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[DESCRIPTION]
This is a protocol from:&nbsp;<br /><br />Suttle, C. A. and J. A. Fuhrman. 2010. Chapter 15: Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy.&nbsp;Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_145.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors:</strong> Curtis A. Suttle<sup>1</sup> and Jed A. Fuhrman<sup>2</sup> <br /><sup>1</sup>Departments of Earth &amp; Ocean Sciences, Microbiology &amp; Immunology, and Botany, University of British Columbia, Vancouver, BC, Canada <br /><sup>2</sup>Marine Environmental Biology Section, University of Southern California, Los Angeles CA, USA<br /><br /><strong>MATERIALS<br /><br />Equipment</strong><br />•Epifluorescence microscope equipped with the following: <br />     •≥100 W Hg-vapor or 150 W xenon lamp (xenon lamps are richer in the blue spectrum and have longer lifetimes and lower lifetime cost) <br />     •100x fluorescence objective (phase contrast objectives typically reduce brightness)<br />     •blue excitation filter (wide bandpass preferred for maximum brightness, e.g., 450-480 or 460-500 nm; excitation peaks 497, 495, and 491 nm for SYBR Green 1, SYBR Gold, and Yo-Pro-1, respectively) <br />     •dichroic mirror (beam splitter), typically 500 or 510 nm <br />     •long-pass sharp cutoff filter (typically 515 nm; emission peaks 520, 537, and 509 nm for SYBR Green 1, SYBR Gold, and Yo-Pro-1, respectively; a long-pass filter allows maximum brightness) <br />     •ocular reticule divided into 100 grid squares <br />     •stage micrometer <br />•Filtration unit, to hold 25-mm-diameter filters<br />•Vacuum pump <br />•Vacuum flask<br />•Pipettes suitable for dispensing 1 μL to 2 mL <br />•Event counter <br />•Filter forceps <br />•Sonicating bath (sediment samples only) <br />•Squeeze bottle (or similar) containing 0.2-μm filtered MilliQ water that will be used to wet the underlay filter. <br /><br /><strong>Reagents and solutions</strong><br />•(SYBR only) SYBR Green or SYBR Gold nucleic-acid gel stain, 10,000x concentrate in anhydrous DMSO (Invitrogen) <br />•(Yo-Pro only) Yo-Pro-1, 1 mM stock solution in a 1:4 solution of dimethyl sulfoxide and water (Invitrogen) <br />•(SYBR only) Antifade solution: p-phenylenediamine dihydrochloride or 1,4-phenylenediamine dihydrochloride (se Sigma P-1519 and not P-6001 [Patel et al. 2007] and store in a tightly capped container away from light) <br />•Spectrophotometric-grade glycerol <br />•Phosphate-buffered saline (PBS): 0.05 M Na<sub>2</sub>HPO<sub>4</sub>, 0.85% NaCl (wt/vol), pH 7.5)<br />•(Sediment samples only) Pyrophosphate (10 mM) <br />•0.02-μm filter autoclaved MilliQ H2O <br />•Tris EDTA (TE) buffer, pH 8 (for acidic samples)<br />•(Yo-Pro only) Aqueous NaCl solution (0.3% wt/vol) <br />•(SYBR only) 25% EM-grade glutaraldehyde, kept at 4°C <br />•Ethanol <br />•DF- or FF-grade immersion oil (refractive index 1/4, 1.516; Olympus). <br /><br /><em>Cautions:</em> Nucleic acid stains, phenylenediamine dihydrochloride and aldehyde-based fixatives are toxic; great care should be taken to avoid inhalation or contact with skin. <br /><br /><strong>Disposable supplies</strong><br />•Anodisc Al<sub>2</sub>O<sub>3</sub> filters, 0.02-μm pore size, 25 mm diameter, with support ring (Whatman) <br />Note: At the time of writing, GE Healthcare, which recently purchased Whatman, is considering discontinuing production of the Anodisc membrane. Currently, there are no other membranes known that are suitable for EFM counts of virus particles. For polycarbonate filters, the background fluorescence is too high (even if stained black) and the porosity is too low to filter an adequate volume of water. <br />•Nitrocellulose membrane filters, 0.45-μm pore size, 25 mm diameter <br />•Precleaned glass microscope slides, 25 x 75 mm <br />•Glass coverslips (25 x 25 mm) of proper thickness (each microscope objective has an optimal coverslip thickness--the cover glass is part of the optics; for example, an Olympus infinity-corrected 100x UVPlanApo is imprinted "∞/0.17," which indicates a 0.17-mm-thick cover glass, also known in the US as #1 1/2) <br />•2.0 mL sterile polypropylene microcentrifuge tubes <br />•2.0 mL screw-cap cryovials <br />•10 μL, 200 μL, and 1 mL sterilized pipette tips <br />•Polypropylene centrifuge tubes (15 or 50 mL) <br />•Petri plates <br />•Kimwipes (Kimberly-Clark) or other lint-free paper wipes <br />•9-cm-diameter paper filters (Whatman #1)<br />•Additional supplies for long-term storage of samples for SYBR staining: <br />     •Liquid nitrogen <br />     •Nylon stockings <br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/8pc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/8pc2xe.png" data-ofn="enumeration fig 1a.png" /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/8qc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/8qc2xe.png" data-ofn="enumeration fig1b.png" /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/8rc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/8rc2xe.png" data-ofn="enumeration fig 1c.png" /><br /><strong>Fig. 1.</strong> Epifluorescence micrographs of natural water samples. Viruses are indicated by solid arrows and bacteria by broken arrows. <br />(A), Yo-Pro 1–stained slide of a water sample from the Arctic Ocean (courtesy of J. P. Payet). <br />(B), SYBR Green I–stained water sample from the coastal waters of Southern California. The large object in the center is a pennate diatom. <br />(C) SYBR Gold–stained sample of a water sample from the Chesapeake Bay (courtesy of F. Chen and K. Wang). The slides emphasize the differences that occur between samples and different staining protocols. <br /><br /><br /><br /><br /><br />

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