Cultivation of M. leprae.

A peculiar yeast-like microorganism, isolated from leprous lesions, was discovered to produce the growth promoting factor for M. leprae. From the mass culture of this organism, the oil substance stimulating M. leprae growth was extracted with hot acid ethanol and purified using organic solvents. The final product was considered to be a kind of siderophore. When inoculating M. leprae on the special solid medium, contained chemically defined materials and supplemented with the growth factor, and incubating for 7 days in 35-36 degrees C, light yellow R type colonies were observed. The colony was composed of the acid fast bacilli which were morphologically indistinguishable from bacilli in the tissues. Successive cultivation of the bacilli was possible only on the special medium used but not on the ordinary media for general mycobacterial species. The whole identification studies are not yet finished, however, the cultivated organism was expected as M. leprae from several view-points including the high frequency of positive cultures.

Failure to validate the growth of Mycobacterium leprae on M-Y 14b agar medium.

Confirmation experiments on colony formation of M. leprae on the M-Y 14b agar medium developed by Murohashi and Yoshida were carried out for two years independently by individual members of an organized research group, according to the method described by Murohashi and Yoshida. The results obtained can be summarized as follows: a) No colony production by M. leprae on M-Y 14b agar medium was seen. b) No increase in the number of cells of M. leprae on M-Y 14b agar medium during cultivation was seen. c) Light and electron microscopic observation indicated that there was an increase in the number of non-solid bacterial cells and ghost cells with time of cultivation. d) It was found by mouse foot pad inoculation that four of six samples of M. leprae used as inocula were definitely viable. e) By means of mouse foot pad inoculation, it was shown that viability of M. leprae inoculated onto M-Y 14b agar medium was lost within approximately seven weeks of cultivation. From these results, can be definitely concluded that there is no evidence indicating that multiplication of M. leprae took place on M-Y 14b agar medium.

Studies towards the standardization of lepromin. Progress and prospects.

Because of the wide range of concentrations of Mycobacterium leprae in existing lepromins the authors studied methods of producing a standardizable lepromin containing 160 million bacilli/ml. The effects of using different dilutions of lepromin on the incidence of false-positive reactions were also studied.Progress reported includes a convenient method for preparing large batches of non-sedimenting lepromin, which is directly suitable for microscopic counting of Myco. leprae cells; and a validation of current methods for microscopic enumeration of Myco. leprae. Skin tests with diluted lepromins have demonstrated that dilutions up to 1:16 increase progressively the ability to distinguish between lepromatous and tuberculoid leprosy. This work has provided further evidence that 20 million bacilli/ml (a 1:8 dilution of the initial lepromin) should produce adequate Mitsuda reactions in general populations, provided that 3-mm reactions are taken as the criterion for 1+ positivity. The net effect of these findings is equivalent to expanding the world supply of lepromin by 8 times.Recommendations for further research are proposed.