Applications of Flow Cytometry in Bacterial Ecology

  • Clive Edwards
  • Julian P. Diaper
  • Jonathan Porter
  • Roger Pickup


Monitoring activity and enumeration of bacteria in natural environments has always posed considerable problems. Conventional plating techniques have normally been employed but have increasingly been shown to be of limited value (Mills and Bell 1986) because only a small proportion of indigenous species (c. 1%) can be isolated by anyone technique (Jones 1977; Pickup 1991). The proposal that some bacteria can adopt a viable but non-culturable state has further complicated the recovery of bacteria from natural environments (Roszak and Colwell 1987). In an attempt to circumvent these difficulties methods have been devised for the direct microscopic enumeration of bacteria; for example, acridine orange staining which stains live bacteria green while debris (and presumably dead cells) appears orange to red. These methods, however, have a number of drawbacks that make them unreliable (Postma and Altemuller 1990; Page and Burns 1991). The requirement for accurate and rapid methods for detecting target species present within diverse and active populations has never been greater.


Flow Cytometry Pyridine Nucleotide Fluorescein Diacetate Acridine Orange Staining Fluorescence Distribution 
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  1. Adams A, Bundy A, Thompson K, Horne MT (1988) The association between virulence and cell surface characteristics of Aeromonas salmonicida. Aquaculture 69:1–14CrossRefGoogle Scholar
  2. Amann RI, Binder BJ, Olson RJ, Chisolm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analysing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedGoogle Scholar
  3. Bercovier H, Resnick M, Kornitzer D, Levy L (1987) Rapid method for testing drugsusceptibility of Mycobacteria spp. and Gram-positive bacteria using Rhodamine 123 and fluorescein diacetate. J Microbiol Meth 7:139–142CrossRefGoogle Scholar
  4. Chrzanowski TH, Crotty RD, Hubbard JG, Welch RP (1984) Applicability of the fluorescein diacetate method of detecting active bacteria in freshwater. Microbial Ecol 10:179–185CrossRefGoogle Scholar
  5. Darzynkiewicz Z, Traganos F, Stainio-Coico L, Kapuscinski J, Melamed MR (1982) Interactions of Rhodamine 123 with living cells studied by flow cytometry. Cancer Res 42:799–806PubMedGoogle Scholar
  6. Gottschal JC (1990) Phenotypic response to environmental changes. FEMS Microbiol Ecol 74:93–102CrossRefGoogle Scholar
  7. Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  8. Harvey J, Gilmour A (1985) Application of current methods for isolation and identification of staphylococci in raw bovine milk. J Appl Bacteriol 59:207–221PubMedCrossRefGoogle Scholar
  9. Jones JG (1977) The effect of environmental factors on estimated viable and total populations of planktonic bacteria in lakes and experimental enclosures. Freshwater Bioi 7:61–97CrossRefGoogle Scholar
  10. Lundgren B (1981) Fluorescein diacetate as a stain of metabolically active bacteria in soil. Oikos 36:17–22CrossRefGoogle Scholar
  11. Matin A (1990) Molecular analysis of starvation stress in Escherichia coli. FEMS Microbiol Ecol 74:185–196CrossRefGoogle Scholar
  12. Matsuyama T (1984) Staining of bacteria with Rhodamine 123. FEMS Microbiol Lett 21:153–157CrossRefGoogle Scholar
  13. Mills AL, Bell PE (1986) Determination of individual organisms and their activities in situ. In: Tate RL (cd) Microbial autoecology: a method for environmental studies. Wiley, New York, pp 27–60Google Scholar
  14. Page S, Burns RG (1991) Flow cytometry as a means of enumerating bacteria introduced into soil. Soil Bioi Biochem 23:1025–1028CrossRefGoogle Scholar
  15. Pickup RW (1991) Development of methods for the detection of specific bacteria in the environment. J Gen Microbiol 137:1009–1019CrossRefGoogle Scholar
  16. Postma J, Altemuller HJ (1990) Bacteria in thin soil sections stained with the fluorescent brightener calcofluor white M2R. Soil Bioi Biochem 22:89–96CrossRefGoogle Scholar
  17. Roszak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365–379PubMedGoogle Scholar
  18. Saylor GS, Layton AC (1990) Environmental application of nucleic acid hybridization. Annu Rev Microbiol 44:625–648CrossRefGoogle Scholar
  19. Shapiro HM (1988) Practical flow cytometry, 2nd edn. Alan R Liss, New YorkGoogle Scholar
  20. Stahl DA, Flesher B, Mansfield HR, Montgomery L (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54: 1079–1084PubMedGoogle Scholar
  21. Steen HB, Skarstad K, Boye E (1990) DNA measurements of bacteria. Meth Cell Bioi 33:519–526CrossRefGoogle Scholar
  22. Steen HB, Boye E, Skarstad K, Bloum B, Godal T, Mustafa S (1982) Applications of flow cytometry on bacteria: cell cycle kinetics, drug effects and quantitation of antibody-binding. Cytometry 2:249–257PubMedCrossRefGoogle Scholar
  23. Zarda B, Amman R, Wallner G, Schleifer K-H (1991) Identification of single bacterial cells using digoxigenin-labelled rRNA-targeted oligonucleotides. J Gen Microbiol 137:2823–2830PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag London 1993

Authors and Affiliations

  • Clive Edwards
  • Julian P. Diaper
  • Jonathan Porter
  • Roger Pickup

There are no affiliations available

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