Pedigreed stocks of Mycobacterium lepraemurium for cultivation and metabolic studies

Abstract

Experience has shown that a prime requirement for investigations with Mycobacterium lepraemurium is pedigreed stocks of cells which provide constant baselines over a long period of time. Biochemical indicator, ATP, was employed to devise a method for preservation of metabolic pools of M. lepraemurium during storage. ATP assays were made by the luciferin–luciferase bioluminescent method. Several cryoprotective agents were compared at −76 and 4 °C. The essential steps have been found to be (i) for prolonged storage and constant supply of material, to freeze the intracellular bacteria within infected cells containing 28% proteins, i.e., to freeze the infected tissue; and (ii) when a large number of diverse experiments are to be undertaken on a single suspension, to stabilize working stocks of refrigerated cells for 12–16 weeks by using bovine serum albumin, fraction V, and Difco yeast supplement B to compensate for the leaching of intracellular cofactors, metabolites, nucleotides, etc.