Morphological and Tinctorial Behavior of B. Leprae During its Adaptation to an In Vitro Habitat

Abstract

Summary

The uncontaminated subcutaneous nodules from 4 cases of human leprosy were cultured upon a variety of media and under various conditions, in order to afford a systematic study of the morphology and tinctorial properties of B. leprae during its period of adaptation to an artificial environment. All changes occurring for the Hansen bacillus in vitro were noted and compared with the morphology and staining properties of the organism in the original human leprous lesion.

During the entire period of observation (6 months) only minor alterations in size and shape of B. leprae were noted, and these were no more than what occurs for the tubercle bacillus under similar conditions of cultivation. Where multiplication occurred in the transplanted leprous tissue the acid-fast rods became longer, thicker, less curved and more distinctly diphtheroidal in appearance, because of the bipolar massing of the chromatin. So striking was this arrangement of the chromatin granules that with Gram's and Loeffler's methods of staining, certain of the in vitro growing forms are indistinguishable from the bipolar metachromatic granular types of B. diphtheriae. Later, when the organism is acquiring the power to grow independently of the host tissue it becomes shorter, more plump and of ten without granules. Still later, after macroscopic growth is well established, the bacilli assume the morphology of those in the transplanted leprous tissue. At no time were branching filamentous and interlacing streptothrical forms encountered.

The carbol fuchsin stained microorganism of leprosy, whether in culture or the living host tissue, is less resistant to decolorization by the ordinarily used agents for this purpose, than is the tubercle bacillus. Failure to realize this fact may account for the reports of non-acid-fast diphtheroidal and streptothrical forms of the Hansen organism. B. leprae in the human lesion is decidedly more resistant to decolorization than it is from macroscopic culture. Where the Ziehl-Neelsen method is employed there occur varying degrees of acid-fastness to complete decolorization of the bacilli from culture. In a given field of the stained preparation there will be rods that have only partially, and still others that have not, retained the stain. On the other hand, where the Ziehl-Neelsen method is used and the time-period for decolorization is greatly lessened, the leprosy bacilli will remain deeply stained.

The Hansen organism of human leprosy is an acid-fast, Grampositive bacterium that possesses much the same morphology and staining properties as the tubercle bacillus.

Finally, in the experience of the author,6 the macroscopic culture of B. leprae is obtained only after months of careful cultivation upon split-protein media, preferably that of the excised, transplanted autolyzing leprorna. Once the organism has successfully passed through the transitional period of in vitro adaptation, growth takes place readily upon the ordinary laboratory nutrients.