A cell wall fraction obtained by sonic oscillation, yielded a Bordetella pertussis O antigen which possessed the ability to both adsorb O agglutinins and to produce the O agglutinins in animals. It was purified by a phenol water extraction and centrifugation method. Physico-chemical and biological properties of the O antigen were similar to that of the so-called endotoxic lipopolysaccharide. It contained 2.6% nitrogen, 1.2-1.5% phosphorus, 9.0-11.0% heptose, 0.3% 2-keto-3-deoxyoctonate (KDO), 13.3-13.4% as total carbohydrate, 16-16.5% hexosamine and 25-32% lipid. Homogeneity of the purified O antigen was confirmed by ultracentrifugation, electronmicrograph and geldiffusion tests. It possessed significant pyrogenicity, adjuvant effect on antibody produc-tion, and toxicity to animals, but not protective antigenicity.

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A diffusion barrier was used in bioautography to prevent the diffusion of compounds separated by chromatography or electrophoresis in a seeded agar toward the separation direction. Two main advantages were demonstrated in the present method; (1) better separation and identification of compounds occurring closely positioned, and (2) more sensitive detection owing to compact growth zones. The present method has proved satisfactory and reliable for the routine detection of unknown growth factors.

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The location of the mucopeptide in the cell wall of Bacteroides convexus was determined by electron microscope after enzymatic and chemical treatment (papain, pepsin, lysozyme and phenol). In the five layered cell wall the innermost electron dense layer (or a part of it) proved to be the mucopeptide. The molar ratio of amino sugar and amino acid components of purified mucopeptide was about 1:1:1:1:1:1 for glucosamine, muramic acid, L-alanine, D-glutamic acid, DL (meso)-diaminopimelie acid and D-alanine.

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In order to identify the antigenic determinant groups of the mannan of C. albicans serotype A, six kinds of manno-oligosaccharides of up to 7 units in chain-length connected by α1→2 linkages were prepared from the partial acetolysate of the parent mannan. In the precipitation-inhibition test of anti-C. albicans serotype A serum with its homologous mannan, inhibitory power of the oligosaccharides was of the followingorder:heptaose_??_hexaose>pentaose>tetraose>triose>biose, and the amounts for 50%-inhibition of the former four oligosaccharides were 0.08, 0.10, 0.50 and 3.0 μmole respectively, and the inhibitory power of the latter two oligomers at 0.5 μmole were 8 and 5% respectively. On the other hand, the cross-inhibition test of anti-C. albicans serotype A serum with the heterologous mannan of C. albicans serotype B afforded the result that the order of inhibitory activities was hexaose>heptaose>pentaose>tetraose>triose>biose, and that the amounts for 50%-inhibition were 0.05, 0.08, 0.1, 0.45, 0.50 and 3.0 μmole respectively. Furthermore, the results of inhibition test on the anti-C. albicans serotype A serum absorbed with the mannan of C. albicans serotype B revealed that the biose, triose and tetraose did not show significant inhibitory power in the range employed, whereas the pentaose, hexaose and heptaose did not significantly affect the inhibitory. activities. Thus, it was concluded that the antigenic determinants of the mannan of C. albicans serotype A are α1→2 linked hexaose or heptaose moieties. Based on the above facts, the serological differences between two antigenic mannans of C. albicans serotype A and B may reside at least in the length of the antigenic determinants in which the former is longer than the latter considering the length of the α-D-mannopyranosyl residue.

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An immune ribonucleic acid (RNA) preparation was extracted with phenol from the spleens of guinea pigs immunized with diphtheria toxoid. Antibody-carrying cells were detected by immunocyte adhesion as rosette-forming cells. When germ-free rats, conventional guinea pigs or mice were injected intraperitoneally with this preparation, the rosette-formers were detected in either peritoneal exudate cells or spleen cells, whereas serum antibodies were unable to be detected thus far in such animals. Two injections with this preparation did not cause any remarkable increase in the number of rosetteformers, and serum antibody was also not detectable. By contrast, a high titer of serum antibody was demonstrated and the number of rosette-formers increased shortly after an injection of a small amount of diphtheria toxoid into guinea pigs which had previously received an injection with immune RNA. This reaction indicates a secondary response of antibody formation. However, secondary responses were not induced by injections of immune RNA preparations in guinea pigs primed with either diphtheria toxoid or immune RNA preparation. These facts suggest that immune RNA preparations did not contain antigens or fragments thereof and the immune response induced by RNA preparation is not the same as that induced by stimulation by the antigen itself. These results moreover can be accounted for by the notion that the immune RNA preparation is able to induce "memory" cells capable of responding to a secondary stimulus with an antigen and producing a high titer of serum antibody.

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A new test method was devised for microbial gluconate oxidation, using an ammonium molybdate reagent. One loopful (about 2 mg wet wt.) of the test organism, grown on a nutrient agar plate for 18 hr, is transferred into 1 ml of the test liquid medium consisting of (NH4)2SO4 0.5 mg, potassium gluconate 10 mg, NaCl 5 mg, KH2PO4 2 mg, MgSO4•7H2O 0.1 mg, and 1 ml of distilled water, incubated at 37 C for 6 hr without shaking, and then mixed with 3 ml of 1% aqueous solution of ammonium molybdate and 0.2 ml of glacial acetic acid. The mixture is heated in boiling water for 5 min, followed by abrupt cooling with running water. A deep blue colour appears in a positive result. A total of 39 strains of Pseudomonas aeruginosa showed positive results by this method, whereas Aeromonas, Vibrio, Proteus group, Klebsiella, Citrobacter and Enterobacter A group were all negative. Though some strains of Enterobacter B group showed a weak blue colour, it could be easily differentiated from the deep blue colour of Pseudomonas. Longer incubation of test microbes in the test medium, and longer heating of the reaction mixture gave unsatisfactory results.210

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Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.

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Attempts were made to modify the formula devised by Brown and Lowbury (1965) for more effective selection of Pseudomonas aeruginosa. An improved formula was devised by the authors as follows: Peptone (Eiken) 2.0%, dipotassium phosphate 0.03%, magnesium sulfate (anhydrous) 0.03%, cetrimide 0.02%, nalidixic acid 15 μg/ml, agar 1.5%, with a final pH 7.4-7.6. On this media the growth and pigment production of Ps. aeruginosa was nearly equal to that of Brown-Lowbury's composition. Strains of Enterobacteriaceae such as Klebsiella, Proteus and Providencia were strongly suppressed on the author's medium although some of them showed a moderate growth on BrownLowbury's medium.

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Infection of mice with a strain of Salmonella typhimurium, known to multiply in macrophages, resulted in a marked alteration of their antibody response to further antigenic stimulation with sheep red blood cells (sRBC) or killed bacterial cells. The mice infected with a wild-type S. typhimurium and simultaneously immunized with sRBC, showed a significantly stimulated immune response to sRBC as revealed by the increased number of hemolytic plaque-forming cells in their spleens during the period following such immunization. Whether the animals were infected intraperitoneally or intravenously did not affect the stimulant effect of the infection on the immune response to sRBC. Injection of killed bacteria in amounts up to 106 organisms, instead of infection with live organisms, did not elicit any effective enhancement of antibody formation to sRBC. In cases of attenuated S. typhimurium infection, the stimulant effect on antibody formation to sRBC was obvious in the mice infected with the organisms simultaneously with or 1 week prior to the sRBC injection, while a slight suppression of antibody formation was observed in mice infected 3 weeks before the injection with sRBC. A similar effect of the infection was observed when the serum anti-bacterial antibody response was examined after stimulation with bacterial antigens. The altered immune responses after Salmonella infection are discussed in relation to the adjuvant effect of bacterial endotoxins and also to the acquired immunity to mouse typhoid.

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