Estimation of Microbial Viability Using Flow Cytometry

Hazel M. Davey1, Douglas B. Kell2, Dieter H. Weichart3, Arseny S. Kaprelyants4

1 University of Wales, Aberystwyth, United Kingdom, 2 University of Manchester Institute of Science and Technology, Manchester, United Kingdom, 3 Max Planck Institut für Molekulare Genetik, Berlin, Germany, 4 Russian Academy of Sciences, Moscow, Russia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.3
DOI:  10.1002/0471142956.cy1103s29
Online Posting Date:  September, 2004
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Abstract

For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

     
 
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Table of Contents

  • The Problem of Determining Viability
  • Fluorescent Stains for Microbial Viability Determination by Flow Cytometry
  • Estimations of Viability of Micrococcus Luteus
  • Use of Cell Sorting in Viability Studies
  • Conclusions
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

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   Yurkow, E.J. and McKenzie, M.A. 1993. Characterization of hypoxia‐dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytomtery: A model for ischemic tissue destruction. Cytometry. 14:287‐293.
Key References
   Amann et al., 1995. See above.
  Important review of microbial diversity and phytogenetics using nucleic acid probe methods that do not require microbial cultivation.
   Davey and Kell, 1996. See above.
  Extensive review of the application of flow cytometry to problems of (mainly) microbiological interest. Includes 1073 literature references.
   Porter, J., Deere, D., Hardman, M., Edwards, C., and Pickup, R. 1997. Go with the flow—use of flow cytometry in environmental microbiology. FEMS Microbiol. Ecol. 24:93‐101.
  A useful review of the applications of flow cytometry in environmental microbiology.
   Shapiro, 1995. See above.
  The Book. A detailed and entertaining overview. All you ever wanted to know about flow cytometry but were afraid to ask.
   Troussellier, M., Courties, C., and Vaquer, A. 1993. Recent applications of flow cytometry in aquatic microbial ecology. Biol. Cell 78:111‐121.
  Another short but useful review of applications of flow cytometry in environmental microbiology, concentrating on aquatic systems.
Internet Resources
   http://pcfcij.dbs.aber.ac.uk/
  The Aberystwyth flow cytometry site has information on microbial flow cytometry, including viability determinations.
   http://www.cyto.purdue.edu/flowcyt/research/micrflow/index.htm
  Microbial flow cytometry section with contributions from several authors on viability measurements.
   http://www.probes.com/handbook/sections/1503.html
  Source for many viability stains, including viability kits.
   http://www.bdbiosciences.com/immunocytometry_systems/application_notes/
  Includes useful application notes on microbial cytometry.
   http://www.partec.de/applications/notes.html
  Application notes on the analysis and enumeration of microorganisms.
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