In the previous communication, it was shown that hyperimmune horse serum injected directly into the brain tissue of mice 24' hours following the inoculation of Japanese encephalitis virus could reduce the mortality of the animals as compared with the untreated controls.
The following experiments were undertaken to elucidate the quantitative aspects of the serum therapy of experimentally infected animals.

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Biochemical reactions of Pasteurella pestis have been investigated by many workers. There are, however, some points of disagreement among them. I had a chance to study a collection of strains of Past. pestis preserved for many years at this institute. The results of biochemical studies with special reference to fermentation reactions of these strains will be described and discussed together with a review of the literature.
Strains used: 69 strains preserved for many years at this institute. Regarding their history refer to my report follwing this paper.
These strains were first plated on horse serum agar, typical coloniq were fished, transplanted on agar slant, and then used in the present experiments.
Morphology: Slide preparations were fixed in a mixture of equal volumes of ether and ethyl alcohol for one minute, wormed by gas flame in order to evaporate the fixative, then stained with Loeffler's methylene blue or Pfeiffer's stain. The organisms were short thor rods with rounded ends, many of them oval, but their shape and size varied considerably, some being elongated as filaments. A few showed bipolar staining. Gram-negative. Degenerated forms in ager slants containing 2.5 per cent NaCl.
Motility: Negative in broth culture at 37°C and 20°C. Tests of motility in the culture at 20°C are said to be important in respect of differentiation from Past pseudo-luberculosis (Arkwright) .
Culture: Growth on ordinary media. Colonies on agar media peculier to Past. pestis with raised center, flatt margin and granular structure. In broth very, slightly turbid, later clear, with floccular deposit and pellicle.

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With advance of protein chemistry, many phenomena of immunity have been explained from viewpoints of physical chemistry. Especially the introduction of the quantitative technique of estimating amounts of antigens and antibodies by Heidelberger has yielded important informations in this field. There are, however, many problems remained yet unsolved, one of which is the role of lipid in immunity reactions.
The effect of lipid extraction from antisera, without apparent denaturation of proteins, was first studied by Hartley, and his observations were extended and complemented more thoroughly by Horsf all and Goodner. According to these authors, the extraction of lipid from anti-pneumococcal horse sera with alcohol and ether in the cold resulted in the complete disappearance of agglutination and precipitation, and also rabbit antisera lost their agglutinating and precipitating ability after the extraction with alcohol, petroleum ether, and ether in the cold. In spite of the disappearance of visible reactions in vitro, the protective power of the extracted antisera was not reduced. Moreover the agglutinating and precipitating ability of the extracted horse serum was restored by the addition of lecithin, while that of the extracted rabbit serum was restored by cephalin. If an extracted serum was injected into the peritoneal cavity of mice and the peritoneal fluid was withdrawn after 30 minutes, this fluid was now able to agglutinate pneumococci.
It appears, therefore, that the extraction process deprives the antisera of their agglutinating and precipitating ability, but it has no influence upon the combining power of antibodies with antigens, and the addition of lipid restores the visible reactions in vitro.
In our experiment we undertook to decide, by means of quantitative method, whether, or not antibodies in the extracted sera were combined with antigens, when their agglutinating power was lost.

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In the field of antibiotic microorganisms streptomyces has been studied most extensively concerning its antibiotic properties. In our laboratory many antibiotic strains have been isolated, and some of their antibiotic have been identified as Actinomycin A, Streptomycin, Streptothricin-group substances, Chloromycetin and Grisein. It seems, however, that there are more other antibiotic substances in this field.
Although the nature of the antibiotic substances of our strains has been and even now is being studied in detail, the characters of the strains have not yet been studied sufficiently. So I undertook the work of classifying these strains. In this paper the characters of our Chloromycetin-producing strain are described.
In regard to the classification of Actinomycetales, a splendid and systhematic description by Waksman was published in Journal of Soil Science (1919) and recently a new classification by him and Henrici has appeared in Journal of Bacteriology (1945) - In my classification studies chiefly these two papers and original descriptions of each species have been referred.

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The fields of antibiotic substances of streptomyces have been examined most in detail, and actinomycin, streptothricin, streptomycin, grisein, chloromycetin, actidione and aureomycin have been isolated and studied in detail. But there seem to be more other antibiotic substances. In general an antibiotic strain of streptomyces develops on the nutrient agar and produces the antibiotic substance which diffuses in the agar and inhibits the growth of bacteria near around its colony. But it is not always that streptomyces showing the inhibiting zone on the agar produces the antibiotic substance in the nutrient broth by shaking or stationary culture. Besides there is not any strict conformity between the kind of species and the kind of antibiotic substances produced. For example, both a streptomycin-producing and a grisein-producing strain chiefly belong to S. griseus, and streptothricin-group substances are produced by various kinds of streptomyces. In these circumstances, it is necessary to identify the kind of antibiotic substances by testing their antibacterial spectra on the nutrient agar, in order to find a new antibiotic substance.
Since many antibiotic strains had been isolated in our laboratory and some of their antibiotic substances had been determined, such as to be actinomycin, streptothricin-group substances, streptomycin, chloromycetin and grisein, so the antibacterial spectra of all the other strains were examined, in order to find whether there were some strains producing a new antibiotic substances. But it is not easy work to test the complete antibacterial spectrum of each strain against many kinds of bacteria. As shown in our previous report, a strain which resistance to one of the antibiotic substances was forcedly increased does not usually become more resistant to other antibiotic substances. streptomycin-fast strain of B. coli is more resistant to streptomycin than the normal strain, but not to streptothricin and other antibiotic substances.. A streptothricin-fast strain of B. coli increases its resistance only against streptothricin-group substances and streptomycin. A grisein-fast strain increases its resistance specifically against grisein. So if the resistances of the fast strains to an antibiotic are tested, then it can be simply determined to which kind of antibiotic substances it belongs.
In stead of testing resistances of many kinds of bacteria, the resistances of the above three fast strains and the normal strain of B. coli were tested against the antibiotic substances produced around the colonies of antibiotic strains of streptomyces. Moreover, as we had already found that B, subtilis is more resistant to streptomycin that B. anthracis and the latter is more resistant to streptothricin-group substances than the former, so the resistances of these two kinds of bancteria were also tested.
According to the antibacterial spectra, antibiotic strains were at first divided into two large groups, the first inhibiting the growth of B. coli and the second not inhibiting B. coli. Furthermore the first large group could be classified into five groups. In this paper the classification of the first group is described. The classification of the second group will be published later, though it is already found that there are more than two kinds of antibiotic substances which do not inhibit the growth of B. coli, that is, actinomycin and other antibiotic substances.

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Antibiotic strains of streptomyces can be classified according to their antibacterial spectra around their colonies growing on the nutrient agar, as described in the previous paper, and it can be easily estimated what kinds of antibiotic substances are produced by them. The strains, isolated by us and inhibiting the growth of E. coli, were classified to streptomycin-producing group, streptothricin-group-substances-producing group, geisein-producing group, chloromycetin-producing group, and the fourth group which chiefly produces streptothricin-group-substances. But during further studies we found a strain showing a new antibacterial spectrum and a yellow crystalline antibiotic substance was extracted. It inhibits both the growth of gram positive and that of gram negative bacteria.
Among antibiotic substances obtained from streptomyces, aureomycin and xanthomycin which were recently found are reported to be yellow colored. Though we do not know the details of these substances, they seem to be different from the substance in this paper. Besides we can not find the species in literature which is identical with the strain No. 26A producing the antibiotic substance described in this paper, though it seems to be most resembling to Actinomyces farcinicus or Streptomyces lipanii. So we decided to call the antibiotic substance Aureothricin.

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As described in our previous report, the antibacterial actions of lichen extracts were studied by some investigators.
We also investigated on the relation between the antibacterial effects and chemical constitution of usnic acid, which is widely distributed in lichen.
The purpose of this paper is to report the results of a systematic study of the antibacterial effect of various types of lichen substances. In this report, we present our studies on antibacterial activities of 23 substances belonging to various groups as classified by Asahina.

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There have been few works on the relationship between growth factor and nutritional sources: carbon and nitrogen sources. Wood et al showed that a given strain of propionic acid bacteria required riboflavin in an ammonium sulfate medium but not in amino acid medium. Kligler and his co-workers showed similar results on the dysentery bacillus about the relationship between nutritional sources and nicotinic acid. Namely, Shigella dysenteriae and S. paratyphi A could grow on the peptone media (or amino acid mixture) without nicotinic acid. When glucose was added, nicotinic acid became essential for growth, whereas, when lactate was added in the place of glucose, nicotinic acid was not necessarily essential. This was also shown in the dehydrogenase activity test by using the resting bacteria. In the case of Proteus vulgaris OX K, it was shown that glucose was ratherr inhibitory when nicotinic acid was absent.
It is the purpose of this paper to report certain quantitative studies on the relationship between nutritional sources and growth factor, nicotinic acid or its amide, in Proteus vulgaris OX 2, both in the growth and in the dehydrogenase activity test.
In the first place, amino nitrogen, asparagine was used as a nitrogen source with and without glucose. In each case, the degree of growth was estimated in relative to the concentration of nicotinic acid or its amide.
Secondly, ammonium nitrogen, ammonium sulfate was used as a nitrogen source. In this case, glucose, sodium lactate or potassium pyruvate was added individually as carbon source. In each case, the degree of growth was also estimated in relative to the concentration of nicotinic acid or its amide.
In dehydrogenase activity test, we adopted the Thunberg's technique, using the bacterial suspensions freshly collected from the liquid culture, having suboptimal amount of nicotinic acid. Nicotinic acid or its amide was added in order to determine whether these growth factors can stimulate the reduction time of methylene blue or not.

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In parts II and III of this paper what may be regarded as the optimum constituents of synthetic medium were determined and now it is the intention of the author to carry out comparative experiments between natural and synthetic media.

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