Laboratory Growth and Maintenance of Streptococcus pyogenes (The Group A Streptococcus, GAS)

Kanika Gera1, Kevin S. McIver1

1 University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9D.2
DOI:  10.1002/9780471729259.mc09d02s30
Online Posting Date:  October, 2013
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Abstract

Streptococcus pyogenes is a Gram‐positive bacterium that strictly infects humans. It is the causative agent of a broad spectrum of diseases accounting for millions of infections and at least 517,000 deaths each year worldwide. It is a nutritionally fastidious organism that ferments sugars to produce lactic acid and has strict requirements for growth. To aid in the study of this organism, this unit describes the growth and maintenance of S. pyogenes. Curr. Protoc. Microbiol. 30:9D.2.1‐9D.2.13. © 2013 by John Wiley & Sons, Inc.

Keywords: Streptococcus pyogenes; the Group A Streptococcus; firmicute; facultative anaerobe; chemically defined medium

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of S. pyogenes (GAS) on Solid Medium
  • Basic Protocol 2: Growth of S. pyogenes (GAS) in Liquid Medium
  • Basic Protocol 3: Growth Curves of S. pyogenes (GAS) Using Klett Tubes
  • Basic Protocol 4: Growth Curves of S. pyogenes (GAS) Performed in Plate Reader
  • Basic Protocol 5: Isolation of S. pyogenes (GAS) from a Mixed Culture
  • Support Protocol 1: Gram Stain
  • Support Protocol 2: Catalase Assay
  • Support Protocol 3: Oxidase Assay
  • Basic Protocol 6: Preparing S. pyogenes (GAS) Freezer Stock
  • Alternate Protocol 1: Growth of S. pyogenes (GAS) in Chemically Defined Medium
  • Alternate Protocol 2: Growth of S. pyogenes (GAS) in Low‐Glucose C Medium
  • Alternate Protocol 3: Growth of S. pyogenes (GAS) in Human Blood
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth of S. pyogenes (GAS) on Solid Medium

  Materials
  • Plate of actively growing viable S. pyogenes colonies or GAS culture from frozen stock (see protocol 9 for frozen stock)
  • TSA supplemented with 5% sheep blood plates (Fisher, cat. no. 221239)
  • Sterile wood sticks or inoculating loops
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Growth of S. pyogenes (GAS) in Liquid Medium

  Materials
  • Plate containing viable S. pyogenes (GAS)
  • THY broth (see recipe)
  • Sterile wood sticks or inoculating loops
  • 15‐ and 50‐ml screw‐cap conical polypropylene tubes (Genesee, cat. no. 21‐101 for 15‐ml; Fisher, cat. no. 430290 for 50‐ml)
  • 37°C incubator
  • Light microscope

Basic Protocol 3: Growth Curves of S. pyogenes (GAS) Using Klett Tubes

  Materials
  • S. pyogenes culture (see protocol 2)
  • THY (see recipe)
  • Antibiotic, optional
  • Concentrated H 2SO 4 (VWR, cat. no. 32920‐042)
  • NOCHROMIX (Godax labs, cat. no. 19‐010)
  • 15‐ml Klett tubes or 125‐ml Klett flasks
  • 37°C incubator
  • Klett‐Summerson colorimeter (Bel‐Art Scienceware, cat. no. 370120000) or spectrophotometer
  • 15‐ml conical tubes
  • Centrifuge

Basic Protocol 4: Growth Curves of S. pyogenes (GAS) Performed in Plate Reader

  Materials
  • Chemically defined medium (CDM; see recipe)
  • Overnight culture in 15‐ml tube (see protocol 2)
  • Saline
  • 24‐well plate
  • Refrigerated centrifuge
  • Transparent optical tape (Bio‐Rad, cat. no. 2239444)
  • Plate reader (Omega)

Basic Protocol 5: Isolation of S. pyogenes (GAS) from a Mixed Culture

  Materials
  • Samples
  • Crystal violet
  • Gram's iodine
  • 95% (v/v) ethanol
  • Safranin
  • Slides (VWR, cat. no. 16004‐430)
  • Staining tray
  • Blotting paper
  • Microscope

Support Protocol 1: Gram Stain

  Materials
  • GAS colonies on plates
  • 10% hydrogen peroxide
  • Sterile inoculating loops
  • Slides

Support Protocol 2: Catalase Assay

  Materials
  • Oxidase reagent (Dalynn Biologicals, cat. no. R095)
  • 18‐ to 24‐hr culture plate of bacteria
  • Sterile cotton tip applicators

Support Protocol 3: Oxidase Assay

  Materials
  • 80% sterile glycerol solution
  • Liquid culture of viable and pure S. pyogenes in medium (e.g., THY)
  • 2‐ml cryogenic vials (Sarstedt, cat. no. 72‐694‐006)
  • −80°C freezer

Basic Protocol 6: Preparing S. pyogenes (GAS) Freezer Stock

  Materials
  • Chemically defined medium (CDM; see recipe)
  • S. pyogenes culture
  • Saline
  • 37°C water bath
  • Refrigerated centrifuge
  • Spectrophotometer
  • Klett tubes
  • Klett‐Summerson colorimeter

Alternate Protocol 1: Growth of S. pyogenes (GAS) in Chemically Defined Medium

  Materials
  • Overnight S. pyogenes culture (see protocol 2)
  • THY (see recipe) with appropriate antibiotic
  • Saline
  • Whole human blood
  • Klett‐Summerson colorimeter or spectrophotometer
  • 37°C rotating incubator
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Figures

Videos

Literature Cited

Literature Cited
  Carapetis, J.R., Steer, A.C., Mulholland, E.K., and Weber, M. 2005. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5:685‐694.
  Kang, S.O., Caparon, M.G., and Cho, K.H. 2010. Virulence gene regulation by CvfA, a putative RNase: The CvfA‐enolase complex in Streptococcus pyogenes links nutritional stress, growth‐phase control, and virulence gene expression. Infect. Immun. 78:2754‐2767.
  Kreikemeyer, B., McIver, K.S., and Podbielski, A. 2003. Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen‐host interactions. Trends Microbiol. 11:224‐232.
  Lancefield, R.C. 1957. Differentiation of group A Streptococci with a common R antigen into serological types, with special reference to the bactericidal test. J. Exp. Med. 106:525‐544.
  Musser, J.M., Kapur, V., Kanjilal, S., Shah, U., Musher, D.M., Barg, N.L., Johnston, K.H., Schlievert, P.M., Henrichsen, J., Gerlach, D., Rakita, R.M., Tanna, A., Cookson, B.D., and Huang, J.C. 1993. Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin). J. Infect. Dis. 167:337‐346.
  Olsen, R.J., Shelburne, S.A., and Musser, J.M. 2009. Molecular mechanisms underlying group A streptococcal pathogenesis. Cell Microbiol. 11:1‐11.
  Opdyke, J.A., Scott, J.R., and Moran, C.P. 2001. A secondary RNA polymerase sigma factor from Streptococcus pyogenes. Mol. Microbiol. 42:495‐502.
  Todd, E.W. and Hewitt, L.F. 1932. A new culture medium for the production of antigenic streptococcal haemolysin. J. Pathol. Bacteriol. 35:973.
  van de Rijn, I. and Kessler, R.E. 1980. Growth characteristics of group A streptococci in a new chemically defined medium. Infect. Immun. 27:444‐448.
  Wood, D.N., Chaussee, M.A., Chaussee, M.S., and Buttaro, B.A. 2005. Persistence of Streptococcus pyogenes in stationary‐phase cultures. J. Bacteriol. 187:3319‐3328.
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