An Improved Method for In Vitro Cultivation of B. Leprae. ∗

Abstract

Summary

The trypsinization of the leprous tissue bits, before attempting the cultivation of the contained B. leprae, affords at once the split-protein products which we consider the necessary nutrient factor. The trypsin apparently exerts no harmful effect upon the Hansen bacillus as evidenced by its unmistakable growth in 4 to 6 weeks. This initial in vitro multiplication occurs in the trypsinized leprous tissue without the aid of other nutrients and under ordinary atmospheric conditions at 37°C. Growth continues in subculture on ordinary agar medium as long as the trypsinized leprous material is transferred. The crucial point in the initial cultivation of B. leprae away from the influence of the host tissue is experienced at this juncture. After the complete utilization of the nutrient material of the trypsinized host tissue, the acid-fast bacillus of Hansen will only multiply in a medium that is enriched with sufficient quantities of protein cleavage products.

Many attempts have been made by various investigators to culture Hansen's bacillus of human leprosy. Undoubtedly, in a number of these attempts the investigators have mistaken the increase in number of acid-fast rods, which occurs in removed autolysing pieces of leprous tissue independent of any specially supplied cultural conditions, for multiplication in vitro upon artificially prepared nutrient medium. The failure to appreciate the facts that B. leprae continues to multiply and that growth ceases when the leprous tissue autolysate is no longer available, explain the increasing difficulty to obtain macroscopic growth of the specific microorganism in transplants through successive generations, and accounts for the absence of growth in subculture in or upon a medium that does not contain the split protein products (amino-acids).

One of us (Duval 1 ) reported that B. leprae seemed unable to utilize whole protein in vivo. This was substantiated by repeated observations that the Hansen bacillus would grow in transplanted leprous tissue after autolysis had commenced. The initial culture away from the influence of the host-tissue was possible if the artificial culture medium contained certain split-products of protein. In our recent studies upon the cultivation of B. leprae, we have modified Duval's 2 earlier method. An improved procedure as described below is recommended.

The uncontaminated subcutaneous leprous nodule, rich in acid-fast bacilli, is the material of choice. Contaminated tissue is unsatisfactory, because one is likely to injure or destroy the viability of the Hansen bacillus when killing the contaminants. The use of sodium hydroxide, or the like, therefore, is not recommended as a germicide. The skin over the selected leprous nodule is cleansed with soap and water and then rinsed with alcohol. Mercurochrome, iodine or other antiseptics should not be employed. The overlying skin is next incised and the exposed nodule is dissected free and removed under strict asepsis.