Growth and Laboratory Maintenance of Pseudomonas aeruginosa

Annette E. LaBauve1, Matthew J. Wargo1

1 Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burlington, Vermont
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6E.1
DOI:  10.1002/9780471729259.mc06e01s25
Online Posting Date:  May, 2012
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Abstract

Pseudomonas aeruginosa is a common, free‐living, Gram‐negative bacterium that can cause significant disease as an opportunistic pathogen. Rapid growth, facile genetics, and a large suite of virulence‐related phenotypes make P. aeruginosa a common model organism to study Gram‐negative opportunistic pathogens and basic microbiology. This unit describes the basic laboratory growth and maintenance of P. aeruginosa. Curr. Protoc. Microbiol. 25:6E.1.1‐6E.1.8. © 2012 by John Wiley & Sons, Inc.

Keywords: Pseudomonas; metabolism; growth; minimal media

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of P. aeruginosa from a Frozen Stock
  • Basic Protocol 2: Growth of P. aeruginosa in Liquid Medium
  • Basic Protocol 3: Preparation of P. aeruginosa Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of P. aeruginosa from a Frozen Stock

  Materials
  • P. aeruginosa frozen stocks (see protocol 3)
  • LB agar plates (see appendix 4A), with antibiotics, if necessary (see Table 6.1.2)
  • Sterile wooden applicator stick
  • 37°C incubator
    Table 6.0.2   Materials   Antibiotic Usage for P. aeruginosa b   Antibiotic Usage for P. aeruginosa

    Antibiotic LB liquid c LB plate MOPS liquid MOPS plate Stock
    Gentamicin 40 µg/ml 50 µg/ml 20 µg/ml 25 µg/ml 50 mg/ml
    Kanamycin 300 µg/ml 300 µg/ml 150 µg/ml 150 µg/ml 100 mg/ml
    Carbenicillin d 900 µg/ml 900 µg/ml 700 µg/ml 700 µg/ml 150 mg/ml e
    Tetracycline 50 µg/ml 100 µg/ml 50 µg/ml 50 µg/ml 50 mg/ml

     bWe report here the antibiotic concentrations we use in our laboratory. However, there are wide ranges of antibiotic concentrations reported in the literature. Some of the variance depends on the age and source of the antibiotic. We recommend titrating your antibiotic during initial trials to get robust growth of resistant (i.e., plasmid carrying) bacteria and no spontaneous resistance from wild‐type.
     cThese concentrations are for selection of cells carrying resistance plasmids. Antibiotic concentrations for plasmid maintenance can be lowered within experiments by half for all of these antibiotics. For transposon mutants, lower concentrations are often used after selection. For example, we use 10 µg/ml gentamicin for PA14 transposon mutants.
     dWe use carbenicillin in place of ampicillin to reduce satellite colonies during cloning with E. coli. Either antibiotic will work for P. aeruginosa and the bla resistance marker codes for detoxification of both compounds.
     eTo add this amount of carbenicillin or ampicilin when making plates, we typically weigh the appropriate amount out, dissolve in water, and filter sterilize before addition.

Basic Protocol 2: Growth of P. aeruginosa in Liquid Medium

  Materials
  • P. aeruginosa freshly grown on agar plates (see protocol 1)
  • LB broth (see appendix 4A) or MOPS minimal medium (see recipe)
  • Antibiotics, if necessary (see Table 6.1.2)
  • Sterile glass tubes (e.g., 18‐mm) or flasks (e.g., 125‐ml)
  • Sterile wooden applicator stick or inoculating loop
  • 37°C incubator

Basic Protocol 3: Preparation of P. aeruginosa Frozen Stocks

  Materials
  • P. aeruginosa grown on LB agar plates (see protocol 1)
  • LB broth (see appendix 4A), with antibiotics if necessary (see Table 6.1.2)
  • 50% glycerol, sterile
  • 37°C incubator
  • 1.2‐ml sterile cryogenic vials
  • Vortex mixer
  • −80°C freezer
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Figures

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Literature Cited

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Internet Resources
   http://www.pseudomonas.com
  The Web site for the Pseudomonas aeruginosa genomes, as well as other sequence species within the Pseudomonas genus.
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