Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Author(s)

Favaro Trombone, Ana Paula ; Barbosa Pedrini, Silvia Cristina ; Diorio, Suzana Madeira ; Fernandes Belone, Andrea de Faria ; Vicenzi Fachin, Luciana Raquel ; do Nascimento, Dejair Caitano ; Rosa, Patricia Sammarco

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 85, p. 7-pg., 2014-03-01.

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. (AU)

• FAPESP's process: 09/06122-5 - Evaluation of the regulatory T and Th17 cells role in the human and experimental leprosy: correlation with leprosy spectrum, metalloproteinases and their inhibitors expression, and interaction with Th1/Th2 paradigm
• Grantee: Ana Paula Favaro Trombone Garlet
• Support Opportunities: Research Grants - Young Investigators Grants