Laboratory Maintenance of Mycobacterium smegmatis

Anil Kumar Singh1, Jean‐Marc Reyrat1

1 Inserm U570, Unité de Pathogénie des Infections Systémiques, Paris, France
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10C.1
DOI:  10.1002/9780471729259.mc10c01s14
Online Posting Date:  August, 2009
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This unit gives background information on Mycobacterium smegmatis, a mycobacterial model system, and covers all the laboratory maintenance for this species including growth in liquid and on solid medium. It also contains recommendations concerning long‐term strain storage. Although M. smegmatis is a Biosafety Level 1 organism, some rare infections in humans have been reported, and, thus all of the required safety measures are discussed here. Curr. Protoc. Microbiol. 14:10C.1.1‐10C.1.12. © 2009 by John Wiley & Sons, Inc.

Keywords: medium; Middlebrook; 7H9; Sauton; LB Miller; strain storage

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of M. smegmatis in Middlebrook Liquid Medium
  • Basic Protocol 2: Growth of M. smegmatis in Sauton Liquid Medium
  • Basic Protocol 3: Growth of M. smegmatis on Solid Medium
  • Basic Protocol 4: Storage of M. smegmatis as Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Growth of M. smegmatis in Middlebrook Liquid Medium

  • Middlebrook 7H9 liquid medium (see recipe)
  • M. smegmatis growing on plate ( protocol 3)
  • 50‐ml (for small‐volume culture) or 500‐ml (for large‐volume culture) Erlenmeyer or conical flask (Borosil, cat no. 4980;
  • Disposable plastic inoculation loops, sterile
  • Porous plugs for flasks
  • Incubator containing platform shaker
  • Additional reagents and equipment for bacterial culture techniques including inoculation of liquid medium ( appendix 4A)

Basic Protocol 2: Growth of M. smegmatis in Sauton Liquid Medium

  • Sauton medium (see recipe)
  • 175‐cm2 Corning Cell Culture Flasks (Sigma, cat no. 431080)
  • Additional reagents and equipment for growth of M. smegmatis ( protocol 1)

Basic Protocol 3: Growth of M. smegmatis on Solid Medium

  • M. smegmatis (mc2155; ATCC no. 70084)
  • Middlebrook 7H10 or 7H11 agar plates (see recipe)
  • Sterile plastic inoculating loops
  • Sterile plastic plate spreaders
  • Heavy‐duty aluminum foil or sterile metal plate canisters
  • Additional reagents and equipments for bacterial culture techniques including spreading and streaking plates ( appendix 4A)

Basic Protocol 4: Storage of M. smegmatis as Frozen Stocks

  • M. smegmatis culture ( protocol 1)
  • Storage medium (see recipe)
  • Sterile 2‐ml cryotubes
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Literature Cited

Literature Cited
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