STUDIES ON PATHOGENIC HALOPHILES:II. SALT REQUIREMENTS FOR GROWTH AND SURVIVAL YUKIO YAMAZI, TSUNEO KOZIMA, TSUYOSHI SHIBA AND TIUICHI ISHIZEKI

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The best growth of N4 and EB102 strains was observed in the presence of 0.4 to 1.2M (2.3 to 7.0 per cent) sodium chloride. This agrees with Takikawa's result(6). Consequently these halophiles can be classified with the facultative halophiles according to Flannery(9).In this experiment, anaerobic culture was performed in almost 100 per cent carbon dioxide or hydrogen, and it was observed that the growth of N4 and EB102 was poor and the peak of the growth curve occurred with lower salt concentrations. The influence of gases upon halophilic bacteria was of special interest to Stuart et at.(10), who made a study in which varying amount of several gases were substituted for air, concluding that red-pigmented halophiles have a preference for reduced oxygen tension. Flannery(9) repeated the work quantitatively with a nonpigmented halophile, and concluded that oxygen tension has little influence on the growth response of the organism.Sodium chloride, magnesium sulfate, potassium chloride, magnesium chloride, sodium bromide, sodium sulfate and sodium nitrate were employed in our investigation of salt requirements of pathogenic halophiles. From the experimental results it is evident that the sodium chloride requirement of the halophile strains is not specific. With salts in which anion substitution was made, chloride, bromide, sulfate and nitrate were found to contribute to the survival of N4 in the presence of sodium. Iodide not to contribute to survival. Chloride and bromide contributed to survival of EB102 in the presence of sodium, but sulfate, nitrate and iodide did not. When cation substitutions were made, sodium, magnesium and potassium contributed to the survival of both strains in the presence of chloride. The degree of the beneficialeffect of ions is described above. Magnesium was more favourable to these organisms than sodium in the presence of sulfate. It was observed that the maximum concentration allowing growth of halophiles was around 1.6M even for the most effective salts. Although our results of salt requirements resemble those of Takikawa(6), some differences are observed which can be ascribed to differences of the experimental methods.Eisenberg(11) and Holm et al.(12) observed that the toxic effect to anions in the presence of sodium for non-halophiles, was in order of toxicity, fluoride, iodide, nitrate, phosphate, bromide, sulfate and chloride ions. A similar order of toxicity was observed in our experiment, except for sulfate and bromide.According to Winslow et al.(13), an antagonistic effect between salts may be explained by antagonism between cations. In our experiment in which halophiles were incubated in media containing one of various salts plus 0.05M sodium chloride, the toxicity of iodium, sulfate, nitrate, and bromide was observed to be antagonized by chloride. This may be due to the supporting effect of a chloride ion concentration as low as 0.05M as an important growth promoting factor for these halphiles. The results of our experiment do not seem to agree with an opinion of Flannery et al.(4, 9) that sodium ions are more important than chloride ion for the growth of V. costicolus, but it must be noted that no cations generally considered to inhibit enzymes were used in our experiment.Lefebre et al.(14) suggested that a certain osmotic pressure may be required by halophilic bacteria, and that if this be so other salts would act similarly and satisfy the requirement. Lamanna et al.(15) insisted on the importance of choosing a measure to separate the effects of osmotic pressure and chemical activity of an environment required by halophiles. Though nonelectrolytes (sugars) were used extensively by investigators of osmotic pressure phenomena, it was found that the sugars would not substitute for the salt required by V. costicolus.(4, 9)
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STUDIES ON PATHOGENIC HALOPHILES:II. SALT REQUIREMENTS FOR GROWTH AND SURVIVAL YUKIO YAMAZI, TSUNEO KOZIMA, TSUYOSHI SHIBA AND TIUICHI ISHIZEKI

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