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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Abstract

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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Materials

Basic Protocol 1:

  Materials
  • 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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Figures

Videos

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.
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   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.
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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
  http://www.probes.com/servlets/pis
  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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