THE POSSIBILITY OF THE GROWTH OF MYCOBACTERIUM LEPRAE IN VITRO

Abstract

Inoculating small amounts of the emulsions of leprous tissues into certain nutrient media, some workers in our country recently said that the leprosy bacilli, though in small amount, seemed to be able to develop in vitro. This may perhaps be the first step towards the solution of the problem, even though these workers did not, however, explain why the succesive cultures were not successful in any way.
Is it possible to verify the growth of leprosy bacilli in vitro, without using the method of transferring succesively in ordinary way? It may be verified, if one recognize 1) more or less considerable increase in number of bacilli, though within microscopic range, in comparison with the control tubes not incubated, 2) characteristic arrangement of the multiplied bacilli; formation of the cords; construction of the colonies, though within microscopic width, and 3) process of development; growth phase.
After being examined, from this point of view, many cultures in fluid and solid media, it became clear that the leprosy bacilli were able to develop in vitro in somewhat characteristic manner. The observation was made with-the cultures in Kirchner's fluid and solid media. The smears made, without stirring, with a drop of the sediments were examined weekly. One recognized that the leprosy bacilli developed with almost similar rapidity as the human or bovine tubercle bacillus showing their maximum growth after 3 to 5 weeks of incubation. They constructed fine acid-fast net-works visible with weak magnification covering many debris found in the smears (Fig. 1). And also in the smears made from the cultures on the slants, the bacilli often appeared forming peculiar round colonies though within microscopic width (Fig. 2).
It was above all striking that the bacilli could multiply only in small amounts; i. e. withh the limited growth ratio.
If one reminds the growth curves of other mycobacteria, it may be possible to suppose by analogy, that the number of viable leprosy bacilli in the cultures also begins to decrease as soon as they show the maximum development after 3 to 5 weeks of incubation. Thus if one try to transfer the cultures, it may be preferable to do at these periods of time. Actually the transfers were, however, not successful, although careful attention was taken to give more or less dense sediments of the primary cultures into new culture media. This might be due to the fact that the leprosy bacilli grow only with the minimum growth ratio as already written.
Some factors being supposed to influence the development of the bacilli were also able to be examined with success by means of the method described above. One inoculated a drop of more or less dense emulsion of tissues into Kirchner's fluid medium and examined, with weak magnification, the smears made with a drop of sediments of the cultures. For example, the bacilli developed better at 37°C than at 30°C, and also better in the medium containing glycerol of 2 to 4% than without it. As to the serum added to the medium, those of rabbit and rat promoted the development better than those of goats, men and guinea pigs.