Small mutant colonies of Vibrio comma and El Tor Vibrio, which were isolated in 1958-1963, were found on the differential plating media containing bile salts, besides the R or rugose type colonies reported by Balteanu and others. Mutant colonies were found most frequently on the Mac Conkey's medium than on the desoxycholate medium, but not on the TCBS medium. The Balteanus rugose and R type colonies did not appear on these media. The mutation rates of strains, subcultured on heart infusion agar slant every week, were relatively higher than frozen dried stock cultures of the same strains. The mutation rates seemed to be affected by the concentration of desoxycholate in the media. Rugose I type colonies appeared about 80% to all colonies on the desoxycholate agar plate without desoxycholate, but decreased with the raise of its concentration, and did not appear on the media containing above 0.07% of desoxycholate. Rugose II and III type colonies appeared very rarely on the media containing above 0.1% of Na. desoxycholate, small I type colonies were found some 40% to all colonies. Small II type colonies were most frequently produced at 0.04% of desoxycholate. Some of the small II type colonies were more exacting in nutrition than other colonies of the parent strains. They could hardly grow on heart infusion agar or peptone water, and all sugar-fermentations and hemolysin production became negative. These mutants showed the decrease in the resistance to alkaline and bile salts. These characters were fixed and did not change by serial transfers. Other mutant strains scarcely showed any changes in biological characters, and these strains did not seem true mutant strains, because back mutation took place in about 70% of the growing cells.
1) Immunospecific antigen consists mostly of protein. Its hydrolysate is observed to contain 20 different kinds of amino acid, to which a small quantity of a polysaccharide consisting of galactose and rhamnose is bound. 2) Non-immunospecific antigen is a polysaccharide consisting mostly of galactose, glucosamine (1: 1) and a microscopic quantity of xylose. It is bound with a small quantity of peptide composed of six kinds of amino acid. 3) Immunospecific antigen was reacted only with anti-anthrax serum but non-immunospecific antigen reacted with anti-cereus and anti-subtilis serum. 4) The antigen-antibody reaction seen in an Ascoli reaction is entirely or nearly a group reaction.
As described in the previous theme, the purified substance, hypoxanthine, was extracted from the circulating blood in the early stage after the second sensitization of typhoid vaccine in rabbits, but it was shown that it has no immunological effects. Then that is why the method of purification was re-examined in this paper. The effective serum separated from blood was treated with Amberlite IR 120 H+ resin (100-200 meshes) or Dowex 50H+ resin (200-300 meshes) and finally, a substance which had been eluted from these resins together with hypoxanthine was separated. This new substance had the immunological properties and an absorption maximum at 24μE like hypoxanthine, but, on the other hand, contained ribose moiety. The substance was re-crystallized from methanol and identified with inosine by the data of paper-chromatography and of intra-red spectrum etc. The purified substance, inosine, as compared with the essential and effective serum (inosine content in serum was calculated by measuring of the optimal density at 249 mμ) separated from the circulating blood in the early stage after the second sensitization of typhoid vaccine in rabbits, was a little weaker in activity. Anyhow, possessing the immunological and biological properties of the antibody promoting factor which had been observed several years before, isonine was indentified with one of the antibody promoting factor itself.