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[DESCRIPTION]
The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously. The method is from:<br />Patel A, Noble RT, Steele JA, Schwalbach MS, Hewson I, Fuhrman JA.<a href="http://www.nature.com/nprot/journal/v2/n2/full/nprot.2007.6.html" target="_blank">Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I</a>. <em><span class="jrnl" title="Nature protocols">Nat Protoc</span>.</em> 2007;2(2):269-76.<br /><br />Please see the full <a href="http://www.nature.com/nprot/journal/v2/n2/full/nprot.2007.6.html" target="_blank">manuscript</a> for more details.

[GUIDELINES]
<strong>Overview:</strong><br /><br />Steps 1 - 8: Sample Collection<br />Steps 9 - 45: Slide preparation<br />Steps 46 - 55:&nbsp;Virus enumeration procedure<br /><br /><strong>Materials (Reagents):<br /><br /></strong>* &nbsp; SYBR Green I nucleic-acid gel stain, 10,000 X concentrate in anhydrous DMSO (500 &mu;l; Molecular Probes-Invitrogen, cat. no. S-7563)<br /><br /><em><span class="caution">Caution:&nbsp;</span>No human mutagenicity or toxicity data are available; however, as the chemical specifically binds to nucleic acids and is supplied in DMSO, it should be treated as a potentially mutagenic substance</em><br /><br /><em>* &nbsp;p</em>-phenylenediamine dihydrochloride or 1,4-phenylenediamine dihydrochloride (Sigma, cat. no. P-1519)<br /><span class="critical">Critical </span>Do not use Sigma, cat. no. P-6001<br /><br /><em><span class="caution">Caution </span>Toxic, avoid inhalation and contact with skin and eyes</em><br /><br />* &nbsp;Glycerol (Sigma)<br /><br />* &nbsp;PBS: 0.05 M Na2HPO4, 0.85% (wt/vol) NaCl (pH 7.5)<br /><br />* &nbsp;0.02 &mu;m filter-autoclaved MilliQ H2O<br /><br />* &nbsp;0.02 &mu;m filtered seawater, formalin preserved, 2% (vol/vol) final concentration<br /><br />* &nbsp;0.02 &mu;m filtered formalin (37&ndash;39% (wt/vol) saturated formaldehyde solution)<br /><span class="caution"><br /><em>Caution </em></span><em>Toxic, avoid inhalation, ingestion or contact with skin, eyes or mucous membranes; dispense inside chemical fume hood if possible, or in an open-air environment with appropriate safety attire and eye protecti</em>on<br /><br />* &nbsp;Ethanol (200 proof)<br /><br />* &nbsp;Non-fluorescent immersion oil for microscopy (refractive index (nd) = 1.516; Olympus)<strong><br /><br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;(Equipment):<br /><br /></strong>* &nbsp;Epifluorescence microscope with filters for blue excitation and green emission with 100 &times; objective<br /><br />* &nbsp;Microscope stage micrometer<br /><br />* &nbsp;Glass 25 mm filter holder, 15 ml filter funnel with fritted glass base and clamp (Millipore, cat. no. XX10 025 00)<br /><br />* &nbsp;0.02 &mu;m pore size, 25 mm diameter Anodisc Al203 filters (Whatman, cat. no. 6809-6002)<br /><br />* &nbsp;0.8 &mu;m pore size, 25 mm diameter AA mixed-ester membrane filter (Millipore)<br /><br />* &nbsp;Filter forceps (Millipore)<br /><br />* &nbsp;Glass microscope slides, 25 &times; 75 mm, standard thickness, frosted at one end (VWR)<br /><br />* &nbsp;Glass coverslips (25 mm &times; 25 mm, squares; VWR or Corning)<br /><br />* &nbsp;2.0 ml clean and sterilized micro-centrifuge tubes (VWR or Eppendorf)<br /><br />* &nbsp;Pipettes suitable for 2&ndash;10 ml volumes (Rainin or Eppendorf)<br /><br />* &nbsp;5 ml and 10 ml clean, sterilized tips (VWR)<br /><br />* &nbsp;50 ml conical centrifuge tubes (polypropylene (PP); Falcon or VWR)<br /><br />* &nbsp;Glass Erlenmeyer filter flask or multiple filter holder<br /><br />* &nbsp;Polystyrene Petri dishes (VWR or Falcon)<br /><br />* &nbsp;Kimwipes (Kimberly-Clark)<br /><br />* &nbsp;Vacuum source, suitable for at least 25 cm Hg vacuum (e.g., diaphragm pump; Gast, cat. no. DOA-P104-AA)<br /><br />* &nbsp;Low-temperature dry-heat block with anodized aluminum heat block (VWR)<br /><br />*1 l filter flask (polypropylene secondary filtrate waste reservoir or 'trap'; Cole-Parmer)<strong><br /><br />Equipment Setup:<br /><br />Epifluorescence microscope&nbsp;</strong>We use a BX60 epifluorescence microscope (Olympus) with a 100 &times; UPlanApochromat objective, 150 W xenon lamp (Optiquip), UMWB blue excitation filter cube (wide band, excitation band pass (BP) 450&ndash;480 nm; dichroic mirror (DM) 500 nm; emission barrier filter (BA) 515) and 10 &times; focusing eyepiece with a 10 &times; 10 square grid reticle.<br /><br /><strong>Glass coverslips</strong> Select the correct thickness to optically match the microscope objective you are using (e.g., our Olympus system uses <span class="mb">#</span>1&frac12; or 0.17-mm thick cover glasses). The correct thickness is usually marked on the oil-immersion objective after a slash, or you can ask the lens manufacturer.<br /><br /><strong>Filtration apparatus</strong> Connect the filter flask or filter manifold to the secondary 1 l filter flask ('trap'/waste reservoir) with appropriate diameter tubing. Connect the waste reservoir filter flask to the vacuum pump with tubing and hose clamps. At the vacuum pump end, we use a vacuum pump-protection filter to prevent liquids entering the vacuum pump, although this is optional. To set up the filter apparatus, first place the glass 25 mm filter holder in its silicone stopper (usually provided with the glass filter holder from Millipore) into the filter flask or a port in the multi-filter holder manifold. Once these are firmly in place, test whether you are getting good suction by turning on the vacuum pump and pipetting H<sub>2</sub>O onto the fritted glass filter base. Use a vacuum of &le; 25 cm Hg (~1/3 atm).<br /><br /><strong>Glass 25 mm filter holder</strong> Note that the inside diameter of the filter funnel and the diameter of the porous fritted area of the glass base should be close to (or slightly larger than) the diameter of the porous part of the Anodisc filter (these filters have a non-permeable plastic outer ring); if the funnel or base is smaller, this might restrict the actual filtered area, which will need to be taken into account when calculating virus abundance (see Step 55).<strong><br /><br />Reagent Preparation:</strong><br /><br /><a href="https://www.protocols.io/view/10-wt-vol-p-phenylenediamine-djh4j5" target="_blank">10% (wt/vol) p-phenylenediamine</a><br /><br /><a href="https://www.protocols.io/view/50-glycerol-50-PBS-1-1-vol-vol-solution-dji4kd" target="_blank">50% glycerol/50% PBS (1:1) (vol/vol) solution</a><br /><br /><a href="https://www.protocols.io/view/1-400-SYBR-Green-I-solution-djj4km" target="_blank">1:400 SYBR Green I solution</a><br /><br /><a href="https://www.protocols.io/view/0-1-vol-vol-p-phenylenediamine-anti-fade-mounting-djv4n5" target="_blank">0.1% (vol/vol) p-phenylenediamine anti-fade mounting medium&nbsp;<br /><br /><br /><br /><br /></a>

[STEPS]
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