Laboratory Maintenance of Mycobacterium tuberculosis

Michelle H. Larsen1, Karolin Biermann1, William R. Jacobs,1

1 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.1
DOI:  10.1002/9780471729259.mc10a01s6
Online Posting Date:  August, 2007
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Abstract

This unit includes protocols for the laboratory maintenance of Mycobacterium tuberculosis, including growth on liquid and solid media as well as recommendations for long-term strain storage. Considerations for working with M. tuberculosis at Biosafety Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.1.1-10A.1.8. © 2007 by John Wiley & Sons, Inc.

Keywords: tuberculosis; medium; Middlebrook; 7H9; 7H10; 7H11; Sauton; strain storage

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of M. tuberculosis in Middlebrook Liquid Medium
  • Alternate Protocol 1: Growth of M. tuberculosis in Sauton Liquid Medium
  • Basic Protocol 2: Growth of M. tuberculosis on Solid Medium
  • Basic Protocol 3: Storage of M. tuberculosis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of M. tuberculosis in Middlebrook Liquid Medium

 Materials
  • Middlebrook 7H9 liquid medium (see recipe)
  • M. tuberculosis
  • 30-ml square medium bottles (Nalgene, cat. no. 2019-0030), sterile
  • Disposable plastic inoculation loops, sterile
  • Incubator containing platform shaker or roller-bottle apparatus
  • 490-cm2 roller bottles (Corning, cat. no. 430195)
  • Additional reagents and equipment for bacterial culture techniques including inoculation of liquid medium (appendix 4A)

Alternate Protocol 1: Growth of M. tuberculosis in Sauton Liquid Medium

 Materials
  • Sauton medium (see recipe)
  • M. tuberculosis
  • 30-ml square medium bottles (Nalgene, cat. no. 2019-0030), sterile
  • Disposable plastic inoculation loops, sterile
  • Incubator containing platform shaker or roller-bottle apparatus
  • 490-cm2 roller bottles (Corning, cat. no. 430195)
  • Additional reagents and equipment for bacterial culture techniques including inoculation of liquid medium (appendix 4A)

Basic Protocol 2: Growth of M. tuberculosis on Solid Medium

 Materials
  • M. tuberculosis
  • Middlebrook 7H10 or 7H11 agar plates (see recipes and Strategic Planning)
  • Vesphene IIse disinfectant (Fisher)
  • Sterile plastic inoculating loops
  • Sterile plastic plate spreaders
  • Heavy-duty aluminum foil or sterile metal-plate canisters
  • Additional reagents and equipment for bacterial culture techniques, including spreading and streaking plates (appendix 4A)

Basic Protocol 3: Storage of M. tuberculosis

 Materials
  • M. tuberculosis culture (Basic Protocol 1 or Alternate Protocol 1)
  • M. tuberculosis strain storage medium (see recipe)
  • Sterile 2-ml cryotubes
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Figures

  •  FigureFigure 10A.1.1 Colony morphology. Colonies of Mycobacterium tuberculosis appear different when grown on solid medium (A) with or (B) without Tween-80. In the presence of a detergent, like Tween-80, M. tuberculosis colonies appear smooth, round, and have a shiny surface as depicted in panel A. If no detergent is present in the medium, the colonies will appear more rough, wrinkled, and matte, as depicted in panel B.
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Literature Cited

Literature Cited
    Cohn, M.L., Oda, U., Kovitz, C., and Middlebrook, G. 1954. Studies on isoniazid and tubercle bacilli. I. The isolation of isoniazid-resistant mutants in vitro. Am. Rev. Tuberc. 70:465-475.
    Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall, A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a biosafety level 3 laboratory. J. Clin. Microbiol. 39:769–771.
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