Numerical classification of slowly growing mycobacteria, including 159 strains received as Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti, was carried out using 88 characters, and the following results were obtained. 1) All 159 strains received as M. tuberculosis, M. bovis, M. africanum, and M. microti formed one cluster, and no clear-cut differentiation among these four species was achieved. These four species should be reduced to one species, M. tuberculosis. 2) Within the cluster, two subclusters appeared, with a number of strains located outside the subclusters. One subcluster was composed, except for two strains, of only M. tuberculosis strains, and another of M. bovis and M. africanum strains only. The subclusters were regarded as subspecies tuberculosis and subspecies bovis, respectively. M. africanum was regarded as a synonym of M. bovis, i.e., a niacin-positive variety of M. bovis (subsp. bovis). 3) An intermediate subcluster was observed outside the M. tuberculosis and M. bovis subclusters. This may be regarded as intermediate between the two subspecies, tuberculosis and bovis. 4) In the last two decades, the characteristics of M. tuberculosis strains isolated from patients seemed to be approaching those of M. bovis strains. The recently isolated strains showed stronger arylsulfatase activity and lower resistance to thiophene-2-carboxylic acid hydrazide than the strains isolated 16 to 19 years ago.

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Variation in the cell respiration rate of pigmented and nonpigmented strains of Serratia marcescens was exhibited. The respiration rate of a pigmented strain decreased earlier than that of nonpigmented strains in the late exponential or early stationary phase. However when prodigiosin synthesis was not induced by exchange of carbon sources in the medium, the decrease in the respiration rate of the pigmented strain was the same as that of nonpigmented strains. Measurement of the oxygen consumption rate in the sonicated cell membrane by adding NADH solution showed that the rate in the pigmented strain was lower than that in nonpigmented strains. Furthermore, the cell membrane of prodigiosin-induced organisms was more sensitive to respiration inhibitors than that of pigment-noninduced organisms of the pigmented strain. These results showed that the respiration activity was decreased by prodigiosin synthesis in S. marcescens.

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A method of rapid susceptibility testing by bioluminescent assay was developed. Correlation between the 50% inhibition dose of antimicrobics for bacterial adenosine triphosphate measured by bioluminescent assay and the minimum inhibitory concentration obtained by the broth dilution method was satisfactory. In the bioluminescent assay the incubation time required was only 90min.

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Heat resistance at 95C, heat activation at 75C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D₉₅-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KCl medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl₂ in the presence of CO₂ without heat activation (A-type). The strains were tested for euterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.

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A rapid micro-test method for mumps virus neutralization was developed. In this method, a 96-well tissue culture plate was used for preparation of cell monolayers and the PAP staining technique was used for visualization of mumps virus infected cells. Clusters of infected cells were observed as a focus and the numbers of foci could be counted by the naked eye 2 days after the infection. A linear relationship between virus dilutions and focus numbers was observed. When neutralizing antibodies in sera from cases of natural mumps infection were assayed, a good correlation was observed between those obtained by the focus reduction method applying the micromethod and those obtained by the ordinary plaque method. Our results indicate that this micromethod is useful in mumps virus neutralization tests and it has many advantages over other methods previously reported.

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Methylcellulose-elicited peritoneal macrophages from BALB/c mice were cultivated in vitro and inoculated with dengue virus (DV). At intervals thereafter portions of the culture fluids were taken and titrated for viral infectivity. Extracts from Ascaris suum and Parascaris equorum, either crude or Sephadex G-100 fractionated, were examined for effects on the multiplication of DV. The macrophage cultures treated with the above substances produced larger amounts of DV compared with untreated control cultures. The enhancing effect of the substances depended on doses added and duration of treatment and was suppressed by co-treatment with carrageenan, a specific macrophage-inhibiting agent, but was not related to the viability of cultured cells. In fluorescent antibody (FA) as well as infectious center assay experiments, it was shown that the DV-infected cells were found more frequently in treated cultures than in untreated control cultures. In the treated cultures phagocytosis by cultured cells was also of a higher magnitude than that in untreated cultures. In cocultures of macrophages and splenocytes from the same line of mice, no additive effect of splenocytes was noted. The limulus amebocyte lysate clotting enzyme reaction (Limulus test) indicated that involvement of bacterial lipopolysaccharides in the enhancement phenomena was negligible. The data so far obtained suggest that the enhancing effect was due to direct action of the parasitic extracts on macrophages. Four Sephadex G-100 fractions from the crude extracts showed similar activities; however, the effects of fractions I and III appeared to be comparatively strong. Significance of the findings in relation to the pathogenesis of DV infection was discussed.

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OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5×10⁵ cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.

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