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[DESCRIPTION]
<strong>Purpose: </strong>This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method. Briefly, fluorescently-stained samples are wetmounted directly onto slides for epifluorescence microscopy after an optional chemical flocculation concentration step used for samples with anticipated virus concentrations of &lt;5&times;10<sup>7</sup> viruses mL<sup>-1</sup> (samples with &gt;5&times;10<sup>7</sup>&nbsp;viruses mL<sup>-1 </sup>do not require this concentration step prior to analysis). Virus concentration in the wet-mounted sample is determined from the ratio of viruses to microsphere beads, which are added at a known concentration. This wet-mount method for enumerating viruses is significantly less expensive than the filter-mount method (i.e., the cost of microsphere beads per sample is ~500-fold lower than the cost of one filter per sample), and is appropriate for rapid, precise and accurate enumeration of aquatic viruses over a wide range of viral concentrations encountered in field and laboratory samples. The only limitation of this method is that samples with virus concentrations &le;1&times;10<sup>6</sup> viruses mL<sup>-1</sup> cannot be enumerated, as the abundance of viruses is too low for efficient enumeration.<br /><br /><br /><img src="https://s3.amazonaws.com/pr-journal/dfc2w6.jpg" alt="" /><br />
<p>Figure 1. Overview of the wet-mount method for enumeration of aquatic viruses.</p>

[GUIDELINES]
<strong>Optional Viral Concentration Method:</strong><br />Samples with &lt;5&times;107&nbsp;viruses mL-1 must be concentrated using this iron chloride&nbsp;flocculation method adapted from John et al. [1] prior to enumeration of viruses with the&nbsp;wet-mount method. The steps for this procedure are displayed in Figure 1A.<br /><br /><strong>Materials Required:</strong><br />- 1.5 mL microcentrifuge tubes<br />- microcentrifuge<br />- vortexer<br />- phosphate buffered saline (PBS)<br />- iron chloride (FeCl3&bull;6H2O)<br />- ultrapure water<br />- magnesium EDTA (Mg2EDTA)<br />- ascorbic acid<br />- sodium hydroxide (NaOH)<br /><br /> <strong>Reagent Preparation:</strong> <br /> <a href="https://www.protocols.io/view/Iron-Chloride-Solution-c8wzxd" target="_blank">Iron Chloride Solution</a><br /><a href="https://www.protocols.io/view/Ascorbic-EDTA-Buffer-c8xzxm" target="_blank">Ascorbic-EDTA Buffer&nbsp;</a> <br /><br /><strong>Virus Counting Procedure<br /> </strong>This part of the protocol starts with either 10 &micro;L of unconcentrated sample (if the anticipated virus concentration is &gt;5&times;107 viruses mL-1 ) or 10 &micro;L of concentrated, resuspended sample (see above). The steps for this procedure are displayed in Figure 1B.<strong> <br /><br /> Materials Required: <br /></strong>- epifluorescence microscope (1000X magnification and ~495 nm excitation) <br />- vortexer <br />- 1.5 mL microcentrifuge tubes <br />- isopropanol <br />- glass slides (25x75 mm) <br />&nbsp;- cover slips (24x60 mm) <br />- microsphere silica beads (Bangs Laboratories, 2.34 &micro;m diameter, conc. 7.845x109 beads/mL, Cat. # SS04N/4186, Inv. # L060320A)<br /> - glycerol <br />- phosphate buffered saline (PBS) <br />- SYBR Gold (Invitrogen) <br />- Kimwipes <br />- ascorbic acid (if analyzing unconcentrated samples) <br /><br /> <strong>Reagent Preparation:</strong><br /><a href="https://www.protocols.io/view/SYBR-Gold-Working-Stock-c82zyd" target="_blank">SYBR Gold Working Stock</a><br /><a href="https://www.protocols.io/view/Working-Bead-Solution-c84zyv" target="_blank">Working Bead Solution</a><br /><a href="https://www.protocols.io/view/Ascorbic-Acid-Antifade-Solution-c87zzm" target="_blank">Ascorbic Acid Antifade Solution<br /><br /></a><strong>Related References</strong> <br />1. John SG, Mendez CB, Deng L, Poulos B, Kauffman AKM, Kern S, Brum J, Polz MF, Boyle EA, Sullivan MB (2011) A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environmental Microbiology Reports, 3:195&ndash;202. doi:10.1111/j.1758-2229.2010.00208.x<br /><br /><strong> <br /></strong>

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