Enumeration of purified suspensions of Mycobacterium leprae.

Previously described methods of counting noncultivable bacteria have a number of drawbacks including unpredictable variation due to differential staining, low reproducibility between replicate sample smears, and inexact estimations of the bacillary population due to the non-normal distribution of bacilli across the counting field. A simple method is described which minimizes the disadvantages of previous methods and allows the application of a stratified sample technique which improves the accuracy of the count by partially compensating for the uneven distribution of bacilli across the counting area. This method of analysis also allows an estimation of the optimum sampling rate in each strata of the counting area and the determination of a sample variance. Different fixation and staining techniques have been compared, and the methenamine silver method which is more likely to visualize either the entire population or a constant proportion of the bacilli is recommended.