Growth and Maintenance of Escherichia coli Laboratory Strains

Mike S. Son1, Ronald K. Taylor2

1 Plymouth State University, Plymouth, New Hampshire, 2 Geisel School of Medicine at Dartmouth, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5A.4
DOI:  10.1002/9780471729259.mc05a04s27
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Escherichia coli is a Gram‐negative bacterium, commonly used in both teaching and research laboratories. This unit includes protocols for the growth and maintenance of E. coli in any teaching‐ or research‐associated laboratory. Curr. Protoc. Microbiol. 27:5A.4.1‐5A.4.9. © 2012 by John Wiley & Sons, Inc.

Keywords: Escherichia coli; growth; maintenance

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of E. coli from Frozen Stocks
  • Basic Protocol 2: Growth of E. coli on Solid Media
  • Basic Protocol 3: Growth of E. coli in Liquid Media
  • Basic Protocol 4: Storage of E. coli Frozen Stocks in Glycerol
  • Basic Protocol 5: Storage of E. coli in Agar Stabs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Growth of E. coli from Frozen Stocks

  Materials
  • E. coli frozen stock cultures
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • Wooden applicator sticks, sterile (Fisher, cat. no. 01‐340)
  • 37°C incubator

Basic Protocol 2: Growth of E. coli on Solid Media

  Materials
  • E. coli overnight liquid cultures (or frozen stock)
  • Luria‐Bertani (LB) broth, sterile (see recipe)
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm; see recipe), supplemented with antibiotics, if necessary (see appendix 4A)
  • 70% ethanol in a glass beaker (to sterilize glass spreader)
  • 2‐ml microcentrifuge tubes, sterile (USA Scientific, cat. no. 1620‐2700)
  • Vortex mixer
  • Glass spreader
  • Bunsen burner (or alternative flame source to burn off the excess ethanol on the spreader)
  • 37°C incubator

Basic Protocol 3: Growth of E. coli in Liquid Media

  Materials
  • Luria‐Bertani (LB) broth (see recipe)
  • E. coli single colonies on solid medium (with appropriate antibiotics, if necessary)
  • Antibiotics (if necessary; see appendix 4A)
  • Capped glass culture tubes, sterile (Tubes—Fisherbrand, cat. no. 14‐961‐32; Caps—Fisherbrand, cat. no. 05‐888) (or sterile Erlenmeyer flasks with caps)
  • Wooden applicator sticks, sterile (Fisher, cat. no. 01‐340)
  • 37°C incubator with a mechanism for rotating or shaking liquid cultures

Basic Protocol 4: Storage of E. coli Frozen Stocks in Glycerol

  Materials
  • E. coli overnight liquid cultures
  • Luria‐Bertani (LB) broth
  • Antibiotics (if necessary; see appendix 4A)
  • 37°C incubator with a mechanism for rotating or shaking liquid cultures
  • 4‐ml glass vials (Wheaton, cat. no. 224‐882) containing 1.5 ml of sterile 50% (w/v) glycerol [alternatively, cryovials (Fisher, cat. no. 12‐565‐167N) containing half the volume can be used]
  • Vortex mixer
  • 2‐ml sterile microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • Microcentrifuge
  • −80°C freezer

Basic Protocol 5: Storage of E. coli in Agar Stabs

  Materials
  • E. coli overnight liquid cultures
  • E. coli streak plate with single, well‐isolated colonies
  • Luria‐Bertani (LB) agar stabs (see recipe)
  • Inoculating loop (Fisher, cat. no. 22‐268‐169)
  • Bunsen burner
  • Inoculating wire needle (Fisher, cat. no. 22‐032‐099) (or sterile double‐pointed, round toothpicks)
  • 37°C incubator
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Boyer, H.W. and Roulland‐Dussoix, D. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459‐472.
   Casadaban, M.J. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541‐555.
   Casadaban, M.J. and Cohen, S.N. 1979. Lactose genes fused to exogenous promoters in one step using a Mu‐lac bacteriophage in vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. U.S.A. 76:4530‐4533.
   Casadaban, M.J. and Cohen, S.N. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179‐207.
   Curtiss, R. III. 1978. Biological containment and cloning vector transmissibility. J. Infect. Dis. 137:668‐675.
   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.
   Fotadar, U., Zaveloff, P., and Terracio, L. 2005. Growth of Escherichia coli at elevated temperatures. J. Basic Microbiol. 45:403‐404.
   Grant, S.G., Jessee, J., Bloom, F.R., and Hanahan, D. 1990. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation restriction mutants. Proc. Natl. Acad. Sci. U.S.A. 87:4645‐4649.
   Komeda, Y. and Iino, T. 1979. Regulation of expression of the flagellin gene (hag) in Escherichia coli K‐12: Analysis of hag‐lac gene fusions. J. Bacteriol. 139:721‐729.
   Lacks, S. and Greenberg, B. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114:153‐168.
   Meselson, M. and Yuan, R. 1968. DNA restriction enzyme from E. coli. Nature 217:1110‐1114.
   Neidhardt, F.C. (ed.) 1996. Escherichia coli and Salmonella—Cellular and molecular biology, 2nd Edition. ASM Press, Washington, D.C.
   Peters, J.E., Thate, T.E., and Craig, N.L. 2003. Definition of the Escherichia coli MC4100 genome by use of a DNA array. J. Bacteriol. 185:2017‐2021.
   Yanisch‐Perron, C., Vieira, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103‐119.
Internet Resources
   http://openwetware.org/index.php?title=E._coli_genotypes&oldid=577711
  This Web site is for various E. coli strains and gene‐specific information.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library