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

Abstract

Mycobacterium leprae cannot 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 to M. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of M. leprae with the course of athymic mouse foot pad infection to optimize the provision of viable M. leprae as a research reagent. The data show that in untreated suspensions of M. leprae there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of M. leprae with 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 of M. leprae declines more than the extent of membrane damage at 37 8C within 72 h, but that they are not significantly affected at 33 8C. Irradiation at 10 4 Gy 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 which M. leprae is killed. This staining method helped us predict reliably that the smaller M. 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.

Viability states of bacteria—Specific mechanisms of selected probes

“…Nevertheless, E. coli was found to not present elevated PI values in the same experiments suggesting that this false positive staining may not only be substrate but also species dependent (65). Nevertheless, PI is a good probe for detecting dead cell numbers in bacterial populations, which is proven by numerous publications (86)(87)(88)(89). However, the user should be aware that the integrity of the bacterial surface can be also disturbed by cell growth and the connected rearrangement of membranes and cell walls.…”

Mycobacterium leprae Inhibits Dendritic Cell Activation and Maturation

“…Bacilli were washed in Middlebrook 7H12 medium and enumerated by direct count according to Shepard's method (11). The relative viability of M. leprae in the suspension was evaluated using the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes) as recently adapted for M. leprae (10). Pure preparations of bacilli free of mouse footpad tissue were obtained by treating the footpad suspension with 0.1 M NaOH for 5 min followed by neutralization with 0.1 M HCl and three washes with PBS.…”

“…The mycobacteria had been maintained in programmed serial passage in the footpads of athymic nu/nu mice infected with 5 ϫ 10 7 freshly harvested M. leprae. Briefly, bacilli were harvested from the footpads 3-4 mo after infection (at mid-log growth) as described previously (10). Bacilli were washed in Middlebrook 7H12 medium and enumerated by direct count according to Shepard's method (11).…”

Optimized Protocols for <em>Mycobacterium leprae</em> Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

“…Our study demonstrates that the protocol of inoculum preparation by filtration and trypsin digestion allows the inocula to be obtained with very little cellular debris and with high viability of bacilli (score 0+). Sodium hydroxide has been used to disaggregate the tissue for purification of bacilli 6 . Studies performed in our laboratory using sodium hydroxide for purification of M. leprae resulted in formation of clumps of bacilli, hampering the homogenization of the suspension for viability determination and animal inoculation (data not shown).…”

“…Most experimental procedures that use M. leprae are set up with bacilli purified from skin lesions of leprosy patients or from experimental animal models, such as armadillos and several strains of mice 5,6 . For many years, researchers have depended on purification of M. leprae from skin lesions of multibacillary leprosy patients for use in experimental procedures.…”