Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.
Total time
~4–6 weeks per propagation cycle (including mouse footpad development and bacterial harvest)
Model organism
Mouse (athymic nude)
Steps
1
Prepare Mycobacterium leprae bacillary suspension
Extract and suspend M. leprae from infected tissue into a standardized bacillary suspension for downstream applications. This suspension serves as the stock material for all subsequent propagation and preservation steps.
▶ 00:20
2
Perform cold Ziehl-Neelsen staining procedure
Apply cold Ziehl-Neelsen staining to visualize and differentiate M. leprae cells in the suspension. This staining method allows microscopic examination of bacterial morphology and distribution.
▶ 04:24
3
Determine bacterial viability status
Assess the viability of M. leprae in the suspension using morphological and staining criteria. This evaluation ensures the stock maintains adequate living bacteria for experimental use.
▶ 06:06
4
Freeze and thaw M. leprae suspension stock
Cryopreserve M. leprae suspension using controlled freezing protocols and document thawing procedures to maintain bacterial viability during long-term storage.
▶ 07:48
5
Inoculate athymic nude mice via footpad
Inject M. leprae suspension into mouse footpads to establish systemic infection and propagate bacilli for subsequent harvest and stock maintenance.
▶ 09:08
6
Analyze propagation and viability results
Review outcomes from mouse inoculation, bacterial recovery, and stock quality to validate protocol effectiveness and confirm successful M. leprae maintenance.
▶ 09:35