Application of a viability-staining method for Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad

Abstract

Mycobacterium lepraecannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied toM. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability ofM. lepraewith the course of athymic mouse foot pad infection to optimize the provision of viableM. lepraeas a research reagent. The data show that in untreated suspensions ofM. lepraethere is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation ofM. lepraewith ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity ofM. lepraedeclines more than the extent of membrane damage at 37 °C within 72 h, but that they are not significantly affected at 33 °C. Irradiation at 10⁴Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by whichM. lepraeis killed. This staining method helped us predict reliably that the smallerM. leprae-infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.