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Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Nature Protocols volume 2, pages 269–276 (2007)Cite this article

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Abstract

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20–200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes–Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-μm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.

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Figure 1: Epifluorescence-microscopy image of a seawater sample filtered onto a Whatman 0.02 μm Anodisc filter stained with SYBR Green I.

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  • 06 September 2007

    In the version of this article initially published, on p. 270, under “Equipment,” “Anodisc Al203 filters” should have read “Anodisc Al203 filters”. On p. 274, the second sentence of Step 21, which ended with “around the entire filter”, should have ended with “around the entire filter; i.e., if 3 boxes resulted in a count of 34 for the first field, then count 3 boxes in each of 9 other areas of the filter (do not count different numbers of boxes in different fields of one filter.” The third sentence ended with “viruses counted”; it should have ended with “viruses counted; therefore some fields may yield counts that are <30. But if the total count for a filter is <200, count extra fields.” In Step 22, the equation (X × 100)/(n × RSF)/V should have been RSF x X x (100/n)/V, and “counted on the microscope eyepiece 10 × 10 reticle grid” should have been “counted within the 10 x 10 reticle grid”. These errors have been corrected in all versions of the article.

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Authors and Affiliations

  1. Marine Environmental Biology Section, University of Southern California, 3616 Trousdale Parkway, AHF-107, Los Angeles, 90089, California, USA

    Anand Patel, Joshua A Steele & Jed A Fuhrman

  2. Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, 28557, North Carolina, USA

    Rachel T Noble

  3. Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, 97331, Oregon, USA

    Michael S Schwalbach

  4. Department of Ocean Sciences, University of California Santa Cruz, 1156 High Street EMS A418, Santa Cruz, 95060, California, USA

    Ian Hewson

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  6. Jed A Fuhrman
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Correspondence to Anand Patel.

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Patel, A., Noble, R., Steele, J. et al. Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nat Protoc 2, 269–276 (2007). https://doi.org/10.1038/nprot.2007.6

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