Cultivation of Mycobacterium Leprae. III. ∗

Abstract

The experiments here recorded were made with the object of confirming in a distant land and with patients of another race our earlier studies 1 on the isolation and cultivation of the leprosy bacillus. A description was given of a slow growing non-chromogenic acid-fast organism isolated from human leprous tissue by incubating the inoculated media in an atmosphere of 40% O₂ and 10% CO₂. There was unequivocal evidence of the proliferation of the germs but it was also true that the quantity was limited. The organisms were removed from the positive tubes and transferred to freshly prepared sterile media; after an incubation period of about 6 weeks the tiny colonies that had developed were subcultured as before. A total of 26 serial subcultures were made but there was no apparent improvement on the part of the cells to assume a saprophytic existence. The culture in the 26th generation showed the same characteristics as at the beginning of the series.

A subsequent communication 2 reported efforts to provide a more suitable substratum. Several of the complex formulae ordinarily employed for the isolation of such species as Johne's bacillus and the tubercle bacillus were prepared and inoculated; in addition amino acids and fresh vegetable tissues were incorporated in several of the usual laboratory media. The results were uniformly disappointing. The overwhelming numbers of organisms in the lesions of the disease and the impoverished growth in test tubes led to the conclusion that some essential food factor was lacking in the nutritive media.

There have been several criticisms directed at the studies carried out in Puerto Rico; the most important of which has been the suggestion, that in making the serial subcultures, tissue debris containing stainable organisms was mechanically transferred from tube to tube, leading to the erroneous conclusion that proliferation had occurred when actually no in vitro multiplication had taken place. The appearance of definite colonies, although of limited size, gave assurance that such was not the case, however in planning the protocols for the studies in the Philippines adequate controls were included to obviate this as well as other objections.