Laboratory Maintenance of Mycobacterium abscessus

Mélanie A. M. Cortes1, Rachid Nessar1, Anil Kumar Singh1

1 INSERM U1002, Unité de Pathogénie des Infections Systémiques, Paris, France
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10D.1
DOI:  10.1002/9780471729259.mc10d01s18
Online Posting Date:  August, 2010
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Mycobacterium abscessus, is an emerging, rapidly growing pathogen, with the ability to cause chronic lung disease. This unit covers background information and laboratory maintenance procedures for this bacterium, including growth in liquid and on solid medium. It also contains recommendations concerning long‐term strain storage. M. abscessus is a Biosafety Level 2 organism, and the required safety measures are also discussed. Curr. Protoc. Microbiol. 18:10D.1.1‐10D.1.12. © 2010 by John Wiley & Sons, Inc.

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

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Table of Contents

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

  • Middlebrook 7H9 liquid medium (see recipe)
  • M. abscessus (ATCC# 19977T) growing on a 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, sterile
  • 37°C incubator containing platform shaker

Alternate Protocol 1: Growth of M. abscessus 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. abscessus ( protocol 1)

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

  • Frozen stock of M. abscessus ( protocol 4) or liquid preculture ( protocol 1)
  • 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
  • 37°C incubator

Basic Protocol 3: Storage of M. abscessus as Frozen Stocks

  • M. abscessus culture ( protocol 1)
  • Storage medium (see recipe)
  • Microcentrifuge tubes, sterile
  • Tabletop centrifuge
  • 2‐ml cryotubes, sterile
  • −70°C freezer
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Literature Cited

Literature Cited
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