Pedigreed stocks of Mycobacterium lepraemurium for cultivation and metabolic studies.
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 degrees 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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001431:Bacteriological Techniques; D003080:Cold Temperature; D004229:Dithiothreitol / Q000494:pharmacology; D005615:Freezing; D009167:Mycobacterium lepraemurium / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology; D011309:Preservation, Biological; D013997:Time Factors
D000255:Adenosine Triphosphate; D010100:Oxygen; D004229:Dithiothreitol