Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.

Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a

Homogeneous fragments of exosporium were extricated in centigram amounts from dormant spores of Bacillus cereus and analyzed for intrinsic constituents. The membrane proved to be chemically complex but not unique, consisting mainly of protein (52%), amino and neutral polysaccharides (20%), lipids (18%), and ash (4%). Seventeen common amino acids were identified by chromatography, and were present in usual proportions except for low levels of cystine-cysteine, methionine, tyrosine, and histidine. Glucosamine was the only amino sugar, and glucose and rhamnose were the principal neutral sugars. The lipid fraction contained 5.5% cardiolipin as the only phospholipid, 12.5% neutral lipids, and at least 19 fatty acids, among which normal C16 and C18 ones predominated. Calcium and phosphorus occurred in the ash. Small amounts of teichoic, ribonucleic, and dipicolinic acids were believed to represent contamination.

Protoplast wet densities (1.315 to 1.400 g/ml), determined by buoyant density sedimentation in Metrizamide gradients, were correlated inversely with the protoplast water contents (26.4 to 55.0 g of water/100 g of wet protoplast) of nine diverse types of pure lysozyme-sensitive dormant bacterial spores. The correlation equation provided a precise method for obtaining the protoplast water contents of other spore tpes with small impure samples and indicated that the average protoplast dry density was 1.460 g/ml.