Antibiotic Susceptibility Testing by Flow Cytometry

David J. Mason1, Fiona C. Mortimer2, Vanya A. Gant3

1 University of Warwick, Coventry, United Kingdom, 2 Formerly of King's College London, London, United Kingdom, 3 University College Hospital and Trust, London, United Kingdom
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.8
DOI:  10.1002/0471142956.cy1108s08
Online Posting Date:  May, 2001
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Abstract

Flow cytometry can provide a rapid indication of antibiotic susceptibility. This unit describes a number of fluorescent dyes that allow the cytometer to distinguish the heterogeneic nature of populations exposed to antibiotics. No single method can be used for multiple organisms, since it is the interference with the organisms growth and structural integrity that signals the impact of the antibiotic so rapidly. This unit discusses membrane structural changes as well as alterations in nucleic acid and the probes that can be successfully used with flow cytometry.

     
 
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Table of Contents

  • Basic Protocol 1: Antibiotic Susceptibility Testing with DiBAC4(3)
  • Alternate Protocol 1: Antibiotic Susceptibility Testing with PI
  • Alternate Protocol 2: Antibiotic Susceptibility Testing with SYBR Green I
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Antibiotic Susceptibility Testing with DiBAC4(3)

  Materials
  • Bacterial cultures grown to log phase in 0.2‐µm‐filtered nutrient broth or Iso‐Sensitest broth (Oxoid)
  • 200 µg/ml gramicidin S solution (see recipe)
  • 100 µg/ml DiBAC 4(3) solution (see recipe)
  • Sample tubes as required by the flow cytometer
  • Flow cytometer with 488 nm excitation and band‐pass filter centered at or around 515 nm

Alternate Protocol 1: Antibiotic Susceptibility Testing with PI

  • 80% (v/v) ethanol, ice cold
  • 0.2‐µm‐filtered broth
  • 100 µg/ml PI solution (see recipe)
  • Band‐pass filter centered at or around 605 nm

Alternate Protocol 2: Antibiotic Susceptibility Testing with SYBR Green I

  • 100 U/ml SYBR Green I solution (see recipe)
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Figures

Videos

Literature Cited

   Boye, E., Steen, H.B., and Skarstad, K. 1983. Flow cytometry of bacteria: A promising tool in experimental and clinical microbiology. J. Gen. Microbiol. 129:973‐980.
   Gant, V.A., Warnes, G., Phillips, I., and Savidge, G.F. 1993. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 39:147‐154.
   Jepras, R.I., Paul, F.E., Pearson, S.C., and Wilkinson, M.J. 1997. Rapid assessment of antibiotic effects on Escherichia coli by bis(1,3‐dibutylbarbituric acid) trimethine oxonol and flow cytometry. Antimicrob. Agents Chemother. 41:2001‐2005.
   Mason, D.J., Allman, R.J., Stark, M., and Lloyd, D. 1994. Rapid estimation of bacterial antibiotic susceptibility with flow cytometry. J. Microsc. 176:8‐16
   Mason, D.J., Power, E.G.M., Talsania, H., Phillips, I., and Gant, V.A. 1995. Antibacterial action of ciprofloxacin. Antimicrob. Agents Chemother. 39:2752:2758.
   Ordonez, J.V. and Wehman, N.M. 1993. Rapid flow cytometric antibiotic susceptibility assay for Staphylococcus aureus. Cytometry 14:811‐818.
   Roth, B.L., Poot, M., Yue, S.T., and Millard, P.J. 1997. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl. Environ. Microbiol. 63:2421‐2431.
   Russell, A.D. and Day, M.J. 1996. Antibiotic and biocide resistance in bacteria. Microbios 85:45‐65.
   Suller, M.T.E., Stark, J.M., and Lloyd, D. 1997. A flow cytometric study of antibiotic‐induced damage and evaluation as a rapid antibiotic susceptibility test for methicillin‐resistant Staphylococcus aureus. J. Antimicrob. Chemother. 40:77‐83.
   Walberg, M., Gaustad, P., and Steen, H.B. 1996. Rapid flow cytometric assessment of mecillinam and ampicillin bacterial susceptibility. J. Antimicrob. Chemother. 37:1063‐1075.
   Walberg, M., Gaustad, P., and Steen, H.B. 1997. Rapid assessment of ceftazidime, ciprofloxacin, and gentamicin susceptibility in exponentially‐growing E. coli cells by means of flow cytometry. Cytometry 27:169‐178.
Key References
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single cell analyses. Microbiol. Rev. 60:641‐696.
  An informative review for anyone applying flow cytometry to microbiology.
   Lloyd, D., ed. 1993. Flow Cytometry in Microbiology. Springer‐Verlag, London.
  A useful compilation of work covering many aspects of the application of flow cytometry to microbiology, including antibiotic susceptibility testing.
   Pore, R.S. 1994. Antibiotic susceptibility testing by flow cytometry. J. Antimicrob. Chemother. 34:613‐627.
  A thorough and critical review of the various techniques applied to flow cytometric susceptibility testing.
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