New method for concentration and quantitation of Mycobacterium leprae

A new method of enumerating Mycobacterium leprae has been developed. Suspensions containing the organisms were filtered through a polycarbonate membrane filter (25-mm diameter, 0.4-micronm pore size, 10-micronm thick; Nucleopore) to concentrate the organisms. The membrane was then mounted on a glass slide and stained with a standard acid-fast stain. Finally, the membrane was treated with a small amount of chloroform to fix it to the slide and make it transparent. This method enabled us to detect M. leprae in quantities as small as 4.98 X 10(2) regardless of the total volume of the original material. Comparison with a standard method for enumerating M. leprae showed that both methods gave similar results when the organisms counted by the standard method were present in sufficient quantity for reproducibility. Because the least number of organisms that can be detected with the standard method is 10(4) ml and because the organisms detected with the new method could be concentrated on the polycarbonate filter from a large amount of infected fluid, a substantial number of suspensions were shown by the new method, but not by the standard method, to contain M. leprae.