Peptone-yeast extract (PY) medium containing 0.035% ferric ammonium citrate as an indicator, 0.05% sulfite as a substrate, 0.05% cysteine as a reducer and 0.5% glucose was found to be suitable for observing the sulfite reduction test. The effect of added cysteine on the test was suppressed by the addition of glucose. In cultures of bacteria grown for 2 days at 37 C in medium containing the above ingredients, 121 among 132 strains of clostridia, including 86 strains of Clostridium perfringens, gave a positive reaction. Although some strains of Salmonella and Proteus were positive, the specificity of the test for clostridia was thought to be relatively high. Positive reactions in a resting cell system were limited to some species of clostridia.

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Germ-free (GF) -ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c : K (B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.

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Treatment of Bacillus cereus T spores with trichloroacetic acid delayed their germination. The extent of retardation depended on the concentration of trichloroacetic acid, and the temperature, pH and duration of treatment. The effect was completely reversed by subsequent heating, and this restoration of germination also depended on the temperature and duration of heat treatment. Fourteen compounds were examined for their ability to suppress germination of spores. The halogenated fatty acids tested, such as trifluoro-, tribromo-, and dichloroacetic acid, caused suppression of germination, whereas other compounds, i.e., free fatty acids and amino acids, did not. It is concluded that the charge distribution of fatty acid molecules is important for their effect in suppressing germination of spores.

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The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamidee. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.

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By using phage P22spl, a deletion mutant of phage P22, the structures of two new transposons on P22 genomes were studied by the electron microscopic heteroduplex method. One of these was the Cm (chloramphenicol) transposon derived from an R plasmid, NR1, and the other the Km (kanamycin) transposon from pNR502. The heteroduplex between P22 phage DNAs with and without the Cm transposon revealed that the Cm transposon was similar in structure to the Tn9 element, a well-known Cm transposon derived from the R plasmid pMS14. On the other hand, the Km transposon of pNR502 was quite different in structure from other Km transposons reported previously. This transposon consists of a 6.8 kilobase (kb) segment of DNA, in which a short inverted repeat is contained. The heteroduplex experiments showed that a 4.5 kb segment of DNA was deleted from the P22 genome in the P22spl genome. Because of a shorter unit length of the genome, phage P22spl is considered to be useful for assaying various kinds of transposable elements.

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The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils.
These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.

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Water-soluble glycopeptides isolated from Lactobacillus plantarum and Staphylococcus epidermidis cell walls elicited a delayed type hypersensitivity (DTH) -like skin reaction in rats previously immunized with Mycobacterium tuberculosis cell walls, but not in unimmunized rats. Histological examination of the skin reaction sites in immunized animals revealed a close similarity of this skin reaction to a typical DTH reaction with respect to the time course of development and the types of cells that infiltrated into the skin reaction sites, which were characterized by a predominant infiltration of mononuclear cells at 48 hr. This DTH-like reaction was also demonstrated by immunizing the rats with the cell wall peptidoglycans of L. plantarum or S. epidermidis and skin testing them with homologous as well as heterologous peptidoglycans. The DTH-like reaction appeared to be caused by peptidoglycans that exist in common in the cell walls of phylogenetically distant bacterial species. Furthermore, it was also suggested that the putative antigenic determinant (s) might include both the glycan chain and part of the peptide moieties of the cell wall peptidoglycan rather than either of the single moieties.

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