Growth and Laboratory Maintenance of Vibrio cholerae

Raquel M. Martinez1, Christina J. Megli1, Ronald K. Taylor1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6A.1
DOI:  10.1002/9780471729259.mc06a01s17
Online Posting Date:  May, 2010
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Abstract

Vibrio cholerae is a Gram‐negative enteric pathogen. This unit includes protocols for the growth and maintenance of V. cholerae in the laboratory. Curr. Protoc. Microbiol. 17:6A.1.1‐6A.1.7. © 2010 by John Wiley & Sons, Inc.

Keywords: Vibrio cholerae; laboratory growth; cholera

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of V. cholerae from a Frozen Stock
  • Basic Protocol 2: Growth of V. cholerae in Liquid Medium
  • Basic Protocol 3: Preparation of V. cholerae Frozen Stocks
  • Basic Protocol 4: Preservation of V. cholerae in Agar Stabs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of V. cholerae from a Frozen Stock

  Materials
  • V. cholerae frozen stock (see protocol 3)
  • LB agar plates ( appendix 4A)
  • Wooden applicator stick, toothpick or inoculating loop, sterile
  • 37°C incubator

Basic Protocol 2: Growth of V. cholerae in Liquid Medium

  Materials
  • V. cholerae, grown on LB agar, containing antibiotics if appropriate ( protocol 1)
  • LB broth (see appendix 4A)
  • Antibiotics, if necessary (Table 6.1.2)
  • Wooden applicator sticks or inoculating loop, sterile
  • Capped test tubes, sterile
  • Vortex
  • 37°C incubator with a mechanism for rotating or shaking cultures
    Table 6.0.2   Materials   Antibiotic Stock Solutions for Use with V. cholerae Cultures a   Antibiotic Stock Solutions for Use with V. cholerae Cultures

    Antibiotic Solvent Stock concentration Working concentration bb , cc Storage
    Ampicillin Water 100 mg/ml 100 µg/ml 4°C
    Chloramphenicol Ethanol 34 mg/ml 34 µg/ml 4°C
    Kanamycin Water 45 mg/ml 45 µg/ml 4°C
    Polymyxin B Water 50,000 U/ml 50 U/ml 4°C
    Streptomycin Water 100 mg/ml 100 µg/ml 4°C
    Tetracyline Methanol 15 mg/ml 15 µg/ml −20°C

     aAll antibiotics should be filter sterilized by passage through a 0.22‐µm filter.
     bDilute antibiotic 1:1000 into media. For example, when working with a 5 ml broth culture, add 5 µl of the antibiotic stock solution.
     cWhen making agar plates, autoclave medium and let cool to 50°C before adding antibiotics.

Basic Protocol 3: Preparation of V. cholerae Frozen Stocks

  Materials
  • V. cholerae grown on LB agar, containing antibiotics if appropriate ( protocol 1)
  • LB broth (see appendix 4A)
  • 50% (v/v) glycerol, sterile
  • 4‐ml freezer vials, sterile (e.g., Wheaton or Cryovial)
  • Vortex
  • 37°C incubator with a mechanism for rotating or shaking cultures
  • −80°C freezer

Basic Protocol 4: Preservation of V. cholerae in Agar Stabs

  Materials
  • V. cholerae grown on agar, containing antibiotics if appropriate ( protocol 1)
  • LB agar stab (see recipe)
  • Inoculating loop or toothpick, sterile
  • 37°C incubator
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Figures

Videos

Literature Cited

   Ansaruzzaman, M., Bhuiyan, N.A., Safa, A., Sultana, M., McUamule, A., Mondlane, C., Wang, X.Y., Deen, J.L., von Seidlein, L., Clemens, J.D., Lucas, M., Sack, D.A., and Balakrish Nair, G. 2007. Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique. Int. J. Med. Microbiol. 297:443‐449.
   Elbing, K. and Brent, R. 2002a. Media preparation and bacteriological tools. Curr. Protoc. Mol. Biol. 59:1.1.1‐1.1.7.
   Elbing, K. and Brent, R. 2002b. Growth on solid media. Curr. Protoc. Mol. Biol. 59:1.3.1‐1.3‐6.
   Chun, J., Grim, C.J., Hasan, N.A., Lee, J.H., Choi, S.Y., Haley, B.J., Taviani, E., Jeon, Y.S., Kim, D.W., Lee, J.H., Brettin, T.S., Bruce, D.C., Challacombe, J.F., Detter, J.C., Han, C.S., Munk, A.C., Chertkov, O., Meincke, L., Saunders, E., Walters, R.A., Huq, A., Nair, G.B., and Colwell R.R. 2009. Comparative genomics reveals mechanism for short‐term and long‐term clonal transitions in pandemic Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 106:15442‐15447.
   Crump, J.A., Bopp, C.A., Greene, K.D., Kubota, K.A., Middendorf, R.L., Wells, J.G., and Mintz, E.D. 2003. Toxigenic Vibrio cholerae serogroup O141‐associated cholera‐like diarrhea and bloodstream infection in the United States. J. Infect. Dis. 187:866‐868.
   Dziejman, M., Serruto, D., Tam, V.C., Sturtevant, D., Diraphat, P., Faruque, S.M., Rahman, M.H., Heidelberg, J.F., Decker, J., Li, L., Montgomery, K.T., Grills, G., Kucherlapati, R., and Mekalanos, J.J. 2005. Genomic characterization of non‐O1, non‐O139 Vibrio cholerae reveals genes for a type III secretion system. Proc. Natl. Acad. Sci. U.S.A. 102:3465‐3470.
   Mohapatra, S.S., Ramachandran, D., Mantri, C.K., Colwell, R.R., and Singh, D.V. 2009. Determination of relationships among non‐toxigenic Vibrio cholerae O1 biotype El Tor strains from housekeeping gene sequences and ribotype patterns. Res. Microbiol. 160:57‐62.
   Nair, G.B., Qadri, F., Holmgren, J., Svennerholm, A.M., Safa, A., Bhuiyan, N.A., Ahmad, Q.S., Faruque, S.M., Faruque, A.S., Takeda, Y., and Sack, D.A. 2006. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J. Clin. Microbiol. 44:4211‐4213.
   Safa, A., Sultana, J., Dac Cam, P., Mwansa, J.C., and Kong, R.Y. 2008. Vibrio cholerae O1 hybrid El Tor strains, Asia and Africa. Emerg. Infect. Dis. 14:987‐988.
   Tobin‐D'Angelo, M., Smith, A.R., Bulens, S.N., Thomas, S., Hodel, M., Izumiya, H., Arakawa, E., Morita, M., Watanabe, H., Marin, C., Parsons, M.B., Greene, K., Cooper, K., Haydel, D., Bopp, C., Yu, P., and Mintz, E. 2008. Severe diarrhea caused by cholera toxin‐producing Vibrio cholerae serogroup O75 infections acquired in the southeastern United States. Clin. Infect. Dis. 47:1035‐1040.
Internet Resources
  http://gsc.jcvi.org/projects/msc/vibrio/index.shtml
  The Web site for the Genomic Sequencing Center for Infectious Diseases.
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