Optimization of Procedures for Counting Viruses by Flow Cytometry

TLDR

The development of sensitive nucleic‑acid stains combined with flow cytometry has enabled virus identification and enumeration in aquatic systems, yet methods have been inconsistent. This study evaluates a broad range of sample preparations to optimize virus counts and promote methodological consistency. The authors tested dyes, fixatives, dilution media, additives, temperature, incubation time, dilution factor, and storage, finding that fixation with 0.5 % glutaraldehyde for 15–30 min, liquid‑nitrogen freezing, storage at –80 °C, thawing, Tris‑EDTA dilution, SYBR Green I staining, 10‑min 80 °C incubation, and 5‑min cooling produced optimal counts. Low storage temperatures (at least –80 °C) are essential, as higher temperatures caused 50–80 % decreases in total virus abundance over time.

Abstract

ABSTRACT The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10 −5 dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.