In preceding papers, several reports were made on the in vitro cultivation of M. lepraemurium. This paper describes some further observations on the primary and secondary cultures of this organism.
1. Gross appearance and transplantability of primary isolates.
Macroscopically the primary cultures of M. lepraemurium isolated on the egg yolk slant may be divided into three: (i) discrete colonial growth; (ii) growth on a debris of the tissue homogenate inoculated, either colonial or membraneous; and (iii) membraneous growth. Although each of the growth was obtained from various kinds of materials, cultures from the tissues with a very large amount of bacilli had a tendency to develop into (ii) or (iii), and the ones from the tissues with a smaller amount into (i).
The positive rate on the first passage of the primary isolates varied over a wide range depending on the type of growth; namely, the rate of those cultures which appeared as (i) colonial growth, (ii) colonial or membraneous growth on the debris, and (iii) membraneous growth was 84%, 50% and 28% respectively.
2. Occurrence of smooth variants during serial passages.
Cultures of M. lepraemurium on the egg yolk slant are originally rough but during serial passages occasionally become smooth. Retrospective analysis on the occurrence of smooth variants was made on a total of 2, 118 subcultures of Hawaiian strain and of 257 subcultures of Keishicho strain in the courses of their respective twenty and sixteen passages. Smooth variants occurred in the 7th to the 14th passages and the rate of occurrence was found to be 0.7% and 2.3% respectively. Gross appearance of the primary isolate and the kind of tissue material used for primary isolation bore no relation to the occurrence of smooth variants.
The in vitro characteristics of smooth variants were the same as those of the original rough ones, except that the formers were more easily emulsified.

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Mice of 6 inbred strains (C3H, CF#1, BALB/C, KK, DDD and C57BL/6) were inoculated subcutaneously in the back with 0.25 ml of a 1:1000 leproma suspension (Hawaiian strain). Spread tissue preparations were made from the inoculation site at about weekly intervals until 10 weeks to observe growth patterns of murine leprosy bacilli in subcutaneous tissues.
There were no remarkable differences among these 6 strains during the first 3 weeks after inoculation. In 1 week, an acute inflammatory reaction with polymorphonuclears disappeared and elongation of the bacilli without increase in number was observed in mononuclears. The bacilli showed longer and thinner forms with a length of about 2 to 3 times the initial size. At 3 weeks, enlarged mononuclears, being crowded with the long bacilli, could easily be demonstrable by low magnification.
Four weeks after inoculation, however, significant differences were observed in growth patterns of murine leprosy bacilli among these mouse strains. In C3H and CF#1 mice, inflammatory cells were consisted mainly of mononuclears, most of which were heavily loaded with the long bacilli. On the contrary, in the other 4 strains, many lymphocytes and polymorphonuclears were found in the subcutaneous inoculation site, surrounding a smaller number of the bacillus-containing mononuclears. Such differences between mouse strains became more remarkable at 5 weeks, because of more pronounced cellular reactions in these 4 strains.
The significant differences between C3H and CF#1 mice were manifested in 10 weeeks. In CF#1 mice lymphocyte infiltration, which surrounded the lesions of mononuclears containing the bacilli, was evident, whereas no host cellular reactions were seen in C3H mice.
From the results of these observations, this experimental method can be recommended for early evaluation of development of mouse leprosy, with special reference to the relation of the host cells to the organism, and these mouse strain differences are presumably due to the cell-mediated immunity developed in the hosts.

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Cytochrome b_l and cytochrome a₂ were detected in the in vivo grown or in vitro grown Mycobaterium lepraemurium, however, cytochrome c and cytochrome a were not found. Cytochrome a₂ is a complex of D and C type cytochrome which is mainly seen in Pseudomonas aeruginosa grown in anaerobic condition and is not seen in the bacteria grown in aerobic condition. Mycobacterium lepraemurium was grown in aerobic condition on 1% Ogawa yolk medium, however, growing place of the organisms might be fairly anaerobic condition because the cytochrome a₂ was found in this organisms. When a few bacteria was inoculated on the 1% Ogawa yolk medium, the organism might be unable to make an optimal anaerobic condition on this medium. Then trial of the cultivation of Mycobacterium lepraemurium under low oxygen tension was made by the author.
Media were put in glass desiccator and were exchanged with gas mixture through sponge gum cap. Mixture gas was added in desiccator once a weeks. As shown in Table 1, the mixture gas composed of 5% CO₂, 1% O₂ and 96% N₂ gave the best result for cultivating with a few organism, but no colonie formation was obtained in case of the inoculation with 10⁵ bacilli. In the primary isolation of Mycobacterium lepraemurium 100% success was not obtained, especially primary isolation from infectious tissue contained relatively little numbers of bacilli was very difficult. As seen in Table 2, the low oxygen condition was better than normal air condition. Primary isolation from ten times diluted inoculum was failed in normal air condition, but succeeded on some tubes in 1% condition. Primary isolation of Mycobacterium lepraemurium from tissue culture of A 31 cells which contained less bacilli than the murine leproma, is also possible in 1% O₂ condition.
Acknowledgment.
In this work, the author was indebted to Miss Saito for making the media. This work was supported in part by the grants from World Health Organization and the USJAPAN Cooperative Medical Science Program.

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Follow-up experiments of Ogawa's method for cultivation of Mycobacterium lepraemurium were succeeded by Kozeki and Mon. This steadfast method of isolation of M. lepraemurium might have been established today. Now it is urgently necessary that this method shold be applied to cultivation of Mycobacterium leprae encouraging by the success in case of M. lepraemurium. As it was already thought from a biochemical study of M. lepraemurium that M. lepraemurium might be injured by the excess of oxygen and Prabharan reported that M. leprae have a diphenol oxidase and the diphenol might have an important role in the metabolism of M. leprae, some suitable reducing agents and diphenol compounds must be used in culture of M. leprae.
Inhibition testes for some reducing agents and diphenol compounds were done by using M. lepraemurium and 1% Ogawa yolk medium. As seen in Table 1 and Table 2, the suitable concentrations of DOPA, cystein, thioglycolate and adrenalin were respectively 3lγ-62γ, 31γ, 15γ-31γ and 10γ per ml. The other redusing and modified reagents were unsuitable to the growth of M. lepramuriume. Then the leproma materials were cultivated for one year at 30°-35°C. on 1% Ogawa yolk media modifield with 33.3γ/ml DOPA, 33.3γ/ml 1-cystein, 17γ/ml thioglycolate and l0γ/ml 1-adrenalin. Eight materials from 8 leprosy patients in National Leprosarium Airakuen, 3 materials from one patient in National Leprosarium Nanseien, one material sent from Professor Nakamura, one material given from National Leprosarium Seishoen and 3 meterials from 3 new patients in our clinic were cultured. The results of all experiments were negative even cultivating under the 5% CO₂, 1% oxygen and nitrogen condition.

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