Sixteen Shigella strains resistant to ampicillin(APC) were isolated from 3760 strains examined in 1965. Five strains among these isolates were resistant to tetracycline(TC), chloramphenicol(CM), streptomycin(SM) and sulfanilamide(SA) in addition to APC, and transferred their APC resistances by conjugation together with the aforementioned four drugs. The 5-drug resistance was transferred to an Escherichia coli strain and the resistance was analyzed by both conjugation and transduction. Three out of the five Shigella strains transferred a single type of R (TC. CM. SM. SA. APC) factor, but the linkage relationship between the resistant markers were different from each other. The ampicillin gene, however, was always linked close to the genes governing both transfer and replication. The 5-drug resistance was transferred to an E. coli strain from the remaining two Shigella strains. According to the genetic studies of E. coli strains which had acquired the 5-drug resistance, it was concluded that the two parent Shigella strains carried two types of R factors in a cell, i. e., fi⁺R (TC. CM. SM. SA) and fi^- (SM. APC). These two R factors in a cell sometimes recombined and formed a recombinant R factor possessing the 5-drug resistance in which the ampicillin gene was linked close to the transfer gene. The enzymological properties of the five penicillinases coded by R factors possessed by the five Shigella strains were all similar to each other and of the type I penicillinase, which was demonstrated from Klebsiella pneumoniae and mediated by most of the R factors.

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An R factor carrying resistant genes, ampicillin (amp), tetracycline (tet), chloramphenicol (cml), dihydrostreptomycin (str), and sulfanilamide (sul), was analyzed by three genetic techniques: spontaneous segregation, transduetion, and recombination. The R factor was stable in Shigella and Escherichia coli, but in Salmonella the (cml. str. sul) genes were spontaneously lost at a high frequency. Almost all of the transductants with P1 contained the five drug markers, but a very small number of them carried both tet and amp genes, or the (amp. cml. str. sul) genes. In conjugal transfer from a strain carrying a single type of R factor to R⁺ recipient, the recombination took place between the amp and tet, amp and cml, and between the cml and str genes, at a frequency of 23.5, 39.5, and 3.0%, respectively. Crossover between the amp and transfer (tra) loci was not observed. A circular genetic model is proposed in which the genes are arranged in the order-tet9-tet1-tra. amp-cml-str-.

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Opsonization of Staphylococcus aureus to phagocytosis by guinea pig peritoneal exudate cells (PEC) was examined with various isolated serum globulins. Significant enhancement of in vitro phagocytosis was afforded by partially purified 7Sγ₁-immunoglobulin. The rate of killing or the rate of ingestion of ³H-thymidine labeled bacteria by PEC was used as an index of phagocytosis. The results indicate that the immunoglobulin can mediate the adsorption of an allogeneic surface independent of a specific antigenic component.

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Factors in the fermentation-inhibition test for the measurement of growth-inhibiting antibody in serum to Mycoplasma pneumontiae were studied. The fermentation-inhibiting antibody titer, as read on the day when the color of the control cup without serum had just changed to yellow, was constant among inoculum dilutions of 10^-2 to 10^-5 (10⁶ to 10³ CFU/ml) of a M. pneumoniae broth culture. The use of 10-3 to 10-5 dilutions (10⁵ to 10³ CFU/ml) was adequate for inoculation, inasmuch as one day delay in reading did not result in a significant decrease in the test. Heat inactivation of the serum gave no significant effect on the titer. The test was simple and reliable. The growth-inhibiting antibody was shown to be detectable in the test, when the growth of M, pneumoniae was suppressed at least to 1/100 of the growth of the control without serum. The growth-inhibiting antibody titer rose later than the complement-fixing antibody titter in some cases after M. pneumoniaeinfection. It is suggested that, when an erythromycin-sensitive strain of M. pneumoniae is used, the titer transiently rises and does not show a real growth-inhibiting antibody titer in sera from patients under erythromycin administration.

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When cells of the Clostridium botulinum A190 strain were subjected to treatment with mitomvcin C or to irradiation with ultraviolet ray and then grown at 37C, an induced lytic agent(s) was produced in the resultant lysate. Production of the lytic agent in the induced bacterial culture was inhibited in the presence of chloramphenicol. A crude preparation of the phage-free lytic agent readily lysed freezethawed or acetone-treated cells of C. botulinum B-NIH19, and heated or chloroform-treated cells to a lesser extent at the optimal pH of 6.8. The lytic agent was stable at or below 37C, but unstable above 37C. A crude preparation of the lytic agent did not exhibit a detectable effect on the viability of vegetative cells.

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An attempt to classify fourteen strains of Clostridium sporogenes and thirty strains of Clostridium botulinum types A, B, C, D, E, and F by numerical taxonomy was made. All proteolytic strains of C. botulinum types A, B, and F and C. sporogenes were classified in the phenon I at a level of 86% S-value. In the phenon I, these strains coexisted in a mixed-up fashion, irrespective of conventional species or type of bacteria. A concentrated lysin solution, which was prepared from an induced lysate of a C. botulinum A190 culture, lysed vegetative cells of all proteolytie C. botulinum and C, sporogenes strains classified in the phenon I, but did not lyse the cells of the other clostridia. However, cells of nonproteolytic C. botulinum F-OSU and four out of five strains of Clostridium tetani were lysed by the concentrated lysin to a limited extent. The phenon II contained all nonproteolytic strains of C. botulinum B, C, D, E, and F, which were mutually linked with S values of more than 85%. Strains of Clostridium histolyticum used as a reference group formed the phenon III. Any strain grouped in one phenon was differentiated from strains grouped in the other phenons by low S-values, that were less than 77%. A striking difference in the number of strains susceptible to mitomycin C was demonstrated between C. botulinum and C, sporogenes. In all thirty-five cultures, except four substrains of C. botulinum B-NIH, of proteolytie and nonproteolytic C. botarlinum, bacterial lysis was consistently induced by the treatment of 1μg per ml of mitomycin C, while only four out of thirty-seven C. sporogenes strains were as sensitive to mitomycin C as C. botulinum. Toxigenic substrains of C. botulinum B-NIH, NIH15 and NIH19, were sensitive to mitomycin C, but nontoxigenic strains, NIH5, and NIH-NP, were not.

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Vesicular stomatitis virus (VSV) plaque-forming virions obtained from mixed infection of HeLa cells vith VSV and HVJ (Sendai virus) were shown to contain two types of phenotypically mixed virions: virions doubly neutralizable with both anti-VSV and anti-HVJ antisera (up to 15% ), and virions which could be neutralized only with anti-HVJ serum and not with anti-VSV serum (up to 0.3%). No evidence for genetic recombination was obtained between these two viruses. Phenotypically mixed virions could adsorb to and elute from chiken erythrocytes, and bullet-shaped virions, typical of VSV, were found to be adsorbed to chicken erythrocytes in electron microscopy. Neuraminidase activity was detected in the virions specifically sedirnented with anti-VSV serum. From these findings, phenotypically mixed virions were suggested to contain hemagglutinin and neuraminidase of HVJ in the envelope. In sucrose gradient centrifugation, phenotypically mixed virions enclosed within HVJ antigens sedimented slightly faster than standard HVJ.

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The scanning electron microscope was used to investigate the alteratiwns in surface morphology of Staphylococcus aureus 209P and Escherichia coli NIH induced by the action of cephalexin known to interfere with cell-Wall synthesis. Exposure to cephalexin produced a series of changes on the surface morphology in proportion to the concentrations of cephalexin added. Untreated S. aureus cells had smootb contours. Exposure to 1μg/ml of cephalexin during the logarithmic phase of growth in S. aureus did not produce any detectable changes. Upon exposure of S. aureus to 5μg/ml or 10μg/ml, some cells were larger than noarmal and showed abnormal cell division-like structures in part. When S. aureus was exposed to 50μg/ml, cell division was completely inhibited, and no formation of grape-like clusters was obsersed. Untreated E. coli cells appeared to have smooth and regular contours. E. coli propagated almost nor-mally upon exposure of the organisms to 1μg/ml of cephalexin. Filamentous structures were observed with the exposure of E. coli to 12.5μg/ml or 25μg/ml, but spheroplast-like structures were not observed. Exposure to, 100μg/ml of cephalexin resulted in the formation of marked filament us cells and sphero-plast-like structures having multiple small saccular outpouehings. Scanning electron microscope demonstrated more completely the morphological abnormalities induced by cephalexin.

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The purification procedures and characteristics of an equine infectious anemia (EIA) antigen reacting specifically in the immunodiffusion test with sera from EIA-infected horses were investigated. Partially purified antigens were extracted from both infected horse leukocyte cultures and the horse spleen principally by dialysis against 0.01M acetate buffer, pH5.0. These extracts were further purified by filtering through a Sephadex G-100 column. The purified antigens had a common antigenic determinant and were heat-labile and soluble at pH5.0. The results of immunoelectrophoresis and gel filtration suggested that the spleen-derived antigen was of a smaller molecule than the culture-derived antigen. The partially purified antigens from both sources were comparatively used for the detection of precipitating antibody in experimentally infected horses and in horses in the field. The two antigens showed an identical reaction with each serum tested. The two partially purified antigens were considered to be useful for diagnosis of EIA.

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Cytoplasmic extracts of BHK21 cells infected with Chikungunya virus were analyzed by sucrose gradient sedimentation. Besides 140S nucleocapsid, 65S component associated with 26S single-stranded ribonucleic acid was labeled with ³H-uridine in acinomycin-treated infected cells. The 65S component, demonstrable in ethylenediaminetetraacetic acid-containing buffers, appeared to accumulate when protein synthesis was inhibited. After glutaraldehyde fixation, this component had a density of 1.48-1.50g/ml in CsCl. Pulse-chase experiments did not indicate that the 65S component was a precursor of the 140S nucleocapsid.

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A Pseudomonas aeruginosa, strain Ps5-70 which was mercury-resistant, was found to have a sex factor. This sex factor, designated as FP5 factor, mediated chromosome transfer by conjugation. Crosses between Ps5-70, FP5⁺ and PAO, FP2⁺ strain of Holloway were fertile, and Ps5-70 acted as the donor. Some of the recombinants between FP5⁺ and FP5^- strains displayed maleness, and those recombinants usually acquired mercury resistance. So, transferability of FP5 factor itself and a very close association between the two determinants of FP5, one for fertility and the other for mercury resistance, were proved similar to the FP2 factor. Female sublines of Ps5-70 which behaved as recipients in intrastrain crosses were obtainable from cultures multiplied in the presence of acriflavin. Those females still retained mercury resistance. An R factor originated in P. aeruginosa interfered with the recombinant formation mediated by FP5 factor when the two episomcs coexisted within a single cell.

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Analysis of nuclei of adenovirus 12-infected cells revealed that viral DNA replicated in association with the nuclear membrane and that complete viral DNA was liberated from the nuclear membrane. Analysis of isolated nuclei in vitro showed that DNA polymerase activity increased in the nuclear membrane of adenovirus 12-infected cells without addition of primer DNA.

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