Laboratory Maintenance of Methicillin‐Resistant Staphylococcus aureus (MRSA)

Nicholas P. Vitko1, Anthony R. Richardson1

1 Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9C.2
DOI:  10.1002/9780471729259.mc09c02s28
Online Posting Date:  February, 2013
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Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin‐resistance (MRSA). Many strains of S. aureus are utilized in the laboratory, underscoring the genetic differences inherent in clinical isolates. S. aureus grows quickly at 37°C with aeration in rich media (e.g., BHI) and exhibits a preference for glycolytic carbon sources. Furthermore, S. aureus has a gold pigmentation, exhibits β‐hemolysis, and is catalase and coagulase positive. The four basic laboratory protocols presented in this unit describe how to culture S. aureus on liquid and solid media, how to identify S. aureus strains as methicillin resistant, and how to generate a freezer stock of S. aureus for long‐term storage. Curr. Protoc. Microbiol. 28:9C.2.1‐9C.2.14. © 2013 by John Wiley & Sons, Inc.

Keywords: Staphylococcus aureus; HA‐MRSA; CA‐MRSA; growth; strain selection; CDM; freezer stock

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of S. aureus on Solid Media
  • Basic Protocol 2: Testing S. aureus for Methicillin Resistance
  • Basic Protocol 3: Growth of S. aureus in Liquid Media
  • Basic Protocol 4: Preparation of S. aureus Frozen Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Growth of S. aureus on Solid Media

  • S. aureus frozen stock ( protocol 3)
  • BHI agar plates (see recipe)
  • Sterile wooden applicators
  • 37°C incubator

Basic Protocol 2: Testing S. aureus for Methicillin Resistance

  • S. aureus streak plate (see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.5 McFarland standard
  • Mueller‐Hinton agar (MHA) plates supplemented with 2% NaCl and oxacillin (2 and 4 µg/ml) (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Inoculating loops, sterile
  • Vortex mixer
  • Spectrophotometer
  • Plastic cuvettes
  • 35°C incubator

Basic Protocol 3: Growth of S. aureus in Liquid Media

  • BHI broth (see recipe)
  • Streak plate of S. aureus (see protocol 1)
  • Inoculating loops, sterile
  • 15‐ to 20‐ml culture tubes (with caps), sterile
  • 37°C shaking incubator

Basic Protocol 4: Preparation of S. aureus Frozen Stocks

  • Dimethyl sulfoxide (DMSO; see recipe), sterile
  • Overnight culture of S. aureus (see protocol 3)
  • 2‐ml cryotubes, sterile
  • Vortex mixer
  • −80°C freezer
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