A calorimetric procedure was applied to the study of the inhibitory action of ethanol on the growth of yeast cells. Heat evolved during growth of cultures in media with different amounts of ethanol was recorded as a function of time by using a multiplex batch calorimeter with 24 units. The presence of ethanol affected both the growth rate constant μ and the time tα necessary for the culture to reach a selected level of calorimetric signal. Methodology is presented to quantitatively analyze the recorded data. Based on the analysis of μ and tα, respectively, the parameters defined are: the concentrations of ethanol which inhibit 50% of the growth activity of the cells (named Kμ and Kθ) and the concentration of ethanol at which the growth activity is completely lost (MICμ and MICθ). The values of these parameters were determined for 10 yeast strains grown at 30°C in presence of ethanol. Significance of results, factors that affect the method, and suitability of the procedure for the study of other drugmicroorganism interactions are also discussed.

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Strain KINI-1 capable of producing an anti-nucleating agent was isolated from a camphor leaf. Strain KINI-1 was identified as Acinetobacter calcoaceticus from its characteristics and taxonomics; the optimum temperature and pH for the production of the anti-nucleating agent were 30°C and 7.0, respectively. As the activity of this agent disappeared with the application of protease treatment, this agent, which was an anti-nucleating protein (ANP) was partially purified from the culture to an electrophoretically main band state by ultrafiltration, acetone precipitate, QA-52 column chromatography and Toyopearl HW 55S and Superose 12 gel filtration chromatography. The molecular weight of the ANP estimated by sodium dodecylsulfatepolyacrylamide gel electrophoresis was 55 kDa. The ANP had various substrate specificities for ice nuclei from various ice-nucleating bacteria and AgI. It was demonstrated that the antinucleating activity of ANP increased in proportion to the protein concentration and the antinucleating activity of ANP against ice nucleus from the cells of Erwinia uredovora KUIN-3 was 2.2°C at a concentration of 12.5μg/ml. Furthermore, we found that this ANP was bound to the surface of the ice nuclei, thereby causing anti-nucleating activity. The surface hydrophobicities of the ice nuclei were increased after binding with ANP, whereas the surface charge was changed from 6.6 to 6.1. Also, we confirmed that this action by ANP could give rise to a supercooling point at -5.0°C in the cell suspension of Erwinia uredovora KUIN-3.

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A novel method of killing bacterial cells by cell autolysis induction, ‘the suicide induction method, ’ was proposed. Cold shock treatment and the addition of a surfactant or a high concentration of monovalent cations used for autolysis induction were found to cause the substantial cell death of Bacillus subtilis. Enumeration with the conventional colony counting of the wild-type B. subtilis and its autolysis-defective mutant suggests that the autolysis induction is responsible for the induced cell death, namely bacterial suicide. Although no direct relationship between the cell lysis detected by the optical density decrease and the reduction in colony counts was observed, the evaluation of cell death by the respiratory activity measurement supported the above suggestion. In a continuous tube-flow system consisting of a cooling-warming cycle, bacterial suicide of B. subtilis in substantial numbers was observed. We suggest that ‘the suicide induction method’ may be applicable for practical control of some bacteria.

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The antimicrobial activity of aqueous solutions of benzalkonium chloride (BAC), chlor-h exidine digluconate (CHG), povidone-iodine (PVP-l), and ethanol (EtOH) were tested against a wide range of microorganisms, including hepatitis B virus (HBV) at the actual concentration employed at medical facilities. The chemical stability of these disinfectants in the presence of human serum was also investigated. BAC, CHG, PVP-I, and EtOH preparations showed potent bactericidal activity against nine vegetative bacteria tested and Candida albicans. Three strains of mycobacteria and four strains of spore forming fungi were more resistant to these disinfectants than vegetative bacteria. The spores of Bacillus subtilis were not killed by all four disinfectants even in 360 min at any concentration tested. However, the spores of Clostridium sporogenes and Clostridium sphenoides were killed by PVP-I within 30 min, though they were not killed by BAC, CHG, and EtOH even in 360 min. BAC, CHG, and PVP-I did not inactivate hepatitis B surface antigen (HBs-Ag) in 120 min at any concentration tested. Although HBs-Ag was inactivated by 75 and 95% (v/v) EtOH in 30 s, it was not inactivated by 35 and 55% (v/v) EtOH even in 120 min. The addition of human serum decreased the concentration of aqueous BAC and PVP-I solutions. The decrease of available iodine in aqueous 0.5% (w/v) PVP-I was particularly large ; namely, compared with that of the control, the concentration was decreased over 24 h by approximately 88 and 100% after the addition of 10 and 30% (v/v) human serum, respectively. In contrast, the concentration of aqueous CHG and EtOH solutions decreased little over 24 h after the addition of human serum.

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The features of Arrhenius plots of the relationship between specific growth rate and temperature for four strains of mesophilic bacteria varied depending on the cultivation temperature of the seed. An inflection point was observed around 20°C for the seed grown at 28 to 30°C but not for the seed grown previously at 15 to 17°C. The inflection temperature coincided with the phase transition temperature (around 23°C) determined by the DSC spectra of phospholipids extracted from the cells of Lactobacillus plantarum and Escherichia coil grown at 30°C. From the comparison of fatty acid compositions of phospholipids from the cells grown at different temperatures, unsaturated fatty acids were thought to be converted to corresponding cyclopropanoic acids by transmethylation at the higher temperature. Thus it was suggested that the increase in the cyclopropanoic acids causes the decrease in the membrane fluidity and the inflection point appears in the Arrhenius plot around 20°C for the seed grown at 28 to 30°C.

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N-Alkylcyanopyridinium derivatives which have a cyano group at the 2-, 3-, or 4-position of the pyridine ring were synthesized from the corresponding cyanopyridines and alkyl bromides at 80°C for 72 h under 80 MPa. The bactericidal and bacteriostatic activities of the bromides against Escherichia coli K12 W3110 were strongly affected by the structures of the substituents. Their activities were enhanced in proportion to the alkyl chain length, or the molecular hydrophobicity. The derivatives with the dodecyl group had the same high activities as the derivatives with longer alkyl chains (carbon number = 14 - 18). The introduction of a cyano group enhanced the bactericidal activity, and the derivative with the substituent at the 2-position showed the strongest activity. All of the three dodecylcyanopyridinium bromides had a higher activity in an alkaline medium than in an acidic medium. According to the measurement of the critical vesiculation concentrations of the bromides and the observation with a scanning electron microscope, the cells treated with the bromides leaked turbid materials. The pKa values of the corresponding cyanopyridines did not correlate with their bactericidal activities, but the NMR spectra of the most effective drug, dodecyl-2-cyanopyridinium bromides, implied the increase of the electron density of the ammonium nitrogen atom.

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N-Dodecylpyridinium iodide derivatives having hydroxyl, amino, methyl, chloro and/or trifluoromethyl groups on the pyridine ring were synthesized from the corresponding pyridines and dodecyl iodide under 80 MPa in order to delineate a quantitative structure-activity relationship. The bactericidal and bacterioclastic activity of the derivatives against Escherichia coli K12 W3110 was strongly affected by the kind of substituent groups these derivatives possesed and their positions. The electron-releasing groups such as amino and methyl groups markedly enhanced such activity, while the electron-attracting groups such as carboxyl and carbamoyl groups reduced it. The bactericidal activity of derivatives was dependent on acidic dissociation constant of the corresponding pyridines and the chemical shift of methylene protons adjacent to the ammonium nitrogen. When bactericidal activity was plotted against bacterioclastic activity, the relationship was found to be linear. There was no correlation, however, between bactericidal activity and hydrophobicity of derivatives. In conclusion, our findings suggest that the bactericidal activity of N-dodecylpyridinium derivatives is dependent on the electron density of the ammonium nitrogen, and also on the bacterioclastic activity.

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For the evaluation of pollutant toxicity in the aquatic environment, a novel principle of a dynamic image analysis of bacterial motion by using a tumbling-ability-defective mutant of Escherichia coil K-12, OW22, isolated as a laboratory indicator organism, is proposed. The average linear motion speed of intact cells of this mutant was approximately 32μ Em/s and decreased markedly by the presence of model chemical pollutants tested. It was suggested that the mutant should be useful as an indicator organism for the evaluation of pollutant toxicity by the dynamic image analysis method.

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The single hypha assay method was applied to the screening of active antifungal volatile components emitted from plant leaf samples. An appropriate hypha was selected from a colony of Aspergillus niger or Rizoctonia solani, and its growth rate was measured. A sample holder containing leaf samples was inserted into the reaction vessel. The hyphal growth was observed on a television monitor and recorded on a video tape recorder. The leaf samples were then removed and the reaction vessel was flushed with air. By using this method, the antifungal activity of volatile components could be quantitatively evaluated within several hours.

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Among various cycloalkane carboxylic acids, cyclopentane carboxylic acid was found to be highly active in vivo as a primer molecule of branched-chain fatty acid biosynthesis in Bacillus subtilis 626. The chain-lengthened products derived from this primer substance were identified as ω-cyclopentane undecanoic acid and ω-cyclopentane tridecanoic acid by mass spectrometry. Furthermore, we found that a mixed ethanol solution of fatty acid solutions containing ω-cyclopentane fatty acids, myristic acid and palmitic acid had high antibacterial activity against Bacillus subtilis, and a mixed ethanol solution of palmitic acid and myristic acid had no activity.

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