Resolution of Viable and Membrane‐Compromised Free Bacteria in Aquatic Environments by Flow Cytometry

Gérald Grégori1, Michel Denis2, Sergio Seorbati3, Sandra Citterio3

1 Purdue University Cytometry Laboratories, West Lafayette, Indiana, 2 Laboratorie d'Océanographie et de Biogéochimie, Marseille Cedex, France, 3 Università di Milano‐Bicocca, Milan, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.15
DOI:  10.1002/0471142956.cy1115s23
Online Posting Date:  February, 2003
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In aquatic environments, free heterotrophic bacteria play an extremely important role because of their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic‐acid double‐staining protocol (NADS) for flow cytometry that can distinguish the fractions of viable, damaged, or membrane‐compromised cells within the free‐bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains, membrane‐permeant SYBR Green and membrane‐impermeant PI. The efficiency of the double staining is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI will identify cells with a compromised membrane, partial quenching will indicate cells with a slightly damaged membrane, and lack of quenching will characterize cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • Natural fresh‐ or seawater samples
  • SYBR Green I (fresh water) or II (seawater) working solution (see recipe)
  • Cells killed by paraformaldehyde fixation, heat, or ozone treatment
  • Freshly harvested cells: freshly harvested natural sample with >95% viability or sample from a bacterial culture in exponential growth phase
  • 1 mg/ml propidium iodide (PI; Molecular Probes): store ≤1 month at 4°C in the dark
  • Fresh water or seawater: filter through a 0.2‐µm filter
  • 10% bleach: dilute standard bleach (5% sodium hypochlorite) 1:10 in H 2O
  • 70% ethanol
  • 100‐µm filter
  • Flow cytometer:
  •  488‐nm argon laser or arc lamp
  •  Filters for collection of 525 ± 15‐nm (green) fluorescence and >620‐nm (red) fluorescence
  •  Sheath fluid: distilled water passed through a 0.2‐µm filter
  • 12 × 75–mm tubes, sterile
NOTE: All water, including fresh water or seawater used to dilute samples, and that used to make solutions, should be passed through a 0.2‐µm filter prior to use.
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Literature Cited

Literature Cited
   Barbesti, S., Citterio, S., Labra, M., Baroni, M.D., Neri, M.G., and Sgorbati, S. 2000. Two and three color fluorescence flow cytometric analysis of immunoidentified viable bacteria. Cytometry 40:214‐218.
   Gasol, J.M. and Del Giorgio, P.A. 2000. Using flow cytometry for counting natural planktonic bacteria and understand the structure of planktonic bacterial communities. Scientia Marina 64:197‐224.
   Gasol, J.M., Zweifel, U.L., Peters, F., Fuhrman, J.A., and Hagström, A. 1999. Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol. 65:4475‐4483.
   Grégori, G., Citterio, S., Ghiani, A., Labra, M., Sgorbati, S., Brown, S., and Denis, M. 2001. Resolution of viable and membrane‐compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining. Appl. Environ. Microbiol. 67:4662‐4670.
   Haugland, R.P. 1998. Handbook of fluorescent probes and research chemicals. Molecular Probes. Eugene, Ore.
   Hobbie, J.E., Daley, R.J., and Jasper, S. 1977. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol 33:1225‐1228.
   Jones, K.H. and Senft, J.A. 1985. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate‐ propidium iodide. J. Histochem. Cytochem. 33:77‐79.
   Joux, F. and Lebaron, P. 2000. Use of fluorescent probes to assess physiological functions of bacteria at single‐cell level. Microbes and Infection 2:1523‐1535.
   Kirchman, D.L., Suzuki, Y., Garside, C., and Ducklow, H.W. 1991. High turnover rates of dissolved organic carbon during a spring phytoplankton bloom. Nature 352:612‐614.
   Kogure, K., Simidu, U., and Taga, N. 1979. A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25:415‐420.
   Lebaron, P., Parthuisot, N., and Catala, P. 1998. Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64:1725‐1730.
   Lloyd, D. and Hayes, A.J. 1995. Vigor, vitality and viability of microorganisms. FEMS Microbiol. Lett. 133:1‐7.
   López‐Amorós, R., Castel, S., Comas‐Riu, J., and Vives‐Rego, J. 1997. Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using Rhodamine 123, DiBAC4(3), propidium iodide and CTC. Cytometry 29:298‐305.
   McFeters, G.A., Yu, F.P., Pyle, B.H., and Stewart, P.S. 1995. Physiological assessment of bacteria using fluorochromes. J. Microbiol. Meth. 21:1‐13.
   Mitchell, P. 1961. Coupling of phosphorylation to electron and hydrogen transfer by chemi‐osmotic type mechanism. Nature 191:144‐148.
   Nebe‐von Caron, G. and Badley, R.A. 1995. Viability assessment of bacteria in mixed populations using flow cytometry. J. Microsc. 179:55‐66.
   Nicholls, D.G. 1982. Bioenergetics. An introduction to the chemiosmotic theory. Academic Press, London.
   Pomeroy, L.R. 1984. Significance of microorganisms in carbon and energy flow in marine ecosystems In Current Perspectives in Microbial Ecology (M.J. Klug and C.A. Reddy, eds.) pp. 405‐411. American Society for Microbiology, Washington, D.C.
   Porter, J., Diaper, J., Edwards, C., and Pickup, R. 1995. Direct measurements of natural planktonic bacterial community viability by flow cytometry. Appl. Environ. Microbiol. 61:2783‐2786.
   Porter, J., Deere, D., Pickup, R., and Edwards, C. 1996. Fluorescent probes and flow cytometry new insights into environmental bacteriology. Cytometry 23:91‐96.
   Sgorbati, S., Barbesti, S., Citterio, S., Bestetti, G., and De Vecchi, R. 1996. Characterization of number, DNA content, viability and cell size of bacteria from natural environments using DAPI PI dual staining and flow cytometry. Minerva Biotec. 8:9‐15.
   Trousselier, M., Courties, C., and Vaquer, A. 1993. Recent applications of flow cytometry in aquatic microbial ecology. Biol.Cell 78:111‐121.
   Williams, S.C., Hong, Y., Danavall, D.C.A., Howard‐Jones, M.H., Gibson, D., Frisher, M.E., and Verity, P.G. 1998. Distinguishing between living and nonliving bacteria evolution of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples. J. Microbiol. Meth. 23:225‐236.
   Zimmerman, R. and Meyer‐Reil, L. 1974. A new method for fluorescence staining of bacterial populations on membrane filters. Kiel. Meeresforsch. 30:24‐27.
   Zweifel, U.L. and Hagström, A. 1995. Total counts of marine bacteria include a large fraction of non‐nucleoid‐containing bacteria (ghosts). Appl. Environ. Microbiol. 61:2180‐2185.
Key Reference
   Barbesti, et al. 2000. See above.
  The original presentation of the NADS protocol.
   Grégori et al. 2001. See above.
  This article describes the adaptation of the NADS protocol described by Barbesti et al. () to natural fresh and marine water samples.
Internet Resources
  This Web site displays technical bulletins on Molecular Probes fluorochromes with useful storage and handling information and suggestions for their use.
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