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Spectroscopic microanalysis of the element-characteristic X rays produced by a scanning electron microprobe was employed to detect calcium and carbon in both intact and thin-sectioned spores of Bacillus cereus T and B. megaterium QM B1551. Linear scan profiles and multilinear scan images of the X-ray emissions for calcium (Ca(Kalpha)) were compared with those for carbon (C(Kalpha)) as an index of mass. Location was accomplished by stereological comparisons with secondary electron images and conventional transmission electron micrographs. Although the elements could be detected at the attogram level theoretically, spatial resolution was limited to approximately 500 to 1,000 nm in an intact spore, e.g., by the primary electron beam diameter, the electron-excited spore microvolume, and the type of specimen support. The resolution was improved to approximately 100 to 200 nm by use of thin-sectioned spores, with precautions to prevent calcium leakage from the specimen during preparations. In both intact and sectioned spores, calcium was distributed throughout the spore, similarly to carbon, and concentrated mainly in a central region corresponding to the spore protoplast.

The form of growth of Mucor rouxii (National Regional Research Laboratory 1894) under nitrogen is dependent on inoculum size. With a large inoculum (10(6) spores inoculated per ml), the morphology consists mainly of swollen spores with some filaments and yeastlike cells. At lower inoculum levels growth is filamentous. The morphology of this strain on incubation under nitrogen and the dependence of the form of growth on inoculum size are similar to those found previously for other strains of M. rouxii.

Lipids were extracted from vegetative cells and spores of Clostridium botulinum. The total lipids extracted averaged approximately 3.8% of the dry weight of vegetative cells and 2.5% of the dry weight of spores of types 61E, "F," and 115B. The fatty acids were analyzed in the form of their methyl esters by gas-liquid chromatography. Infrared spectroscopy, mercuric acetate fractionation, and silver nitratethin layer chromatography served as complementary means of analysis. The total fatty acids included straight chain, saturated, unsaturated, and cyclopropane acids. Hexadecanoic and tetradecanoic acids were the predominant acids in both the spores and vegetative cells. Together, they comprised over 50% of the total fatty acids. Unsaturated acids were the second major group. These were primarily 7,8-tetradecenoic, 9,10-hexadecenoic, 7,8-hexadecenoic, 11,12-octadecenoic, and 9,10-octadecenoic acids. Nonproteolytic types 61E and "F" possessed an 18-carbon diunsaturate, which was not found in the vegetative cells or spores of proteolytic type 115B. A mechanism for the synthesis of unsaturated and cyclopropane acids was proposed.

Six major oligosaccharides were released from the peptidoglycan of spores of Bacillus subtilis by lysozyme treatment. They were isolated and characterized as a disaccharide, tetrasaccharide, and hexasaccharide composed of equal amounts of muramic acid and glucosamine and containing two, three, and four acetyl groups, respectively. Three of the compounds were substituted by a single L-alanine residue, and the other three by a single tetrapeptide substituent on the acetylmuramic acid residue at the reducing end of each compound. The other muramic acid residue in the tetrasaccharides (and two of the three in the hexasaccharides) were shown to be present as muramic lactams, a sugar not previously found in nature and, hence, a unique spore constituent. Other features of the structure of spore peptidoglycan are discussed.

In spores of Bacillus stearothermophilus produced at 45 and 55 C, branched-chain fatty acids predominated in the former and straight-chain acids in the latter.

Dipicolinic acid-less spores of a Bacillus cereus T mutant in 33 mm sodium bicarbonate (pH 8.5) underwent pseudogermination (germination-like changes) accompanied by ribonucleic acid leakage and loss of viability.

The relative virulence was determined for 14 species of aspergilli, by inoculating normal mice intravenously with graded doses of spores. Eleven were found to possess some degree of virulence, whereas three others were avirulent. Members of the Aspergillus flavus group were the only species that consistently killed mice with doses as low as 10(4) viable spores. When the in vivo fate of spores was compared for a virulent and an avirulent strain of Aspergillus, spores of the latter were cleared rapidly from the liver and spleen but grew in the kidneys and brain, producing progressive disease. Mice which inhaled spores did not succumb, but macrophages washed from their lungs contained spores. A relationship of virulence to spore characteristics such as germination time, size, shape, and external markings could not be established. Virulence could not be related to aflatoxin production inasmuch as at least one virulent strain did not produce aflatoxin in vitro.

A self-inhibitor of spore germination has been isolated from spores of Dictyostelium discoideum, a cellular slime mold, and chemically characterized as 2-dimethylamino-6-oxypurineriboside.

A variety of analytical techniques was employed to study the composition of the chlamydospore of Candida albicans. The outer, thin, electron-transparent layer was found to be composed of glucan, together with a small amount of chitin. The inner, thick, electron-dense layer is proteinaceous. The central structure is composed largely of ribonucleic acid and lipid globules. In addition to being acid-fast, the chlamydospore was found to contain glycolipids and to lose the property of acid-fastness on extraction with ethanol-ether.

The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, "mesosome-like" bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10(-5)m. Eserine (10(-5)m) and Paraoxon (10(-7)m) inhibited B. megaterium enzyme. Sodium acetate at 10(-2)m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.

Two germination inhibitors from bean rust uredospores were identified as the cis and trans isomers of methyl 3,4-dimethoxycinnamate. They appear to be the "self-inhibitors" previously described from these spores.

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.

A sulfonic acid found to be a major constituent of spores of Bacillus subtilis was provisionally identified as 3-l-sulfolactic acid. This compound was completely absent from vegetative cells during growth, but large amounts accumulated in sporulating cells just before the development of refractile spores. Essentially all of the accumulated sulfolactic acid was eventually incorporated into the nature spore, where it may represent more than 5% of the dry weight of the spore. Germination resulted in the rapid and complete release into the medium of unaltered sulfolactic acid. This compound was not found in spores of Bacillus megaterium, B. cereus, or B. thuringiensis.

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.

The lipids of Rhizopus arrhizus Fischer mycelia and sporangiospores were extracted, isolated, and separated by thin-layer, liquid, and gas chromatography. Structural confirmations of the compounds were made by a gas chromatographmass spectrometer combination. The n-heptane fraction contained squalene (1%) as a major hydrocarbon constituent. Other major lipid classes detected were free fatty acids, naturally occurring methyl esters of fatty acids, triglycerides, sterols, and polar lipids. The polar lipids (44.4%) were found in the highest concentrations, and the triglycerides (22.1%), sterols (16.7%), and free fatty acids (11.7%) were present in lesser concentrations. This is the first report of naturally occuring methyl esters of long-chain fatty acids being present in fungal mycelium. There appears to be a preference for incorporation of unsaturated acids into the complex lipids, with the exception of the triglycerides. The major saturated fatty acids in the mycelium were palmitic (C(16)) and arachidic (C(20)), whereas the major unsaturated acids were oleic (C(18:1)) and linoleic (C(18:2)), respectively.