Laboratory Maintenance of Clostridium difficile

Joseph A. Sorg1, Sean S. Dineen1

1 Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9A.1
DOI:  10.1002/9780471729259.mc09a01s12
Online Posting Date:  February, 2009
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Abstract

Clostridium difficile is a Gram‐positive, spore‐forming, anaerobe and is the leading cause of antibiotic‐associated diarrhea, pseudomembranous colitis, and toxic megacolon. Essential to the lifestyle of C. difficile is the ability to form a metabolically dormant spore, germinate, and grow out upon appropriate signals and elicit disease with the secretion of two toxins. To aid in the study of this organism, this unit describes the growth and maintenance of C. difficile. Included are methods to isolate C. difficile from environmental samples, grow in laboratory medium, and produce and purify spores. Curr. Protoc. Microbiol. 12:9A.1.1‐9A.1.10. © 2009 by John Wiley & Sons, Inc.

Keywords: Clostridium difficile; spore; anaerobe; firmicute; germination; anaerobic chamber; environmental sampling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of C. difficile on Solid Medium
  • Basic Protocol 2: Growth of C. difficile in Liquid Medium
  • Basic Protocol 3: Isolation of C. difficile from the Environment or Stool
  • Basic Protocol 4: Sporulation of C. difficile and Recovery of Spores on Agar Medium
  • Basic Protocol 5: Maintenance of Clostridium difficile Spore Stocks
  • Basic Protocol 6: Maintenance of Clostridium difficile Freezer Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Growth of C. difficile on Solid Medium

  Materials
  • BHIS agar plate (for actively growing cultures; see recipe) or BHIS agar plate containing 0.1% taurocholate (for spore stocks or frozen stocks)
  • Viable Clostridium difficile colonies grown on a plate, from chopped meat medium (see protocol 5), or from frozen stock (see protocol 6)
  • Anaerobic chamber
  • Sterile, disposable inoculating loops or cotton swabs

Basic Protocol 2: Growth of C. difficile in Liquid Medium

  Materials
  • BHIS liquid medium (see recipe)
  • Plate containing viable C. difficile
  • Culture tubes
  • Anaerobic chamber
  • Sterile, disposable inoculating loops

Basic Protocol 3: Isolation of C. difficile from the Environment or Stool

  Materials
  • Taurocholate‐cycloserine‐cefoxitin‐fructose agar (TCCFA) petri plate (see recipe)
  • Environmental or stool sample
  • BHIS agar petri plate (see recipe)
  • Anaerobic chamber
  • Sterile swab
  • Sterile inoculating loops
  • PRO discs (Carr‐Scarborough Microbiologicals)

Basic Protocol 4: Sporulation of C. difficile and Recovery of Spores on Agar Medium

  Materials
  • Overnight liquid culture of C. difficile
  • BHIS liquid medium (see recipe)
  • 6‐well tissue culture dish containing 5 ml BHIS agar/well (see recipe for BHIS agar)
  • 20% and 50% (w/v) HistoDenz (Sigma‐Aldrich)
  • BHIS agar medium containing 0.1% (w/v) taurocholate (see recipe)
  • Spectrophotometer and cuvettes
  • Anaerobic chamber
  • Sterile, disposable inoculating loops
  • 1.5‐ml microcentrifuge tubes
  • Microscope slides
  • Coverslips
  • Phase‐contrast microscope
  • Centrifuge tubes

Basic Protocol 5: Maintenance of Clostridium difficile Spore Stocks

  Materials
  • Plate containing viable Clostridium difficile
  • Chopped meat medium tubes (Fisher Scientific)
  • BHIS agar medium containing 0.1% (w/v) taurocholate (see recipe)
  • Anaerobic chamber
  • Sterile inoculating loops

Basic Protocol 6: Maintenance of Clostridium difficile Freezer Stocks

  Materials
  • 50% glycerol
  • Liquid culture containing viable Clostridium difficile
  • 1‐ml cryogenic vials (Eppendorf) or screw‐cap tubes
  • Anaerobic chamber
  • −70°C freezer
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Literature Cited

Literature Cited
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