Culture of Escherichia coli and Related Bacteria

Sean P. Riley1, Michael E. Woodman1, Brian Stevenson1

1 University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 4.2
DOI:  10.1002/9780470089941.et0402s00
Online Posting Date:  October, 2008
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Abstract

This appendix provides general techniques, equipment, and media used for the growth of many commonly encountered bacteria. For specific growth conditions, readers should refer to units regarding the laboratory maintenance of the organism of interest.

Keywords: bacterial medium; bacterial culture techniques; bacterial culture equipment

     
 
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Table of Contents

  • Overview and Principles
  • Safety Considerations
  • Commonly Used Tools
  • Reagents
  • Culturing Bacteria on Solid Media
  • Growing Bacteria in Liquid Culture
  • Analysis and Purification of Plasmid DNAs
  • Basic Protocol 1: CaCl2‐Induced Competence and Transformation with Plasmid DNA
  • Basic Protocol 2: Electroporation of E. coli with Plasmid DNA
  • Basic Protocol 3: Screening Bacteria for Plasmid DNA (Colony PCR)
  • Basic Protocol 4: Small‐Scale Plasmid Isolation by Alkaline Lysis
  • Basic Protocol 5: Small‐Scale Plasmid Isolation by Boiling Lysis
  • Basic Protocol 6: Additional Purification of Plasmid DNA
  • Storage of Cultures
  • Commonly Used Bacterial Media
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: CaCl2‐Induced Competence and Transformation with Plasmid DNA

  Materials
  • LB broth (see )
  • E. coli strain
  • CaCl 2/Tris buffer: 50 mM CaCl 2/10 mM Tris⋅Cl, pH 8.0 (sterilize by filtering through a 0.22‐µm filter; store indefinitely at 4°C), ice cold
  • LB plates with appropriate antibiotic(s) (see )
  • 15‐ml culture tubes
  • 37°C incubator
  • Spectrophotometer (unit 2.1)
  • Refrigerated centrifuge (unit 5.1)
  • Vortex
  • 37° to 42°C heating block or water bath

Basic Protocol 2: Electroporation of E. coli with Plasmid DNA

  Materials
  • LB broth (see )
  • E. coli strain
  • 10% (v/v) glycerol (filter sterilize using a 0.22‐µm filter and store indefinitely at 4°C), ice cold
  • DNA in water or TE buffer
  • SOC broth (see )
  • Solid culture medium plates with appropriate antibiotic(s) (e.g., LB medium with antiobiotics; see )
  • 37°C incubator
  • Spectrophotometer (unit 2.1)
  • Refrigerated centrifuge (unit 5.1)
  • Vortex
  • 1.5‐ml polypropylene tubes
  • 0.2‐cm gap electroporation cuvette
  • Electroporator
  • 17 × 100–mm capped test tube

Basic Protocol 3: Screening Bacteria for Plasmid DNA (Colony PCR)

  Materials
  • Oligonucleotide primers
  • DNA polymerase, heat‐stable
  • dNTPs
  • PCR buffer (units 3.3& 10.2)
  • PCR tubes
  • Sterile, new toothpicks
  • PCR thermal cylcer (e.g., Perkin‐Elmer 9700)

Basic Protocol 4: Small‐Scale Plasmid Isolation by Alkaline Lysis

  Materials
  • LB broth (see )
  • E. coli strain
  • Buffer 1: 50 mM glucose, 10 mM EDTA, 25 mM Tris⋅Cl, pH 8.0 (store 3 to 6 months at 4°C); immediately before use, add lysozyme to stock solution to a final concentration of 2 mg/ml
  • Buffer 2: 0.2 N NaOH, 1% (w/v) sodium dodecylsulfate (store ≤1 month at room temperature)
  • Buffer 3: 3 M sodium acetate, pH 4.8; adjust pH as necessary with glacial acetic acid
  • 0.1 M sodium acetate/ 0.05 M Tris⋅Cl, pH 8.0
  • 70% and 95% ethanol
  • TE buffer (unit 3.3)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge

Basic Protocol 5: Small‐Scale Plasmid Isolation by Boiling Lysis

  Materials
  • LB broth (see )
  • E. coli strain
  • STET, per 100 ml:
    • 1 ml 1 M Tris⋅Cl, pH 8.0 (unit 3.3)
    • 10 ml 0.5 M EDTA, pH 8.0 (unit 3.3)
    • 500 µl Triton X‐100
    • 8 g sucrose
    • Add water to a final volume of 100 ml
  • Lysozyme solution: 10 mg lysozyme/ml of 10 mM Tris⋅Cl, pH 8.0, prepare fresh daily
  • Isopropanol
  • 70% ethanol
  • TE buffer (unit 3.3)
  • 1.5‐ml microcentrifuge tubes
  • Vortexer
  • Boiling water bath
  • Sterile toothpicks

Basic Protocol 6: Additional Purification of Plasmid DNA

  Materials
  • Nucleic acid solution
  • TE buffer (unit 3.3)
  • 3 M sodium acetate, pH 7.0 (units 3.1& 5.2)
  • Phenol (previously saturated with 10 mM Tris⋅Cl, pH 7.5)
  • 95:5 (v/v) chloroform/isoamyl alcohol
  • 70% and 90% ethanol
  • 1.5‐ml microcentrifuge tubes
  • Vortexer
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Figures

  •   FigureFigure 4.2.1 Making an inoculating loop.
  •   FigureFigure 4.2.2 Making a spreader.
  •   FigureFigure 4.2.3 Proper technique for streaking bacteria on solid medium.
  •   FigureFigure 4.2.4 Template for organization of Petri plate.
  •   FigureFigure 4.2.5 Bacterial growth curve.
  •   FigureFigure 4.2.6 Example illustrating a gridded counting chamber, a hemacytometer slide (Improved Neubauer), and a coverslip. The coverslip is applied to the slide and the cell suspension is added to the counting chamber using a mechanical pipettor or a Pasteur pipet. Each counting chamber has a 3 × 3–mm grid (enlarged). The four corner squares (1, 2, 4, and 5) and the central square (3) are counted on each side of the hemacytometer (numbers added).
  •   FigureFigure 4.2.7 Serial dilution.

Videos

Literature Cited

Literature Cited
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513‐1523.
   Coico, R. and Lunn, G. 2005. Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr. Prot. Microbiol. 0:1A.1.1‐1A.1.8.
   Dower, W.J., Miller, J.F., and Ragsdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16:6127‐6145.
   Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193‐197.
   Mandel, M. and Higa, A. 1970. Calcium‐dependent bacteriophage DNA infection. J. Mol. Biol. 53:159‐162.
   Wright, S. 1986. Recombinant DNA technology and its social transformation, 1972‐1982. Osiris 2:303‐360.
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