Genetic studies on the cycloserine-resistance system of Mycobacterium tuberculosis var. hominis (strain Aoyama-B) have been conducted.
It has been suggested that there are two genotypes determining cycloserine-resistance, one determines resistance until 60 to 70 mcg and the other determines resistance until 100 to 200 mcg of cycloserine. Organisms having the wild genotype are able to survive on media containing below 20 mcg of cycloserine. Such mutants as capable of growing on media containing above 200 mcg of cycloserine have not been observed.
The authors wish to express appreciation to Dr. R. Katsunuma, Director of the Obuso National Sanatorium, and Prof. S. Hibino, Nagoya University, for their kind encouragement in the conduct of the study. The authors also wish to express their appreciation to Prof. I. Uchikawa for his kind advice.

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Five kinds of purified tuberculin protein, Seibert's PPDs, IP48 of Bretey and Lamensans, Takeda's TA2 and our π and πT, were prepared from the same lot of culture filtrate of tubercle bacilli for comparative evaluation of the methods of purification.
1. The yield of π was rather small, and the others about equally large.
2. In chemical determination, the content of DNA and polysaccharides was smallest in PPDs and about equal in the rest. The DNA content of PPDs was also found to be small by absorption spectra and electrophoresis.
3. Electrophoretic analyses showed the presence of some protein components of slow mobility besides the main protein component in PPDs and TA2, though it was not recognizable in π or πT, and recognizable only in a small amount in IP48.
4. The amount of potency recoverable in purified tuberculin protein was about 65 to 100 per cent of that of its original culture filtrate.
5. Certain problems suggested by these results concerning a method of purification to be adopted in the preparation of tuberculin protein for routine use have been discussed.

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Genetic studies on the 4-acetylaminobenzaldehyde-thiosemicarbazone (tibione) resistance system of Mycobacterium tuberculosis var. hominis (strain Aoyama-B) have been made with the following conclusions:
There are two genotypes (genes) determining tibione resistance; one determining high resistance and producing individual mutants resistant to 10μg/ml to 50μg/ml (or above) of tibione and the other determining low resistance and producing individual mutants resistant to 1μg/ml. but not resistant to 10μg/ml. of tibione. There appears to be no graded degrees of resistance between these two genotypes (genes). Mutants of these two genes occur independently. Therefore, these two genes appear to be duplicate factors and to be produced by a single gene mutation.
There may possibly be a gene producing wrinkled and large or elevated colonial morphology and there appears to be a close relationship between the gene and the gene determining high resistance. (It may be also possible that both high resistance and wrinkled colonial morphology are produced by manifold effects of a single gene.)

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N4 and EB102 strains isolated respectively by Takikawa and Fujino from cases of food-poisoning are reasonably identified as Pseudomonas or Vibrio, and they differ only in their activity with arabinose, in their morphological and biological characteristics. Their biochemical activities were strengthened in media containing 3 per cent sodium chloride.
In survival studies of N4 and EB102 strains, it was found that magnesium sulfate contributed more favourably to their survival than sodium chloride, but even the former supported the survival for only 2 days, and not for 5 days. On the contrary, these organisms were alive more than 67 days in 30 to 100 per cent marine water. ZoBell⁽¹⁾ reported that natural marine water was more adequate for cultivating freshly isolated marine bacterial strains than isotonic salt solution and an artificial marine water. MacLeod et al. ⁽¹³⁾ suggested that the favourable effect of marine water contributing to the growth of marine bacteria was attributed to inorganic ions contained in it. Our experimental results seem to agree with the former report. Yasukawa⁽¹⁴⁾ observed that V. cholerae survived in sterilized marine water for 2 to 4 months at room temperature in the summer. This information of Yasukawa's suggests a notable resemblance between the pathogenic halophiles and V. cholerae.

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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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1) The author has succeeded in devising a complement fixation test on filter paper using simple equipment devised by the auther.
2) This method is almost equally as sensitive as the AMSGS complement fixation test.
3) The results of this reaction can be preserved for a long time on filter paper.

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1. Zinc and mercury ions appeared to be toxic to Serratia marcescens; copper, nickel, iodide and bromide ions interefered with the production of 2, 3-butanediol by these organisms.
2. Iron and molybdenum ions exerted a beneficial effect on 2, 3-butanediol formation but with increased consumption of sugar.
3. Sulphide and borate ions decrease the formation; and thiosulphate and and sulphite cut the formation of the diol per 100 ml of the culture to one-half.

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Using the autolysate of bacteria which possess the antigens of subgroup E₃, an antigenic transformation E₁→E₃ and E₂→E₃ has been observed. The transformation E₁→E₃ may also occur in the direction E₁→E₂→E₃. Two strains of subgroup E4 were transformed by the process of E₄→E₂→E₃. Antigen-transforming and bacteriolytic powers of the autolysate were demonstrated in parallel and it was supposed that O-3.10→O-3.15 and O-3.15→O-(3).(15).34 can be ascribed to two different bacteriophages.
When antigenic transformation was attempted on E3 strains with anti-O-34 or anti-O-15 serum, O-(3).(15).34→O-3.15 or O-(3).(15).34→O-3.10 was observed.

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In the previous paper, the author reported that in Salmonella group E the strains having the antigen of subgroup E₂ were transformed into E₃ by the autolysate of bacteria of E₃ and that in this phenomenon two different bacteriophages were thought to participate. This paper is an investigation of the relationship between these two bacteriophages and the transforming agent. Their properties will be compared with those of e phage reported by Iseki and Sakai.
Two bacteriophages participating in antigenic transformations O-3.10→O-3.15 and O-3.15→O- (3). (15).34 were purified to discover the transforming agent, which was concluded to be the phage itself.
The two phages were completely different and unrelated both biochemically and serologically. ε15, which was considered responsible for O-3.10→O-3.15 was found to be identical with ε. The other phage found by the author and considered responsible for O-3.15→O- (3). (15).34 was designated as ε34. This is a spherical particle 26-40 mμ in diameter, which resists 80°C for 20 minutes, but is inactivated at 80°C for 30 minutes. It is stable at pH 2.4-12.0 and inactivated at pH below 2.2 and above 12.5. The host range was limited to strains possessing antigens of subgroup E₂. ε34 transformed the strains having antigen of subgroup E₂ into that of E₃. It could not directly transform the strains having antigens of E₁ and E₄ into that of E₃, but could by two steps with the help of ε15. The percentage of cells which had antigen transformed by the infection of ε34 was from 2.6 to 10.3%.
This antigenic transformation can not be explained by spontaneous mutation selection by phage and evidently differs from transformation in the pneumococcus and transduction. Bacteria having the antigen of subgroup E3 were doubly lysogenic. Prophages of ε15 and ε34 in the cell are considered to govern the production of O-15 and O-34 antigens.

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1) A fluorescent antibody technique using 1-dimethylaminonaphthalene 5-sul-phonylchloride and Xylene Red B has been described. The conjugation procedure for each dye with protein is simple without any deterioration of the antibody activity.2) The quality of each conjugate was determined by use of a Beckman spectro-photometer and spectrofluorometer. 3) The viral antigen synthesized in Ehrlich ascites tumor cells infected with ED virus was distinctly visualized by staining of each conjugate, and the specificity of fluorescent staining of antigen was confirmed with the systematic control tests.
XRB-conjugated protein was preferable for the detection of antigen, because it showed brilliant orange fluorescence and could be clearly discriminated from the auto-fluorescene of cells. 4) The viral antigen was detected in the cytoplasm of the infected cells within 6 hous after virus challenge, and increased continuously until a maximum was reached at 24 hours. 5) The observation by mean of fluorescent antibody technique confirms the the previous results obtained by immunochemical and cell-fractionation studies.

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The growth-promoting action of Tween 80 on Ery. incidiosa was investigated with the following results: 1. When the concentration of Tween 80 in the medium was 0.1% the greatest growth promotion was noted regardless of whether the medium was liquid or solid.2. The growth-promoting effect of Tween 80 was dependent on the kind of peptone used. 3. Tween 80 had a more remarkable growth-promoting action than horse serum, but the turbidity of medium containing the former was lower than that of medium containing the latter. 4. A growth-promoting effect of Tween 80 in casamino acids medium was nut observed when yeast extract was added, but definitely observed when glucose was added. 5. Tween 80 did not show an inhibitory effect even when a minute inoculum of bacteria used. 6. Cultures grown in agar medium containing Tween 80 showed considerable changes both in morphology and colony form. 7. Tween 80 had a growth-promoting effect not only on Ery. incidiosa but also on Corynebact. pyogenes. Tween 60 was accelerative on Ery. incidiosa, but was weaker than Tween 80. 8. The mechanism of growth-promoting action of Tween 80 on Ery. incidiosa was discussed. It was assumed that partly it may neutralize the toxicity of the medium, in addition to its wetting and spreading action.

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1. Killifish (Oryzias latipes) died when they were kept in water in which ground fish suspension had been added in various concentrations. Death was related to the growth of Proteus vulgaris in the water. 2. Killifish also died in water in which preparations of “peptone” or tryptic digest of casein had been dissolved. In this case also the growth of Proteus vul-garis was involved. 3. The bacteria themselves had no toxic action. Some toxic factor produced by the bacteria resulted in the death of fish. The production of the factor was not directly proportional to the degree of bacterial growth. 4. Various amino acids gave no support to toxic factor production. Some de-composition product of protein, probably some polypeptide, appeared to be changed into the toxic factor by the action of the bacteria. The toxic factor was produced sooner and to a greater degree as the peptone concentration was increased. 5. Toxic action of the factor was greater as the temperature increased. 7. Fish exposed to sub-lethal doses of Proteus vulgaris toxic factor and reex-posed after two days to a lethal concentration, died in the same manner as the fish which had never been exposed to the factor, showing that there is no possibility to produce in the fish any immunity to the factor. 7. The production of toxic factor affected by the shape of vessel in which the bacteria were incubated. Petri-dishes, which had far greater surface area than test tubes, were generally more suited for the production than test tubes.

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