Isolation and purification of Mycobacterium tuberculosis from H37Rv infected guinea pig lungs

Abstract

Evidence suggests that Mycobacterium tuberculosis grown invivo may have a different phenotypic structure from its invitro counterpart. In order to study the differences between invivo and invitro grown bacilli, it is important to establish a reliable method for isolating and purifying M.tuberculosis from infected tissue. In this study, we developed an optimal method to isolate bacilli from the lungs of infected guinea pigs, which was also shown to be applicable to the interferon-γ gene knockout mouse model. Briefly, 1) the infected lungs were thoroughly homogenized; 2) a four step enzymatic digestion was utilized to reduce the bulk of the host tissue using collagenase, DNase I and pronase E; 3) residual contamination by the host tissue debris was successfully reduced using percoll density gradient centrifugation. These steps resulted in a protocol such that relatively clean, viable bacilli can be isolated from the digested host tissue homogenate in about 50% yield. These bacilli can further be used for analytical studies of the more stable cellular components such as lipid, peptidoglycan and mycolic acid.