title: Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.
abstract: Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
authors.affiliation: Department of Pathology, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Ana Paula Fávaro
authors.initials: AP
authors.lastname: Trombone
authors.affiliation: Laboratory Animal House, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Sílvia Cristina Barbosa
authors.initials: SC
authors.lastname: Pedrini
authors.affiliation: Department of Microbiology, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Suzana Madeira
authors.initials: SM
authors.lastname: Diório
authors.affiliation: Department of Pathology, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Andréa de Faria Fernandes
authors.initials: Ade F
authors.lastname: Belone
authors.affiliation: Department of Pathology, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Luciana Raquel Vicenzi
authors.initials: LR
authors.lastname: Fachin
authors.affiliation: Department of Pharmacology, Instituto Lauro de Souza Lima (ILSL).
authors.forename: Dejair Caitano
authors.initials: DC
authors.lastname: do Nascimento
authors.affiliation: Laboratory Animal House, Instituto Lauro de Souza Lima (ILSL); prosa@ilsl.br.
authors.forename: Patricia Sammarco
authors.initials: PS
authors.lastname: Rosa
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D004195:Disease Models, Animal; D005615:Freezing; D007918:Leprosy / Q000382:microbiology; D051379:Mice; D008819:Mice, Nude; D009166:Mycobacterium leprae / Q000166:cytology* / Q000254:growth & development / Q000302:isolation & purification; D013535:Suspensions
chemical_list: D013535:Suspensions