Protocol abstract


Nature Protocols 2, 269 - 276 (2007)
Published online: 1 March 2007 | Corrected online: 6 September 2007 | doi:10.1038/nprot.2007.6

Subject Categories: Imaging | Model organisms

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

Anand Patel1, Rachel T Noble2, Joshua A Steele1, Michael S Schwalbach3, Ian Hewson4 & Jed A Fuhrman1


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-mum 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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  1. Marine Environmental Biology Section, University of Southern California, 3616 Trousdale Parkway, AHF-107, Los Angeles, California 90089, USA.
  2. Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, North Carolina 28557, USA.
  3. Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, Oregon 97331, USA.
  4. Department of Ocean Sciences, University of California Santa Cruz, 1156 High Street EMS A418, Santa Cruz, California 95060 USA.

Correspondence to: Anand Patel1 e-mail: anandp@usc.edu