Most amino acids, except cysteine, protected the infectivity of phenol-extracted poliovirus ribonucleic acid against inactivation during incubation at 37 C. Of these amino acids, histidine was most effective, whereas cysteine enhanced inactivation. Besides cysteine, three sulfhydryl compounds were also examined. Thioglycolic acid exhibited a similar effect as cysteine, but glutathione and mercaptoethanol were protective or enhancing in the inactivation of poliovirus RNA depending on the concentration of the drug. The effect of metal ions was diverse. Cu⁺⁺ and Hg⁺⁺ reduced the infectivity extensively, whereas Fe³⁺ and Al³⁺ were protective. Some of other metal ions were more or less inactivating, while the other metal ions examined were not. A protector and an inactivator of infectious RNA antagonized each other.

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Ten strains of Proteus morganii were selected and the production of β-lactamase was studied. The cellular level of β-lactamase from P. morganii was regulated by the concentration of the inducer in the growth media, and the enzyme activity appeared within 10 min while its highest value was ob-tained 2 hr after the addition of benzylpenicillin as the inducer. The resistance level to cephaloridine was generally related to the amount of β-lactamase activity among the ten strains of P. morganii, but no relation was found between the ampicillin susceptibilities and the amounts of β-lactamase in their cell-free preparations. The maximum rate of hydrolysis of cephalosporin C and several other deriva-tives of 7-aminocephalosporanic acid by a crude enzyme was five times higher than that of benzylpeni-cillin. Semisynthetic penicillins were resistant to hydrolysis and exhibited competitive inhibition. Among the semisynthetic penicillins, dicloxacillin and carbenicillin were powerful competitive inhibitors of the β-lactamase from P. morganii.

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In the chick tracheal organ culture of Bordetella pertussis phase I organisms, marked bacterial growths were observed in the culture medium and on the tracheal fragments. However, serum-free fresh or conditioned medium alone as well as an emulsion of tracheal tissues prepared in the condi-tioned medium supported little growth of the bacteria. The need for the existence of the organ fragment was inevitable. Fluorescence microscopy of the infected tracheal fragment and a time course study of bacterial growth in the medium with the organ culture suggested that the principal site of bacterial growth appeared to be on the surface of the tracheal fragments and the increase in the culture medium might be brought about mainly by the release of bacteria therefrom.

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C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hyper-sensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensi-tized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hyper-sensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day -21) and in CFA (day -7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pre-treatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.

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A method which permitted counting viable cells of Bifidobacterium bifidum N4 in a solid medium vas developed. A piece of the solid medium (0.7 ml) was quantitatively obtained with the aid of an agar-puncher device and was homogenized in a Potter-Elvehjem homogenizer after the addition of 9.3 ml of sterile physiological saline. A 10-fold dilution of the homogenate was repeated several times to make a series of dilutions. An aliquot (0.2 ml) of the appropriate dilution was used for counting the viable cells using a capillary tube method. The accuracy and the reproducibility of the method were comparable with those of the conventional plate counting method. By using estab-lished procedures the behaviors of B. bifidum N4 in a solid medium were studied. Viability of the organism in a solid medium lacking an energy source (lactose) was generally correlated to the period of preculture; the longer the period of preculture, the shorter was the span of cell life.

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Linkage relationships among loci conferring resistances to neomycin-kanamycin (nek), vio-mycin-capreomycin (vic) and streptomycin (str), as well as auxotrophic markers, were analyzed employing the conjugation system of Mycobacterium smegmatis strains Rabinowitchi and PM5. Nek and vic loci were found to be closely linked to each other and also linked to argA-6 and argB-1. No linkage was observed between str and nek or vic. The locations of other loci, met-5, his-13 and leu-11, also could not be determined. In certain recombinant types, nek and vic alleles formed stable hetero-genotes. When nek^r and nek^s or vic^r and vic^s alleles existed in one cell, sensitivity to those drugs was dominant over resistance. Segregants of nek^r or vicr from the heterogenotes were detected at a fre-quency of about 10^-4 to 10^-5. Such a stable heterogenote was not detected in str allele.

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The antibiotic susceptibility of laboratory strains, Mac and FH, and isolates of Mycoplasma pneumo-niae was determined in broth media. With the Mac strain, the minimum inhibitory concentration (MIC) increased with the increase in inoculum size for most antibiotics and the final minimum in-hibitory concentration as determined during a month's incubation did for all antibiotics. The antibiotics studied were classified by the MIC into three groups; the first group with the order of 1/100 μg/ml (erythromycin, josamycin, and leucomycin), the second with the order of 1/10 μg/ml (oleando-mycin, tetracycline, and spiramycin), and the third with the order of 1 or 10 μg/ml (lincomycin, chloramphenicol, and streptomycin). With the FH strain, the MIC of streptomycin was much lower, showing that the Mac strain was resistant to streptomycin. The susceptibility of 152 isolates from 99 patients to erythromycin, josamycin, tetracycline, and chloramphenicol was essentially similar to that of the Mac strain except for two isolates, which had been isolated from a patient during erythromycin therapy and exhibited resistance to erythromycin and josamycin. The MIC of strepto-mycin was distributed at 1 μg/ml in 93% of the isolates and at 100 μg/ml or over in 6%. It was considered that streptomycin-resistant strains occur in M. pneumoniae. Of nine patients who had been given erythromycin, tetracycline, chloramphenicol, or Clotaon for more than a week, the iso-lates after therapy showed decreased susceptibility to erythromycin in one patient and did not to the antibiotics given in the other eight patients.

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Mycoplasma pneumoniae developed high degrees of resistance to streptomycin and dependence on streptomycin after serial subculture in broth media containing streptomycin. The streptomycin-dependent organisms failed to grow, as indicated by a color change of the medium, in the absence of streptomycin but grew in limited concentrations of streptomycin in broth media. The minimum growth-inhibitory concentration of streptomycin for the parent sensitive strain was very close to the minimum concentration of streptomycin which permitted the growth of the streptomycin-dependent organism. The broth cultures showing streptomycin dependence were shown to consist of streptomycin-dependent organisms or a mixture of streptomycin-dependent and-resistant organisms, as tested on agar media containing varying concentrations of streptomycin.

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L-MS cells, adapted to grow in suspension, were obtained by selection from a high interferon (IF)-producing line of mouse L cell monolayers. A large volume of L-MS cells (20 liters or more; 1-2×10¹⁰ cells) was readily grown in a spinner culture, retaining their ability to produce high yields of IF in serum-free medium following induction with Newcastle disease virus (NDV). The optimal condition for the production of IF in the suspension culture of L-MS cells was established.The system also proved itself to be susceptible to IF induction by polyinosinic-polycytidylic acid (Poly I. Poly C) and by NDV inactivated with ultraviolet light (NDV-UV). By employing the present system, large quantities of mouse IF of a high titer could be routinely prepared.

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The initiating mechanism in the germination of Bacillus thiaminolyticus spores was studied with 14C-L-alanine. A characteristic pattern of incorporation of L-alanine into the spores was observed during the early stages of germination with two incorporation peaks, one occurred just after contact with L-alanine (first incorporation) and the other 5 min later (second incorporation). L-Glutamine, L-valine, or L-serine substituted for the incorporation of L-alanine during the first stage of germi-nation. Although, L-alanine taken up during the first incorporation phase was extractable with trichloroacetic acid (TCA), that taken up during the second incorporation phase was not extractable. The distribution of radioactivity showed that incorporated L-alanine was located in the spore coat, mainly in the paracrystal fraction. The radioactive material which remained in the germination medium or was extractable from the spore coat fraction with TCA treatment or pronase digestion was identified as alanine. Significance of incorporation of L-alanine and its location in the spore in reference to the initiation of germination is discussed.

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The inhibitory mechanism of D-alanine on L-alanine-induced germination in spores of Bacillus thiaminolirticus was studied. D-Alanine completely inhibited every L-amino acid-induced germinations and had the strongest inhibitory effect among all D-isomers. A kinetic analysis of the inhibitory effect indicated that D-alanine competitively inhibited the incorporation of L-alanine during the initial stages of germination.

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