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Growth and Laboratory Maintenance of Vibrio cholerae

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

¹Dartmouth 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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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 Basic 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 (Basic Protocol 1)
LB broth (see appendix 4A)
Antibiotics, if necessary (Table 6A.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 6A.1.2 Antibiotic Stock Solutions for Use with V. cholerae Cultures^a

Antibiotic	Solvent	Stock concentration	Working concentration^b,c	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 (Basic 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 (Basic Protocol 1)
LB agar stab (see recipe)

Inoculating loop or toothpick, sterile
37°C incubator

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Figures

Figure 6A.1.1

Growth curve for V. cholerae O1, classical strain O395, in LB broth.

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Literature Cited

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.