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Loblolly pine ( Pinus taeda ) pollen was found to contain about 7·5 to 9 per cent lipids, most of which were triglycerides of oleic, linoleic, and palmitic acids. The triglycerides were not easily extracted from pollen unless the grains were fractured. Lipids, which were easily extracted and were probably part of the outer spore coat, comprised 1·6 per cent of the pollen. These were mainly octaconsanol and hexacosanol with smaller amounts of wax esters and free acids.

1. When Bacillus subtilis was grown in a medium in which sporulation occurred well-defined morphological changes were seen in thin sections of the cells. 2. Over a period of 7·5hr. beginning 2hr. after the initiation of sporulation the following major stages were observed: axial nuclear-filament formation, spore-septum formation, release of the fore-spore within the cell, development of the cortex around the fore-spore, the laying down of the spore coat and the completion of the corrugated spore coat before release of the spore from the mother cell. 3. The appearance of refractile bodies and 2,6-dipicolinic acid and the development of heat-resistance began between 5 and 6·5hr. after initiation of sporulation. 4. The appearance of 2,6-dipicolinic acid and the onset of refractility appeared to coincide with a diminution of electron density in the spore core and cortex. 5. Heat-resistance was associated with the terminal stage, the completion of the spore coat. 6. The spore coat was composed of an inner and an outer layer, each of which consisted of three or four electron-dense laminae. 7. Serial sections through cells at an early stage of sporulation showed that the membranes of each spore septum were always continuous with the membranes of a mesosome, which was itself in close contact with the bacterial or spore nucleoid. 8. These changes were correlated with biochemical events occurring during sporulation.

The spore coat layers of Bacillus cereus T can be at least partially restored by mixing spores stripped of their coat with solubilized coat protein.

1. It might be better to treat this material as a new variety of O. concatenatum, so far as the dimensions of the oogonium and oospore and the diameter of the vegetative and suffultory cells are concerned, because there is not enough difference in dimensions to establish a new species. We cannot overlook, however, the difference existing in the length of the vegetative cells and of the suffultory cells in both species. 2. The marking of the median spore coat of the oospore of O. nebraskensis is different from that of O. concatenatum f. Hutchinsioe in being "punctate," while the latter is "scrobiculate." 3. The most distinguishing character of this species is the position of the pore in the oogonium, in the supramedian position instead of the superior.

New data are given concerning the strata of the spore coat. It is possible to count the strata, but often the spore-coat strata of different plant families cannot be put in morphological relationships. A rule is found in the phylogenetic evolution of the saccus. In fossil spores no prothallium is seen. Proximal germination at the tetrad scar has occurred for a longer time than was believed (cf. the Cycadofilicales, in part the Cycadeoidales, and perhaps the Coniferales).

The mechanisms of bacteriocidal action of hydrogen peroxide were studied by using E. coli, Vibrio parahaemolyticus and spore of Bacillus subtilis. When various enzymatic activities of cell-free extracts obtained from H2O2 treated cells of E. coli and V. parahaemolyticus were compared with those of control cells, it was found that the activities of all tested enzymes were inhibited by H2O2 treatment and the inhibitory effect increased with increasing amount of hydrogen peroxide per mg of cellular nitrogen.Spores of B. subtilis were resistant to ordinary H2O2 treatment but completely killed by the treatment of 400mg H2O2/mg Spore N at 80°Cfor 30min. During the treatment, it became clear that dipicolinic acid was released from the spores. When the spore coat fractions obtained from H2O2 treated spores were compared chemically and physically with those of control cells, it was found that remarkable changes occurred in the inner coat fraction by H2O2 treatment.

The mature spore of Nosema apis possesses a thick spore coat and a particle-bearing spore membrane. Within the spore membrane, in the anterior portion of the spore, is the highly laminated polaroplast. The fractured face of the lamella is granular. The convex face of the polar filament membrane carries few particles, while the concave face bears many densely packed particles. The nucleus of the mature spore is centrally located, and pores were observed on the nuclear envelope.

The mature spore possesses a thick spore coat and a particle-bearing spore membrane. The highly laminated polaroplast membranes are located at the anterior pole of the spore. Close to its base, the polar filament is surrounded by the polaroplast membrane. The polar filament runs spirally towards the posterior pole of the spore. A large portion of the polar filament is arranged in two layers. A similar arrangement was also observed in immature spores and in the sporoblast stage, although it was not so orderly arranged in the latter. The developing polaroplast membrane was observed in the immature spore, but not in the sporoblast. The sporoblast wall is much thinner than the spore coat, but has the same texture. Endoplasmic reticulum is the most prominent cytoplasmic organelle in the developing stages of Nosema apis. Porous nuclear envelopes are also observed in developing stages. The role of the endoplasmic reticulum in the formation of the polar filament, polaroplast and spore coat, and the function of the spore membrane, are discussed.

Summary: Thin sections, freeze-etch preparations and carbon replicas of Thermoactinomyces vulgaris sporangia and endospores were examined in the electron microscope. The outer spore coat contained parallel arrays of long fibrous striations spaced at about 5 nm; there appeared to be several layers with a different orientation of the fibres in each. The outer coat was polyhedral with slightly concave pentagonal and hexagonal faces. A combination of geometrical considerations, analysis of the patterns of faces seen in the replicas and freeze-etchings, and model building suggested that the polyhedron had 12 pentagonal and approximately 12 hexagonal faces.

SUMMARY: Bacillus fastidiosus, an aerobic spore-forming bacterium able to utilize uric acid or allantoin as sole source of carbon and nitrogen, has been re-isolated and its fine structure described. Anatomical features of vegetative cells are similar to those of other members of the genus Bacillus. The mature endospore has typical core and cortical regions. The spore coat is highly laminated and surrounded by a tight-fitting exosporium to which conspicuous exosporial hairs are attached.

SUMMARY A number of factors of possible significance in elucidating the mechanism of inactivation of spores exposed to the alkylating agent, ethylene oxide (ETO), were investigated in this study. The sterilant system consisted of a mixture of ethylene oxide (12%) end dichlorodifluoro-methane (88%). Spores were exposed to ETO end suspended in distilled water overnight to determine if the ETO treatment would effect release of protein, RNA, DNA, or DPA. Only DPA release was effected, it being appreciably greeter from exposed than unexposed spores. Pretreatment of spores with agents postulated to effect the intactness of the spore coat was noted to alter ETO resistance characteristics. The resulting survivor curve exhibited en unusually long death lag period followed by an increased first-order death rate. Such an alteration, however, could be counteracted by increasing the time between pretreatment end ETO exposure. A comparison of lyophilized vegetative cells, germinated spores, and heat-activated spores revealed that the ETO resistance of germinated spores was closer to that of heat-activated spores than to that of vegetative cells. To obtain a logarithmic death rate for heat-activated spores, it was necessary to precondition at about 98% R.H. prior to the normal preconditioning et 33% R.H. Vegetative cells end germinated spores, however, did not require preconditioning other then et 33% R.H. The effect of increasing time of ETO exposure on germination end postgerminative end vegetative development of spores in a rich medium end in a deficient medium was evaluated. No significant change in rate or efficiency of germination was observed for either medium. However, the postgerminative phase of development of treated spores was notably lengthened over that of untreated spores in either medium. Treated spores which were incubated in the rich medium entered the vegetative phase of growth before those incubated in the deficient medium, this difference becoming more pronounced with increasing periods of exposure.

The development of the spore began at the sporoblast stage. The profile of the developing polar filament was defined clearly in the late sporoblast, but its internal structure was still lacking. During this stage, both a vesicular type of endoplasmic reticulum and an endoplasmic reticulum containing cisternae in a parallel arrangement were abundant in the cytoplasm of the sporoblast. Numbers of large, expanded vacuoles were also observed in the cytoplasm. As spore development proceeded, the spore coat increased gradually in thickness. The expanded vacuoles started to take up positions around the anterior portion of the polar filament and to become incorporated into the developing polaroplast, which eventually became a highly-laminated structure in the mature spore. In the mature spore, the fully-developed polar filament possessed a central tube surrounded by 12 cylindrical structures, and the spore coat attained its maximum thickness.

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO4, OsO4, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO4 in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO4 fixation appeared to provide much better definition of the boundaries of various structures than did OsO4. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO4 fixation) as two thin lines. In cells fixed first with OsO4 solution, and then refixed with a mixture of KMnO4 and OsO4 solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO4. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.

SUMMARY: Germination of heat-activated spores of Clostridium pasteurianum was rapidly induced by treatment with calcium dipicolinate; 90% of the spores lost their refractility within 20 min at 37°C. The sequence of ultrastructural changes during germination and subsequent outgrowth in a nutrient medium, was followed by electron microscopy. Dissolution of the spore cortex was accompanied by escape of cortical material via numerous perforations in the spore coat. The spore core underwent pre-emergent swelling to fill the space vacated by the cortical material, and eventual rupture of the spore coat led to emergence of the germling cell which invariably occurred via the open base of the exosporium. Thus, specimens of residual spore ‘husks’, prepared by the critical point drying procedure, consisted of intact exosporia enclosing surprisingly large fragments of the original spore coats.

Summary Electron microscope investigations of Pithomyces chartarum spores fixed in Osmium tetroxide and embedded in methacrylate have shown the association of a cytoplasmic granule with septal pores. Unusual concentrations of mitochondria and rhibosomes have been noted, and a complex system of layers has been seen in the spore coat. The general cell structure is discussed.

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at a prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.

The fine structure of conidiospores of Aspergillus niger has been studied by means of ultrathin sectioning and electron microscopy. Permanganate fixation of the conidia preserved the details of their inner structural components and enhanced the general contrast within the sections. The conidiospores are characerized by their peculiar surface structures composed of three distinct components; an outer hull, a spore coat or wall, and an intermediate space. As a consequence of the swelling of the conidia, both the outer hull and the intermediate space almost completely disappear, and a variety of the intracytoplasmic organelles becomes visible in large numbers. The mature conidia at the resting stage appear to possess a more electron-dense cytoplasm than that of the parent hyphae of the organism. They also contain a nucleus and a few mitochondria within the cytoplasm. The significance of these structures and their relationship to the parent hyphal cells are discussed.

The ultrastructural changes occurring during defined stages of the transition of dormant Bacillus cereus T spores into heat-sensitive forms were investigated. The coat of the heat-activated spores displayed conspicuous striations across its middle layer. Electron microscopy of thin sections of heat-activated spores revealed the presence in the spore of a layer consisting of hexagonally oriented subunits. It was demonstrated that the subcoat region, but not the cortex, disappears rapidly during germination of B. cereus T spores. The fibrous structures apparently associated with the spore coat remain virtually unchanged during the entire course of activation and germination.

Spores of Clostridium sp. N1 are characterized by numerous broad ribbon-like appendages attached to one end. The appendages are two to three times the length of the spore and, at their maximal dimension, may be two-thirds the width of the spore. They are attached to the spore body by a common trunk which is continuous with the outer spore coat. Each appendage is a multilayered structure and is enclosed in an amorphous material. Details of spore and appendage formation are described, and appendage ultrastructural features are presented. The function of the appendages is not known.

Bacitracin is produced by strains of Bacillus licheniformis. Two strains have been employed extensively in research on biosynthesis of the antibiotic. The strain used by Anker et al. (1948), Hendlin (1949), Snoke (1960, 1961), Aida (1962), Kuramitsu and Snoke (1962), Cornell and Snoke (1964), Shimura et al. (1964), and Snoke and Cornell (1965) is designated ‘Tracy I’ or American Type Culture Collection (ATCC) 10716. The strain employed by Hills et al. (1949), Bernlohr and Novelli (1960, 1963), and Bernlohr and Sievert (1962) s listed as Oxford University Collection A-5.

SUMMARY: Spore coat of Bacillus subtilis ATCC6051 was fractionated as described by Kondo & Foster (1967). X-ray diffraction, infrared spectra, amino acid analyses and N-terminal amino acid studies showed that most of the fractions resembled each other in physical and chemical structure and constituents. Electron micrographs of sections and shadowed replicas of the coat fractions at various stages showed that the inner part of the coat disappeared during treatment; no drastic morphological change was observed throughout the fractionation. The amounts of cystine in the coat and the coat fractions were less than in keratins. This result and solubilizing experiments suggested that the spore coat does not chemically resemble keratins.

Publisher Summary The chapter discusses the involvement of those spore components, both at the molecular and structural level, which may be related to the property of thermoresistance. Loss of spore viability during heating may not be analogous to the heat-induced loss of viability of vegetative forms. It seems most likely that heat normally kills these spores by inactivating enzymes necessary for the initiation of germination, whilst leaving the protoplast viable but trapped within the spore. It is known that lysozyme will cause germination of spores of diverse species, but normally some treatment of the spores is first necessary to make the spore coat permeable to the enzyme. The measured heat resistance of such spores can clearly depend very much on the media and methods used to enumerate the survivors. Spores may be scored as “dead” on conventional media simply because their mechanism for initiating germination has been inactivated, although we know that within the spore the protoplast remains fully viable. It is commonly observed that heat resistance develops late— that is, following synthesis of dipicolinic acid, sulpholactic acid in those organisms that produce it, and the incorporation of calcium, and formation of the cortex. As expected, the mutant spores were found to be heat-sensitive when compared with wild-type spores. The chapter also considers the available data selectively and tries to indicate those factors for which research provides strong evidence for an association with heat resistance. The chapter tries to indicate factors that have at times been considered important in heat resistance, but which may in the light of more recent work, be fortuitous associations and appraise those hypotheses, which have, perhaps, been given insufficient consideration in the past, and briefly speculate regarding their relevance to the mechanisms of spore heat resistance.

IN Haplosporidium chitonis the method of formation of the spore tails, which grow to a great length after the completion of the spore coat, has always been rather difficult to understand; it seemed unlikely that the material for these tails could be provided from the interior of the spore, owing to the thickness of the spore wall, at least toward the end of their growth. On a recent intra vitam examination of some of these spores in Janus green, I discovered that, in the younger stages, each is surrounded by a very distinct thick cytoplasmic envelope (Fig. 1, e). The appearance at this stage gives the impression that the spore is being formed inside a cell, but the absence of nucleus outside the spore shows, of course, that this is not the case.

THE mechanisms involved in the germination of bacterial spores have been studied extensively from the biochemical and morphological points of view11,2. Details of morphological changes occurring during germination, however, have not yet been demonstrated clearly, It is not known whether or not, when a new vegetative cell emerges from the spore, the spore coat splits by some specific mechanism.

WE have already reported by chemical and X-ray diffraction investigations1,2 that the spore coat of Bacillus subtilis is composed of a keratin-like protein with a crystalline structure. This communication reports the results of an electron diffraction investigation designed to give further information on the structure of the spore coat.