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Optimization of procedures for counting viruses by flow cytometry.

Corina P D Brussaard1

  • 1Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, NL-1790 AB Den Burg, The Netherlands. corina.brussard@nioz.nl

Applied and Environmental Microbiology
|March 10, 2004
PubMed
Summary

Standardizing flow cytometry methods for aquatic virus enumeration is crucial. Optimal protocols involve glutaraldehyde fixation, SYBR Green I staining, and -80°C storage for accurate virus detection.

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Area of Science:

  • Environmental Microbiology
  • Aquatic Virology

Background:

  • Flow cytometry enables virus identification and enumeration in aquatic systems.
  • Inconsistent methodologies hinder reliable virus quantification.

Purpose of the Study:

  • To evaluate sample preparation techniques for optimizing flow cytometric virus counts.
  • To establish consistent methods for aquatic virus enumeration.

Main Methods:

  • Tested various dyes, fixatives, dilution media, incubation conditions, and storage temperatures.
  • Examined diverse viruses including bacteriophages and marine virus communities.
  • Optimized fixation, staining, dilution, and storage protocols.

Main Results:

  • Optimal conditions: 0.5% glutaraldehyde fixation (15-30 min), liquid nitrogen freezing, -80°C storage.

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  • Recommended staining: SYBR Green I (5 x 10^-5 dilution), 10 min incubation at 80°C.
  • -80°C storage is critical to prevent significant loss of virus abundance over time.

    • Conclusions:

    • Standardized protocols enhance accuracy and consistency in aquatic virus enumeration.
    • Proper sample preparation and storage are vital for reliable flow cytometry results.
    • Established methods improve the understanding of aquatic viral dynamics.