Laboratory Maintenance of Bordetella pertussis

Robin R. Hulbert1, Peggy A. Cotter2

1 California Polytechnic State University, San Luis Obispo, California, 2 University of California Santa Barbara, Santa Barbara, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4B.1
DOI:  10.1002/9780471729259.mc04b01s15
Online Posting Date:  November, 2009
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Abstract

The causative agent of the respiratory disease whooping cough, Bordetella pertussis, is a nutritionally fastidious microorganism but can be grown with relative ease in research laboratories. Stainer‐Scholte synthetic broth medium and Bordet‐Gengou blood agar both support growth of B. pertussis and are commonly used. B. pertussis prefers aerobic conditions and a temperature range of 35° to 37°C. Appropriate laboratory safety protocols are required to prevent the generation of aerosols, which could potentially spread this highly infectious agent. Curr. Protoc. Microbiol. 15:4B.1.1‐4B.1.9. © 2009 by John Wiley & Sons, Inc.

Keywords: Bordetella pertussis; Stainer‐Scholte; Bordet‐Gengou

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of B. pertussis on Solid Agar Streak Plates
  • Basic Protocol 2: Establishing Broth Cultures of B. pertussis
  • Basic Protocol 3: Preservation of B. pertussis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of B. pertussis on Solid Agar Streak Plates

  Materials
  • B. pertussis frozen stock (see protocol 3)
  • BG blood agar plates (see recipe for Bordet‐Gengou solid medium)
  • Sterile wooden applicators
  • Plastic bag
  • 37°C incubator

Basic Protocol 2: Establishing Broth Cultures of B. pertussis

  Materials
  • Stainer‐Scholte broth (see recipe)
  • 100× Stainer‐Scholte supplement stocks (see recipe)
  • 100× heptakis stock (see recipe)
  • Streaked plate of B. pertussis strain (see protocol 1)
  • Sterile wooden applicators
  • Sterile 9‐ml culture tubes
  • 37°C incubator with roller or shaker

Basic Protocol 3: Preservation of B. pertussis

  Materials
  • Streak plate of B. pertussis strain (see protocol 1)
  • Stainer‐Scholte broth (see recipe for Stainer‐Scholte liquid medium) containing 15% (v/v) glycerol (see recipe)
  • Sterile Dacron swabs
  • 2‐ml cryotubes
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Figures

Videos

Literature Cited

   Ahmad, F. and Calder, M.A. 1984. Isolation of Bordetella pertussis: Benefits of using both Bordet‐Gengou and charcoal agar media. J. Clin. Pathol. 37:1071‐1077.
   Caro, V., Bouchez, V., and Guiso, N. 2008. Is the sequenced Bordetella pertussis strain Tohama I representative of the species? J. Clin. Microbiol. 46:2125‐2128.
   Cotter, P.A. and Jones, A.M. 2003. Phosphorelay control of virulence gene expression in Bordetella. Trends Microbiol. 11:367‐373.
   Imaizumi, A., Suzuki, Y., Ono, S., Sato, H., and Sato, Y. 1983. Heptakis (2,6‐O‐dimethyl)β‐cyclodextrin: A novel growth stimulant for Bordetella pertussis phase I. J. Clin. Microbiol. 17:781‐786.
   Martinez de Tejada, G., Miller, J.F., and Cotter, P.A. 1996. Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica. Mol. Microbiol. 22:895‐908.
   Stainer, D.W. and Scholte, M.J. 1970. A simple chemically defined medium for the production of phase I Bordetella pertussis. J. Gen. Microbiol. 63:211‐220.
   Stibitz, S., Aaronson, W., Monack, D., and Falkow, S. 1989. Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two‐component system. Nature 338:266‐269.
Key References
   Mattoo, S. and Cherry, J.D. 2005. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 18:326‐382.
  A thorough review of Bordetella that integrates information from previous reviews with recent molecular and clinical data.
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