Vi antigen fails to agglutinate when heated at 60°C for 1 hour, thus, it has been considered that the antigen is destroyed by heating.
Stuart and Kennedy stated, in 1948, that upon heating a bacterial suspension, nearly all of the Vi antigen contained is separated from the bacterial cells and the supernatant fluid obtained after its centrifugation has an inhibitory effect on Vi agglutination because the removed free Vi antigen absorbs Vi antibody prior to cause agglutination reaction.
At the Group Meeting sponsored by the National Institute of Health and the Institute for Infectious Diseases held on May 1951, this author reported that the reason why Vi antigen, when heated, fails to agglutinate, is not because it is destroyed but due to its swift isolation into the medium in a large quantity in proportion to the temperature of heating. For instance, when a bacterial suspension heated at 56°C for 1 hour is used in the agglutination test, the reaction is positive at lower dilutions of the Vi serum where there is an abundant amount of Vi antibody, while it is negative at higher dilutions where there is only but a small amount of the antibody. As the bacterial suspension is in a constant concentration, probably, all the Vi antibody contained in the higher dilutions of serum is absorbed by the free Vi antigen in the suspension, whereas, at lower dilutions, there may still some amount of antibody left over to cause the agglutination. Accordingly, when the agglutination is made either with the bacterial suspension removed of free Vi antigen by centrifugation or with the suspension prepared from the culture grown at 22°C inhibiting the development of Vi antigen, it is practicable to cause the agglutination with extremely high serum dilutions.
No appreciable difference was observable, upon running Vi precipitation tests, between the heating at 56°C for 1 hour and at 100°C for 2 hours. Thus, saline suspensions of typhoid bacilli V form were heated at varying temperatures of 56°C, 100°C and 120°C for 1 hour and Vi precipitation tests were made with the supernatant fluids obtained by their centrifugation. Precipitin titers of the supernatant fluids subjected to different temperatures of heating were practically equal, whereas, upon running Vi agglutination tests with the bacterial precipitates of the above suspensions, a strange phenomenon was observed which was the precipitate of the suspension heated at 120°C showed slight agglutination.

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Dysentery cases have been increasing in Japan since the end of the second world war. Especially in the year 1951, reported cases were largest during these years. The present paper is dealing with the typing of Shigella strains isolated in Japan during the year 1951, and forwarded to our laboratory mostly for type-identification and partly to meet our request. But the strains examined were not of the exact representation of true type distribution.

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Several Japanese investigators have described of the cystophorous cercaria from snails, Semisulcospira spp. in Japan, and its first description was made by Osafune (1899), who recovered it in Okayama Prefecture. Seno (1903), Miyagawa (1913) and Ando (1915, 1918) have also made its descriptions later respectively, but these were very incomplete. Following these reports, Yoshida (1917) made a more detailed description of this cercaria which is called “Cercaria F” by him, obtained in various areas in Japan, and Cort and Nichols (1920) named it Cercaria yoshidae. During the past few years the author have recovered many Semisulcospira spp, in various areas in Japan, some of which are from the same spots where Yoshida has collected it before. These snails are parasitized with some 18 species of trematode larvae, one of which being a cystophorous cercaria presented here. In the following chapter, its morphology, geographical accounts and seasonal distributions in detail are reported.

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While investigating the growth inhibitory influence of aromatic acids on various strains of microorganisms, Momose observed and reported in 1930 that pseudodysentery bacilli when grown on agar medium containing hydrocinnamic acid or its sodium salt, stained the medium in reddish brown, while those strains of shigella, typhoid, paratyphoid A, B, and coli groups did not show this coloring reaction in spite of the cultivation done in the same manner. No follow-ups have been made on this reaction and it appears that the problems on pigment formation of dysentery bacilli have been ignored.
While transplanting stock cultures on agar medium since 1943, in our laboratory, some strains of dysentery bacilli were found to color their medium in reddish brown. Upon investigating the cause for this coloring, it was found that the coloring appeared only when Japenese meat extract “Render” was used in the preparation of the medium.
Being hinted by the above mentioned Momose's paper, the presence of hydrocinnamic acid was suspected. When its isolation was attempted a certain substance, the melting point of which was the same as that of hydrocinnamic acid, was obtained.
The agar medium added with this substance was recognized to present exactly parallel coloring reaction as that observed with the medium containing Render meat extract or hydrocinnamic acid. Therefore, the coloring reaction of Render meat extract was found, as reported by Momose, due to the existence of hydrocinnamic acid.
The medium used was prepared in the following manner in order to facilitate the observation of this coloring reaction :
Pepton 20 gm, sodium chloride 5 gm, 10% alcohol solution of hydrocinnamic acid 1.5 ml (approximately 1/1000 Mol), agar 20-30 gm, aqua distillata 1000 ml and pH was adjusted to 7.2-7.4.
When dysentery bacilli are grown on this medium for approximately 20 hours at 37°C and left standing for additional 10 hours at room temperature, the coloring of medium becomes distinct. The coloring also appears but rather weakly when meat extract or meat infusion is added to the above medium. The coloring of medium starts with light pink color surrounding colonies and, following the elapse of time, becomes red spreading all over the slant surface. The coloring was the most intense when left standing for 2 to 3 days at room temperature. Thereafter, changing gradually into brown disappeared by the end of 10 days. Further, the colonies themselves were found stained in faint brown.
Under anaerobic cultivation, this coloring reaction does not take place, but as soon as this medium is exposed to the air, it shows red coloring. The coloring reaction is not influenced by the brightness of light. Optimal cultivation temperature for the coloring reaction is 30°C rather than 37°C. Optimal initial pH of the medium stands between 7.0 to 7.5. Presence of any saccharides, which will subsequently be decomposed by the bacilli, retards the coloring reaction. This is because the medium is acidified, so that, as soon as the medium is alkalized the coloring appears. The pigment produced is easily soluble in water, slightly in methanol and ethanol but absolutely insoluble in any other organic solvents. The pigment loses its color and changes into yellow when the medium is strongly acidified or alkalized, but returns to reddish brown again when the medium is reduced to weak alkaline. Thus, the reaction is reversible. No appreciable change in the coloring will be produced by boiling of the pigment for 30 minutes at 100°C.

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Several papers have already been published for answering the question whether or not the tricarboxylic acid cycle or the C₄-dicarboxylic acid cycle plays a rôle in microorganisms, and a summarizing description of the subject is seen in Ajl's article. Karlson and Barker, for example, stated that the tricarboxylic acid cycle does not operate in Azotobacter agilis, Barron, Ardao and Hearon claimed, however, Corynebacterium creatinovorans to oxidize acetate via the dicarboxylic acid cycle. In the case of Escherichia coli, it has been insisted by Ajl that not the tricarboxylic but the dicarboxylic acid cycle with the Knoop-Thunberg condensation is operating in an adapted way.
In the present paper we are dealing with some adaptive growth patterns of Salmonella typhi using carbon compounds found in Krebs cycle pathway and a certain aspect of the problem with this organism will be discussed.

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