Growth and Laboratory Maintenance of Staphylococcus aureus

Dominique M. Missiakas1, Olaf Schneewind1

1 Department of Microbiology, University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9C.1
DOI:  10.1002/9780471729259.mc09c01s28
Online Posting Date:  February, 2013
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Staphylococcus aureus is a facultative anaerobic Gram‐positive coccus and a member of the normal skin flora as well as the nasal passages of humans. S. aureus is also the etiological agent of suppurative abscesses, as first described by Sir Alexander Ogston in 1880. Ever since, studies on S. aureus have focused on the complex battery of virulence factors and regulators that allow for its swift transition between commensalism and pathogenic states and escape from host immune defenses. The success of this pathogen is further evidenced by its ability to acquire antibiotic resistance traits through mechanisms that often remain poorly understood. Curr. Protoc. Microbiol. 28:9C.1.1‐9C.1.9. © 2013 by John Wiley & Sons, Inc.

Keywords: Staphylococcus aureus; Gram positive; coccus; MRSA

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Table of Contents

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

  • S. aureus frozen stock (see protocol 3)
  • TSA plates with antibiotics, if necessary (see Table 9.1.1)
  • Protective laboratory coat, eye goggles and disposable latex gloves
  • Disposable sterile loop
  • 37°C incubator
    Table 9.0.1   MaterialsAntibiotic Usage for S. aureus

    Antibiotic a Range of final concentration (µg/ml) b Stock (mg/ml) c
    Chloramphenicol 10‐20 20
    Tetracycline 5‐10 10
    Spectinomycin 10‐20 20
    Erythromycin 5‐50 50
    Kanamycin 20‐25 25

     aList of the most commonly used antibiotics.
     bThe specific concentration of antibiotic may vary depending on the strain (owing to intrinsic resistance) and increases with the copy number of the cognate resistance marker (chromosome: single copy; plasmid: multiple copies).
     cAll antibiotics are acquired as powder products; they are weighed, dissolved, filtered, and stored. Antibiotics stocks may be stored frozen at −20°C for several months or kept at 4°C for several weeks. Stocks should not be re‐frozen and should be kept on ice when not stored at 4°C. Chloramphenicol and erythromycin are dissolved in 100% ethanol. Tetracycline is dissolved in 70% ethanol and should be kept in the dark because it is light‐sensitive. Spectinomycin and kanamycin are both dissolved in water. Methods to prepare and store antibiotic solutions can be found in manuals for molecular genetics and molecular biology work (Miller, ; Sambrook and Russell, ).

Basic Protocol 2: Growth of S. aureus in Liquid Medium

  • Sterile medium (e.g., TSB, see recipe)
  • Antibiotics, if necessary (see Table 9.1.1)
  • S. aureus colonies freshly grown on TSA (see protocol 1)
  • Sterile glass tubes (e.g., 18‐mm) or flasks (e.g., 125‐ml)
  • Disposable sterile loop
  • 37°C incubator with a shaker at 200 rpm or a roller drum (tube only)

Basic Protocol 3: Preparation of S. aureus Frozen Stocks

  • S. aureus freshly grown on TSA (see protocol 1)
  • S. aureus freshly grown in TSB
  • 50% and 10% glycerol, sterile
  • 1× or 2× cryopreservation solution (see recipe)
  • Sterile pipets
  • 2‐ml sterile cryogenic vials
  • Sterile loop
  • −80°C freezer
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Literature Cited

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