A numerical classification using fifty-two characters was done on 72 strains of mycobacteria, including Mycobacterium avium, M. terrae, the pathogenic nonphotochromogens and the pathogenic scotochromogens. The results indicated that the test strains could be divided into two subgroups, the first consisting of M. avium and M. terrae and the second of the nonphotochromogens and the scotochromogens. Distinguishing characters for differentiation between these organisms were described, and taxonomic position of these organisms was discussed.

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Forty-nine strains of Mycobacterium fortuitum, three strains named M. runyonii and three rapid-growers from sputa of patients with pulmonary disease were subjected to numerical classification based upon 101 characters. Fifty strains formed a large cluster and were considered to belong to M. fortuitum. Within this cluster, two subclusters could be differentiated. One subcluster consisted of three strains identified as M. runyonii, three rapid-growers and eight strains identified as M. fortuitum. This subcluster, which was partially connected with the other subcluster, was named M. fortuitunz subsp. runyonii. The other subcluster was considered to be typical M. fortuitum; M. fortuitum subsp. fortuitum. The subspecies runyonii could be differentiated from the typical M. fortuitum by the following characteristics: The strains often (i) were unable to reduce nitrate; (ii) lacked allantoinase; (iii) did not utilize ethanol, propanol, propylene glycol and 1, 3-butylene glycol as sole carbon sources; (iv) did not utilize acetamide as the sole, simultaneous nitrogen and carbon sources. A variety of M. fortuitum capable of growing at 45C could also be differentiated from the usual strains that did not grow at 45C by other characters. This variety utilized neither mannitol nor sorbitol as sole carbon sources, formed no acid from trehalose and mannitol, and usually did not utilize propylene glycol as a sole carbon source. Biochemical characters of the typical M. fortuitum (M. fortuitum subsp. fortuitum) are described in detail.

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Successive cellular changes leading to damage induced in human epithelial FL-line cells by poliovirus infection were followed by means of phase cinematography and described on the basis of tine records of living cells and fixed-stained specimens. The first visible change appeared as acidophilic inclusions in nuclei of stained specimens as early as 5 hours after infection. Following distortion of the nucleus, a marked increase in basophilia and the appearance of α paranuclear mass were noted in the cytoplasm. With the development of this mass, the nucleus located in the peripheral area of the cell became intensively wrinkled and distorted. Then several vacuoles appeared in the cytoplasm, and a marked blebbing of the cell surface followed, resulting in shrinkage of the cell. The cell damage was characterized morphologically 20 to 24 hours after infection by an eosinophilic cytoplasmic mass, severe distortion of the nucleus and fragmentation of nuclear material leading to pycnotic degeneration of nuclei. The nucleoli and acidophilic intranuclear inclusions remained unchanged during the course of pycnotic degeneration of cells. Degenerative changes leading to cell damage proceeded rather slowly in infected cells.

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Known numbers of Mycobacterium lepraemurium were inoculated into the foot-pads of 10 mice. Thirty minutes after inoculation, the bacteria were harvested from each mouse and counted by the microspot method. The per cent of the original inoculum recovered ranged from 82.5 to 105.4 among the 10 mice. The difference between the number of bacilli inoculated into a mouse and the number recovered was analyzed statistically. Of the 10 samples, this difference was not significant in 7, and significant in the remaining 3. The facility with which M. lepraemurium can be counted by the microspot method should eliminate much of the laboriousness from experimental leprosy research.

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The nature of the cross reaction of the mycelial mannan of Trichophyton rubrum and galactomannan isolated from the culture medium of Aspergillus fumigatus with antisera of Saccharomyces cerevisiae and Candida albicans is described. Cross-reactivity of polysaccharides of both T. rubrum and A. fumigates was weak with antisera of C. albicans and S. cerevisiae, but the galactomannan of A. fumigatus showed slightly stronger activity than the mannan of T. rubrum which possesses more closely related chemical structure of the mannans of S. cerevisiae and C. albicans.

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The fine structure of a strain of Bacteroides insolitus has been studied by ultrathin sectioning and electron microscopy. Logarithmically growing cells were fixed both by osmic acid and potassium permanganate, and embedded in Epon. Thin sections were stained with uranyl acetate and examined. The periphery of the cell was composed of a wavy three-layered outer membrane (ca. 80A), an intermediate layer (50-200A), and three layered cytoplasmic membrane (ca. 80A). Single or double bridges which connected the outer membrane with the cytoplasmic membrane were observed. Invagination of the cytoplasmic membrane was observed in no occasion. Independent, distinct, and uniform particles were not the main component of the cytoplasm. The cytoplasm was filled with more or less beaded reticulum-like structures. The nucleoplasm with fine fibrils was mainly dispersed continuously in rather regular cubic masses in an intermediate region between the center and the periphery of the cell. Contacts of the nucleoplasm with the cytoplasmic membrane were occasionally observed.

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A survey was made of mycoplasma contamination in cell lines and transplantable tumor lines maintained in laboratories in Japan. A total of 60 cell lines were tested and 52 (86.7%) of which were contaminated by mycoplasmas. These mycoplasmas were isolated from the cell lines of human origin 10/13 (77%), from monkeys 3/3 (100%), from rats 14/19 (74%) and from mice 25/25 (100%). Mycoplasmas were not found in any of the 99 samples of tumor materials, Mycoplasma colonies, upon primary isolation from cell lines, could be differentiated into three morphological types. However, all colonial types showed similar biological properties. All but one of the isolated mycoplasmas could be identified as Mycoplasma hominis type 1 by both growth inhibition tests and by fluorescent antibody technique.

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Resistance of staphylococci to desiccation, dry heat, wet heat and ultraviolet irradiation was studied using 128 strains obtained from patients in our surgical clinics. Staphylococci of phage group I including type 81, particularly of type 80/81, showed significantly high death rates after desiccation, dry heating, and wet heating, and significantly low death rates after ultraviolet irradiation. In contrast, group-II strains responded inversely to these physical agents. The mean death rate of group-III strains approximated that of group I after desiccation, dry heating, and ultraviolet irradiation, but after wet heating it was lower than that of group II. The results indicate that transmissibility of staphylococci might be influenced to a great extent by physical factors in the environment outside the body.

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In order to confirm the data reported in the previous papers, variously prepared pertussis vaccines were employed in the present investigation. Pertussis organisms grown either on α solid or in a liquid semi-synthetic medium were treated by: (1) heating at 56C for 30min, (2) storage in 0.1% formalin at 37C for 5 days, (3) storage in 0.1% formalin at 25C for 5 days, and (4) simple addition of sodium ethyl-mercuri thiosalicylate (merthiolate) as a preservative. Freeze-dried vaccines were made from these preparations four and a half months later. Mice were immunized intraperitoneally and challenged intracerebrally with a virulent strain of Bordetella pertussis 10 or 17 days later. The data were analyzed statistically assuming the probit corresponding to the percentage of survivors at any dose be a linear function of the logarithms of the dose. The 50% effective doses (ED₅₀) and slopes of each vaccine were found to be uniform. More accurate estimates of ED₅₀ were obtained by employing a pooled slope in each experiment. From these ED₅₀ values, the relative potency was estimated by comparing the value of a vaccine to that of a dried merthiolate-vaccine. For vaccines derived from solid cultures, with an immunization period of 17 days, the relative potency of the vaccine heated at 56C was 0.63 (95% fiducial limits=0.52 to 0.76); the value for the formalinized vaccine at 37C was 0.40 (0.30 to 0.53) and one at 25C was 0.51 (0.34 to 0.77). Vaccines derived from liquid cultures showed a relative potency of 20 to 50% less than that of corresponding vaccine derived from α solid culture. The potency obtained for the 17 day immunization period was usually higher than that for the 10 day period. Using the overallpooled slope, an experimental design which will be appropriate for statistical analysis is discussed.

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The present review summarizes most of the data obtained in experimental anticancer studies with hemolytic streptococcus in our laboratories and in others during these past 12 years.
The principal results are as follows:
(1) Streptococci possessing streptolysin S-forming ability act against a wide range of transplantable animal tumors.
(2) Bernheimer's basal medium (BBM) differs from other simpler media, such as 0.85% NaCl and phosphate-buffered saline, in that the streptolysin Sforming and anti-cancer abilities of cocci suspended in BBM is resistant to heating at 45C for 30 minutes.
(3) A procedure consisting of preincubation of live cocci suspended in BBM containing penicillin (2-4×10⁴units/ml) at 37C for 20 minutes followed by heating at 45C for 30 minutes, yields a potent anticancer preparation of having neither streptolysin S-forming ability nor virulence for mice and rabbits. The preparation is about 20 times more effective than the nontreated live coccus suspended in 0.85% NaCl in inhibiting the growth and metastasis of Ehrlich ascites carcinoma in mice, but it is somewhat pyrogenic.

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