by heat. Heating of spores of B. megaterium for 10 min at temperatures from 52.5 to 70 C reduces the concentration of glucose or L-alanine required for their germination (figure 1). Since the maximal effect was obtained by heating spores at 60 C, only this temperature was used in subsequent studies. Heating for 60 min further reduces germination requirements, but there is no spontaneous germination, without added substrate (Powell and Hunter, 1955), even when more concentrated spore suspensions (25 mg per ml) are heated at 65 C for 60 min and then centrifuged and resuspended. Supernatant fluids of centrifuged heated spores have no stimulatory effect on the germination of unheated spores, suggesting that the enhancing effect of heat shock on germination is on some compound, structure, or system within the spore itself, and is not due to stimulatory substances secreted into the medium. (1) Glucose:-We consider that unheated or 204 on May 9, 2020 by guest
doi:10.1128/jb.81.2.204-211.1961 fatcat:o6toot2p6ve5xjux3j3b45ciae