The composition of the insoluble "integuments" and soluble "contents" fractions of spores of four Bacillus species of widely differing heat resistance were compared. Electron microscopy of thin sections was also used to determine and compare the morphological structures in the integument preparations. The soluble fractions of the thermophiles, B. coagulans and B. stearothermophilus, had a higher content of hexose and dipicolinic acid. The hexose content of both fractions of the four species was related to heat resistance. Integument fractions consisted chiefly of protein together with variable amounts of the mucopeptide constituents, a, e-diaminopimelic acid (DAP) and hexosamine. In the thermophiles the DAP and hexosamine were found chiefly in the insoluble integuments fractions, while in B. cereus and B. subtilis most of this material was soluble. Integument preparations, containing mainly protein with little mucopeptide, consisted chiefly of outer and inner spore coats, while preparations having more mucopeptide contained also residual cortical material and a cortical membrane (possibly the germ cell wall). The results suggest that spore integuments consist of mainly proteinaceous outer and inner coats together with variable amounts of residual cortex and cortical membrane which contain the mucopeptide material.

The exosporium of Bacillus cereus T was first observed as a small lamella in the cytoplasm in proximity to the outer forespore membrane (OFSM) near the middle of the sporangium. Serial sections, various staining methods, and enzyme treatments failed to show any connections between the small lamella and the OFSM. The advancing edge of the exosporium moved toward the polar end of the cell until the spore was completely enveloped. The middle coat was formed between the exosporium and the OFSM from a three-layered single plate or “belt,” consisting of two electron-dense layers separated by an electron-transparent layer. This “belt,” usually first observed toward the center of the sporangium, developed without changing thickness or appearance over the surface of the forespore. Between the middle coat and the OFSM, a layer of cytoplasm about 50-nm thick was enclosed by the developing coat; this became the inner coat. Electron-dense material was deposited on the outer surface of the middle coat to form the outer coat.

A B S T R A C TSpore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed. I N T R O D U C T I O NFollowing certain nuclear events (38, 39) the early stage in spore formation involves the enclosing of the spore nucleus by two membranes derived from the centripetal invagination of the sporangial cytoplasmic membrane (5). This invaginatlon to form the initial spore septum is associated with considerable mesosome development (5). The association of certain peripheral bodies with cell division and the early stages of spore formation was observed by several workers (Figs. 2 and 3, reference 2;3;7; Fig. 14, reference 19;33) before the cytological structure of these bodies (mesosomes) was clearly revealed by Fitz-James (5) and Giesbrecht (7). Although glimpses of certain stages in the development of the initial spore septum and "wall" of the forespore had been obtained (17,(32)(33)(34), it was Young and Fitz-James (38, 39) who clearly demonstrated the origin of the septum and FitzJames (5) who showed the association of mesosomes with septum development in several Bacilluf cereus strains, Bacillus megaterium, and Bacillus medusa. Hashimoto (16) has shown a similar development of the septum in Clostridium sporogenes. Thus, the origin of the spore septum is now clear, and considerable detail is available on the structure of the mature spore in several species (2,4,14,16,20,26,32,34). However, until quite recently very little was understood of the stages between the early development and the complex structure of the mature spore, and of the mode of development of the structural components. In 1962 Young and Fitz-James (40) described in B. cereus var. alesti the formation of the cortex between the two membranes of the forespore. The spore coat apparently was formed external to the outer membrane but within the exosporium.This study of Bacillus coagulans confirms the origin of the forespore membranes and the development of the cortex between the two membranes. It also reveals several differences in structure and perhaps in method of formation. M A T E R I A L S A N D M E T H O D SThe observations reported in this study were made with a very heat resistant strain of B. coagulans 111 on

Spore integuments of Bacillus coagulans were prepared containing nearly all the hexosamine and α, ϵ-diaminopimelic acid (DAP) present in intact spores. Subsequent autolytic action resulted in the destruction and removal of the residual cortical structure and "cortical membrane" leaving the appearance of the inner and outer spore coats unchanged in electron micrographs. Concurrently, all the hexosamine and DAP in the preparation was released mainly as non-diffusible mucopeptide containing alanine, glutamic acid, DAP, and all the glucosamine and muramic acid. Some diffusible peptides containing alanine, glutamic acid, and DAP were also present but there was little protein or carbohydrate. Lysozyme digestion of integument preparations from heated spores of Bacillus 636, B. subtilis, B. coagulans, and B. stearothermophilus specifically removed the residual cortex and cortical membrane with the release of the mucopeptide. In B. cereus T, only the residual cortex and part of the mucopeptide were solubilized by lysozyme. The effect of several reagents and enzymes upon the appearance and removal of hexosamine from B. coagul ans spore integuments is reported. The results show that spore mucopeptide is mainly located in the residual cortex and cortical membrane and suggest that these structures consist essentially of mucopeptide. The implications of these results in relation to the "contractile cortex" theory of heat resistance in spores are discussed.

Ten strains of Clostridium botulinum type E have been studied at 12 temperatures between 2�5 and 45�0. Growth proceeded consistently from spore inocula at temperatures between 5 and 37�5�0, but some strains developed slightly at ~empera� tures up to 45�0. Maximum rates of growth occurred at 35�0. The upper and lower temperature limits were several degrees lower than for type A and B strains.

SUMMARYA new species of the genus Bacillus was isolated from Macquarie Island soil. The organism is psychrotrophic, producing spores at temperatures down to and including 0". The maximum temperature for growth is 25". Apart from its temperature relations, it most closely resembles B. pulvifaciens, but differs from that organism in its ability to produce acid from carbohydrates with ammonium salts as sole nitrogen source and in its inability to hydrolyse casein or gelatin, to grow on soybean agar or to grow in 5 yo NaC1. The name Bacillus macquariensis is proposed.Vegetative organisms stained Gram-negative a t all stages of growth. Electron micrographs revealed cell-wall structures typical of Grampositive bacteria; on the other hand, chemical analyses demonstrated in cell-wall preparations a wide range of amino acids and relatively low amounts of amino sugars, as commonly found in Gram-negative species.

Ten strains of Cl. botulinum, type A, and 10 of type B have been studied at 12 temperatures between 10 and 50�C., and rates of growth measured nephelometrically on sealed cultures. Growth proceeded from spore inocula at temperatures from 15 through to 42.5�C., but not at 12.5 or 45�C. When young, actively growing cultures were transferred to temperatures outside the range permitting spore germination, rates of growth were measured at 12.5, 45, and 47.5�C. After transfer to 10 or 50�C. no sustained growth was observed

Photoreactivation of ultraviolet-irradiated Bacillus cereus T declined markedly during the development of stage IV forespores. During ultraviolet irradiation of a culture containing early and late stage IV forespores, both vegetative- and spore-type photoproducts were formed. The formation of vegetative-type photoproducts (mainly thymine dimers) decreased to nearly half during late stage IV, remaining constant until lysis of the mother cells began, when it fell to zero. Spore-type photoproducts were first observed during late stage IV and increased with the increase in numbers of late stage IV forespores. The occurrence of spore-type photoproducts preceded the development of refractile forespores by about 1 h. At stage III the nuclear material occupied a central position, and the ribosomes were at the periphery of the forespore protoplast. During stage IV the deoxyribonucleic acid (DNA) occurred in a peripheral position, and bundles of fibers (“transition” DNA) could be seen. By stage V, all of the DNA appeared to be of the spore type and was peripheral, and the forespore protoplast center was packed with ribosomes. Forespore stages II, III, and IV were classified by light and electron microscopy. The curve for electron microscope classifications preceded that for light microscope classifications by approximately one stage. The formation of spore-type photoproducts preceded differentiation of DNA by about 1 h, the latter coinciding with the development of refractility. Spore-type photoproducts have been associated with DNA in the A state, and the progressive change of the forespore DNA into this state is discussed in relation to the spore differentiation process.