Laboratory Growth and Maintenance of Haemophilus influenzae

Jason W. Johnston1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6D.1
DOI:  10.1002/9780471729259.mc06d01s18
Online Posting Date:  August, 2010
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Abstract

Haemophilus influenzae is a non‐spore forming, non‐motile, pleomorphic Gram‐negative coccobacilli. H. influenzae is a strict human pathogen that can cause a variety of systemic and localized infections, depending on the strain. The three Basic Protocols in this unit will describe how to culture and prepare H. influenzae for the use in biological studies of the organism. Curr. Protoc. Microbiol. 18:6D.1.1‐6D.1.5. © 2010 by John Wiley & Sons, Inc.

Keywords: Haemophilus influenzae; brain heart infusion; sBHI

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of H. influenzae on Solid Medium
  • Basic Protocol 2: Growth of H. influenzae in Liquid Medium
  • Basic Protocol 3: Preparation of H. influenzae Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Growth of H. influenzae on Solid Medium

  Materials
  • H. influenzae freezer stock ( protocol 3)
  • sBHI plates (see recipe)
  • Sterile inoculating loop (disposable plastic or flame‐sterilized wire loop)
  • −80°C freezer
  • 37°C, 5% CO 2 incubator

Basic Protocol 2: Growth of H. influenzae in Liquid Medium

  Materials
  • H. influenzae overnight plate ( protocol 1)
  • Sterile 125‐ml culture flasks
  • Broth medium [e.g., supplemented brain heart infusion broth (sBHI); see recipe]
  • Sterile inoculating loop (disposable plastic or flame‐sterilized wire loop)
  • 2‐ml microcentrifuge tubes, sterile
  • Vortex
  • Disposable cuvettes
  • Shaking incubator set at 37°C
  • Spectrophotometer set to 600 nm

Basic Protocol 3: Preparation of H. influenzae Frozen Stocks

  Materials
  • H. influenzae overnight plate
  • BHI broth (see recipe)
  • 60% glycerol, sterile
  • 2‐ml cryovial
  • Micropipettor and sterile tips
  • Sterile inoculating loop (disposable plastic or flame‐sterilized wire loop)
  • Vortex
  • −80°C freezer
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Figures

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Literature Cited

Literature Cited
   Coleman, H.N., Daines, D.A., Jarisch, J., and Smith, A.L. 2003. Chemically defined media for growth of Haemophilus influenzae strains. J. Clin. Microbiol. 41:4408‐4410.
   Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.F., Dougherty, B.A., Merrick, J.M., et al. 1995. Whole‐genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496‐512.
   Harrison, A., Dyer, D.W., Gillaspy, A., Ray, W.C., Mungur, R., Carson, M.B., Zhong, H., Gipson, J., Gipson, M., Johnson, L.S., Lewis, L., Bakaletz, L.O., and Munson, R.S. Jr. 2005. Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: Comparative study with H. influenzae serotype d, strain KW20. J. Bacteriol. 187:4627‐4636.
   Herriott, R.M., Meyer, E.Y., Vogt, M., and Modan, M. 1970. Defined medium for growth of Haemophilus influenzae. J. Bacteriol. 101:513‐516.
   Kaplan, B., Wandstrat, T.L., and Cunningham, J.R. 1997. Overall cost in the treatment of otitis media. Pediatr. Infect. Dis. J. 16:S9‐S11.
   Murphy, T.F. and Apicella, M.A. 1987. Nontypeable Haemophilus influenzae: A review of clinical aspects, surface antigens, and the human immune response to infection. Rev. Infect. Dis. 9:1‐15.
   Smith‐Vaughan, H.C., Sriprakash, K.S., Leach, A.J., Mathews, J.D., and Kemp, D.J. 1998. Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic. Infect. Immun. 66:3403‐3409.
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