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[DESCRIPTION]
This protocol describes a technique to recover viruses from natural waters using iron- based flocculation and large-pore-size filtration, followed by resuspension of virus- containing precipitates in a pH 6.5 buffer. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (&gt; 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered if using oxalic acid in the resuspension buffer. Particles lose infectivity if resuspended with ascorbic acid in the buffer.

[GUIDELINES]
<strong>PCR inhibition</strong>: Note, that due to the likely issue with the EDTA in the resuspension buffer interacting with the MgCl in a PCR reaction, direct PCR is ineffective. To circumvent this, extract DNA using Wizard columns from Promega (see <a href="https://www.protocols.io/view/Cesium-Chloride-DNA-Extraction-of-Viruses-using-Wi-c26yhd" target="_blank">Tucson Marine Phage Lab&rsquo;s DNA Extraction of Viruses Using Wizard Columns protocol</a>). <br /><br /><strong>Materials:</strong><br />- Seawater<br />- Tris-base (FW=121.14)<br />- Na2-EDTA dihydrate (FW=372.24)<br />- MgCl2 hexahydrate (FW=203.3)<br />- Ascorbic acid (FW=176.12) and/or Oxalic acid (FW=126.07)<br />- NaOH, 5N<br />- HCl<br />- FeCl<sub>3</sub>&middot;6H<sub>2</sub>O (FW=270.3)<br />- MilliQ water<br />- pH paper<br />- Whatman glass fiber filter GF/A (cat. 1820-150; 1.6&mu;m retention; 150mm<br />diameter)<br />- Millipore Express PLUS filter (cat. GPWP14250; 142mm, 0.22&mu;m)<br />- Pall Supor-800 filter (cat. 60114; 142mm, 0.8um)<br />- GE Polycarbonate Membrane filter (cat. K10CP14220; 142mm, 1.0&mu;m),<br />Available through Midland Scientific Inc. (cat. MAINE 1216611)<br />- pH meter<br />- 142mm filtering towers (Need 2 for efficiency)<br />- Peristaltic pump with a pressure gauge<br /><br /><strong>Notes:</strong>&nbsp;<br />Alternative Pre-filtering Method: An alternative method for preparing seawater is to pre-filter the seawater through a Whatman GF/D glass fiber filter (cat. 1823-150; 2.7&mu;m retention; 142mm diameter) to remove large particles. The filtrate is filtered again using a Millipore Steripak GP10 or GP20 (cat. SPGPM10RJ or SPGPM20RJ; 0.22&mu;m). The GP 10 works for &lt;10L volumes and GP20 works for &lt;20L volumes. Larger capsule filters will easily filter 100-200 liters: Pall Acropak 200, 500, 1000 and 1500. The cellular fraction is retained on the filter while the virus fraction is in the filtrate. <br /><br />Filtration should be done at a maximum pressure of 15 psi. A peristaltic pump applies the pressure and a stainless steel Millipore filter rig (PN YY3014236) holds filters. For less expense, use an acrylic or polycarbonate 142 mm filter holder (Geotech Environmental Equipment, acrylic or PC) and pressurize carboy with an air pump. Most lab vacuum pumps have an air-pressure outlet on the other side that can easily be adjusted to provide 15 psi pressure. Nalgene sells caps for their 20L carboys with fittings in the top that can be used for pressurization. <br /><br /><strong>FeCl<sub>3</sub> Formulation</strong>: The solution of ferric chloride hexahydrate is calculated based on the amount of iron, not on the amount of the salt. The stock solution has 10g Fe per liter. For precipitation, the final optimal concentration of Fe in seawater (SW) is 1mg Fe per liter SW which is equal to 2.9mg FeCl<sub>3</sub> per liter SW or to 4.83mg FeCl<sub>3</sub>&middot;6H<sub>2</sub>O per liter SW.<br /> <br /><strong>Equipment Care</strong>: Carboys and collection bottles should be washed with 1N HCl and rinsed with MilliQ water before use. Checking tubing before use and replace if worn. Always rinse stainless steel filter rigs with MilliQ water after use and air dry to prevent corrosion.&nbsp;<br /><br /><strong>Original Author(s)</strong>: Seth John, Bonnie Poulos<br /> <strong>Revision Author(s)</strong>: Bonnie Poulos, Christine Schirmer&nbsp;<br /><br /><strong>Citation</strong>: John, S.G., Mendez, C.B., Deng, L., Poulos, B., Kauffman, A.K.M., Kern, S., Brum, J., Polz, M.F., Boyle, E.A., &amp; Sullivan, M.B. (2011). A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environ Microbiol Rep. 3(2), 195-202. doi:10.1111/j.1758-2229.2010.00208.x.

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