title: Isolation and purification of Mycobacterium tuberculosis from H37Rv infected guinea pig lungs.
abstract: Evidence suggests that Mycobacterium tuberculosis grown in vivo may have a different phenotypic structure from its in vitro counterpart. In order to study the differences between in vivo and in vitro 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.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Libin
authors.initials: L
authors.lastname: Shi
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Ryan
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Suresh
authors.initials: S
authors.lastname: Bhamidi
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: JoLynn
authors.initials: J
authors.lastname: Troudt
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Amin
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Angelo
authors.initials: A
authors.lastname: Izzo
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Anne J
authors.initials: AJ
authors.lastname: Lenaerts
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: McNeil
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Belisle
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: dean.crick@colostate.edu.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Crick
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: delphi.chatterjee@colostate.edu.
authors.forename: Delphi
authors.initials: D
authors.lastname: Chatterjee
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000378:metabolism; D015169:Colony Count, Microbial; D003850:Deoxyribonuclease I / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D006168:Guinea Pigs; D007371:Interferon-gamma / Q000172:deficiency / Q000235:genetics; D008168:Lung / Q000201:enzymology / Q000382:microbiology*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000302:isolation & purification*; D011402:Pronase / Q000378:metabolism; D014397:Tuberculosis, Pulmonary / Q000235:genetics / Q000382:microbiology*
chemical_list: D007371:Interferon-gamma; D003850:Deoxyribonuclease I; D017364:Collagenases; D011402:Pronase
keywords: Bacterial isolation; Guinea pig infection; Mycobacterium tuberculosis