title: Effects of purification and fluorescent staining on viability of Mycobacterium leprae.
abstract: Over the years, researchers have carried out experiments with Mycobacterium leprae obtained from either human multibacillary lesions, or infected armadillo tissues, or infected footpad tissues of conventional mice as well as athymic nu/nu mice. In general, these sources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies, but less than satisfactory for studies in cell biology and immunology where contaminating host tissues pose a serious problem. We examined the utility of a procedure for eliminating mouse footpad tissue from M. leprae suspension using sodium hydroxide solution and its subsequent effect on the viability of the organism by determining the rate of palmitic acid oxidation, bacterial membrane integrity, and growth in the mouse footpad. We found that treating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1N NaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely affecting the viability of the organism. This is a simple and rapid method to get suspensions of nu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a research reagent for studying the host-pathogen relationship in leprosy. We also report here a method for labeling M. leprae with the fluorescent dye PKH26, without compromising on the viability of the organism. This method may be useful in intracellular trafficking studies of M. leprae or in other cell biology studies that require tracking of the bacteria using fluorescent tag. We observed the staining to be stable in vitro over considerable lengths of time and did not affect the viability of the bacteria.
authors.affiliation: Laboratory Research Branch, National Hansen's Disease Programs, Louisiana State University, Baton Rouge, 70803 USA.
authors.forename: Ramanuj
authors.initials: R
authors.lastname: Lahiri
authors.forename: Baljit
authors.initials: B
authors.lastname: Randhawa
authors.forename: James L
authors.initials: JL
authors.lastname: Krahenbuhl
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D004195:Disease Models, Animal; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005528:Foot / Q000382:microbiology; D007918:Leprosy / Q000382:microbiology; D017737:Macrophages, Peritoneal / Q000382:microbiology; D051379:Mice; D008819:Mice, Nude; D008855:Microscopy, Electron, Scanning; D009166:Mycobacterium leprae / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D009930:Organic Chemicals / Q000494:pharmacology; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000378:metabolism; D012972:Sodium Hydroxide / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; D019308:Palmitic Acid; D012972:Sodium Hydroxide