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 reciperecipes and )
  • 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 ( protocol 1 or protocol 2)
  • M. tuberculosis strain storage medium (see recipe)
  • Sterile 2‐ml cryotubes
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Figures

Videos

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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