An Updated Method for the Purification of Mycobacterium Leprae and other Mycobacteria From Infected Tissues

Abstract A “two step” method, consisting of centrifugation in self forming Percol R gradient followed by either unit gravity (1 g) sedimentation (UgS) or 40 g centrifugation (40 gC) on a 5-15% sucrose gradient in “Tulp Chamber” has been developed to purify Mycobacterium leprae (M1) from infected armadillo (A) and human leproma (LL) skin biopsy tissues. The method features large yield and can also be used to purify other in vitro culturable mycobacteria such as Mycobacterium tuberculosis (Mtb) and Mycobacterium lepraemurium (M1m) from infected animal tissues. Viability and the in situ conservation of antigenecity appeared to remain unaffected by the present purification method. Criteria of purity of the isolated bacteria were evaluated by Polyacrylamide gel electrophoresis (PAGE), light microscopy (LM), transmission electron microscopy (TEM), radioimmunoabsorption spot test (RIST) and pyrolysis mass spectrometry (Py-MS).