Chapter 6: Mycobacterium: Isolation, Maintenance, Transformation, and Mutant Selection

Nancy D. Connell

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


This chapter discusses methods for culturing Mycobacterium, isolating genetic mutants, and moving DNA in and out of the cells. Experimental methods for culturing and genetically manipulating M. avium, M. tuberculosis, M. smegmatis, and bacille Calmette Guerin (BCG) is discussed in the chapter. The isolation of mycobacteria from contaminated sources requires decontamination because specimens usually contain mixed bacterial flora and some degree of organic debris such as body fluids, blood cells, and tissue fragments. There are a number of isolation protocols but all require digestion, decontamination, and concentration steps. The mycobacteria must be recovered undamaged while organic debris is liquefied and contaminating micororganisms are killed. The chapter discusses protocol for treatment of sputum samples and is suitable for use in clinical biosafety level (BSL-2) and experimental BSL-3 laboratories for the treatment of clinical samples. The mycobacteriophages have been isolated and developed for the purpose of strain typing. Their use in transfer of genetic material for the construction of strains is discussed.

Original languageEnglish (US)
Pages (from-to)107-125
Number of pages19
JournalMethods in cell biology
Issue numberC
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology


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