Enumeration of Mycobacterium leprae for the standardization of lepromin

1- Optimal conditions for declumping and dispersing mycobacteria in turbid suspensions and tissue homogenates for microscopic purposes have been defined. 2- Shaking 10 per cent leproma suspensions, or ordinary lepromins with tissue in 3-5 per cent concentrations, for 3 minutes with 10 per cent chloroform by volume releases M. leprae from clumps and globi. Further dilution to 1 per cent tissue, with shaking in the presence of 1 per cent serum, permits the preparation of stained films containing suitable numbers of uniformly distributed bacilli. Methods for microscopic enumeration and for comparing the concentrations of bacillui have been given. 3- It has again been shown that the numbers of M. leprae in lepromas may vary over a 10-fold range. 4- Even with simple methods for counting and adjusting the bacterial content of lepromin, the pooling of lepromas must be continued in order to obtain reasonable uniformity in the quality of the bacilli and the concentration of autoclaved human tissue components.

Quantitative aspects of sampling leprosy skin lesions by the scraped incision method

1- The problem of obtaining more useful and convenient estimates of the numbers of M. leprae in material from scraped skin incisions has been investigated by demonstrating: (a) that the spreading of small samples on glas slides causes very rapid dilution; (b) that the areas filmed should be proportional to the volume of samples; (c) that variability in present practical results may often be due to attempts to spread the variable samples on standard areas; and (d) that this practice results in over-dilution and in irregular distribution of components within the films. 2- A study of the art of preparing uniform films on slide areas which are proportional to unknown volumes of sample leads to the suggestion that each sample be spread to the maximal limit which permits a natural redistribution of the cellular components before drying begins. Enumerations reveal that this modification of habit results in a predictable distribution of bacilli. 3- Increased sensitivity, when desired, may be achieved by increasing the thickness of film to 4 times that recommended for routine purposes. Such films may be on the order of 14 times more sensitive than average films, and 56 times more sensitive than the thinnest clinical films which were available for analysis in this study. A second means of increasing sensitivity is to examine all tissue fragments and cell clusters in each film.

Enhancement of resistance to murine leprossy by BCG plus specific antigen

The importance of specific immune response, and a means of enhancing specific protection against leprotic infections, have been investigated. Rats were challenged with M. leprae murium after having received the following antigens: attenuated tubercle bacilli of the BCG and R1Rv strains, heat-killed suspensions of M. leprae murium, and the attenuated tubercle bacilli accompanied by smaller amounts of M. leprae murium antigen. The results indicate that specific antigen plays a distinctive role in protection and that superior protection is afforded by small amounts of specific antigen combined with the tubercle bacilli as stimulators of immune response. Significant protection was achieved by single administrations of antigen, on a basis adopted to practical application. The liabilities which may attend large and repeated doses of mycobacterial antigens have been emphasized as peculiar to those individuals most in need of protection. The theoretical basis of enhancing response to modest amounts od specific antigen has been indicated.

Relationship between the metabolic capacity and the infectiousness of M. leprae murium; refrigeration studies

1- The unfavorable affect of serum from rats and other animals on the infectiousness of M. leprae murium was first observed in very dilute solutions. The adverse effect is here shown to occur within the time during which it is known that inoculated mycobacteria lie in serous exudate prior to phagocytosis. Because of the progressive, seriuos damage to the endogenous metabolism of the bacilli, it is concluded that this exposure to natural extracellular inhibitors lowers significantly both their viability and their infectiousness when inoculated into animals. 2- Similar inhibitors...

A study of the bacilli in tissue cultures of lepromata in serum media

1- Tissue cultures from four lepromata were grown and maintained in a viable condition in serum media for periods of from 14 to 34 days. 2- The infected macrophages died early in the history of the cultures and deposited their bacilli within the necrotic mass of the original explants. 3- The only cells that persisted throughout the life of the cultures were fibroblasts, which ordinarily contained no bacilli or only a few. 4- Quantitative rating of the explants with respect to their bacterial content did not reveal an increase in the total mass of bacilli. 5- The development of turbidity inthe plasma of older cultures was found not to be attributable to multiplication of bacilli in this site as claimed by earlier workers.