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[DESCRIPTION]
This protocol outlines and evaluates the filtration steps needed for the preparation of samples (viral concentrates) containing a density of viral particles concentrated from large volumes of natural water samples. (<a href="http://www.aslo.org/books/mave/MAVE_110.pdf" target="_blank">Fig. 2A and B</a>)

[BEFORE_START]
All filters should be cleaned and rinsed according to manufacturer’s recommendations before use in any application during the concentration procedures.

[GUIDELINES]
I. List of materials <br />   A. Large-scale concentration. <br />        i. Tubing: silicone, various sizes <br />        ii. Tubing: PharMed for pump heads only <br />        iii. Connectors to tubing and filters <br />        iv. Diaphragm pump (Jabsco Industrial Diaphragm pump #31801, 12 L per min capacity) <br />        v. Two 50-L plastic carboys vi. 25-L carboy<br />        vii. 25-µm wound polypropylene sediment filter (pool filter) and housing <br />        viii. Large peristaltic pump with 2 pump heads <br />        ix. 0.22-µm TFF filter x. 30- to 50-kD TFF filter <br />        xi. 0.22-µm syringe filters <br />        xii. 60-mL syringes <br />        xiii. 50-mL conical tubes<br />     B. Small-scale concentration. <br />        i. Tubing: silicone, various sizes <br />        ii. Tubing: PharMed for pump heads only <br />        iii. Connectors to tubing and filters <br />        iv. Small, reversible peristaltic pump with 1 pump head <br />        v. Two 2-L polycarbonate bottles <br />        vi. 500-mL polycarbonate bottle <br />        vii. 30- to 50-kD compact spiral filter <br />    C. Postconcentration. <br />        i. Ultracentrifuge <br />        ii. Swing bucket rotor <br />        iii. Polyallomer tubes <br />        iv. Balance <br />        v. Waste container <br />        vi. Final collection tubes of needed size <br />        vii. 200-µL pipettman and tips<br />II. List of reagents <br />    A. Sterile 60% glycerol<br /><br /><strong>Concentration of virioplankton (Small-Scale)</strong><br /><br />The steps involved in the concentration of viruses from smaller water samples (i.e., &lt;2 L) are similar to those for large samples. However, the TFF filters used for concentration of viruses from small-scale samples are usually 10-fold smaller in filter area. The smaller size of these filters, and the tubing connected to them, results in a coordinately smaller minimum hold-up volume. In our experience, small-scale TFF filtration results in viral concentrates of ca. 250 mL in volume. Small-scale concentration of viruses from ambient water samples will require prefiltration to remove cells and particulates larger than 0.22 µm in size. If small-scale TFF filtration is used as a second step viral concentration following a large-scale (50 L to 2 L) procedure no prefiltration is required. After this two step process, theoretical viral concentration ratios of 200 fold (50 L to 0.25 L) can be achieved. Actual viral concentration ratio will depend on overall filtration efficiency. Oftentimes, large- and smallscale TFF concentration of viruses is performed in the field, and the final 250 mL concentrate is frozen (preferably snap frozen in LN<sub>2</sub>) for transport back to the lab. Some investigators have reported better viral preservation by adding glycerol to a final concentration of 10% prior to snap freezing (Glass and Williamson pers. comm.).<br /><br /><strong>Postconcentration procedures </strong><br /><br />Oftentimes, it is desirable to reduce the volume of viral concentrates below the ca. 250 mL minimum hold-up volume of most small-scale TFF apparatus. In particular, fingerprinting of viral assemblages by pulsed-field gel electrophoresis (Steward 2001; Wommack et al. 1999) or preparation of viral concentrates for metagenomic sequencing (Bench et al. 2007; Breitbart et al. 2002) requires viral particle densities of ≥10<sup>9</sup> mL<sup>–1</sup>. By and large, investigators have produced highdensity viral concentrates using either spin filters, which rely on centrifugal force to push water through a 30- to 50-kD molecular sieve, or an ultracentrifuge to pellet viruses followed by resuspension in a smaller volume of UF or buffer. Disposable spin filters are provided by a number of manufacturers and require only a benchtop swinging bucket rotor for filtration (Bench et al. 2007). Pelleting of viruses can only be done in an ultracentrifuge at centrifugal forces exceeding 100,000g, a requirement that can be cost prohibitive or unavailable at smaller research facilities (Wommack et al. 1992). Recently, Colombet and coworkers (2007) adapted polyethylene glycol (PEG) precipitation of viruses, for postconcentration of viruses within 1 L viral concentrates derived from 20-L water samples. The PEG protocol showed a greater than 2-fold increase in the recovery efficiency of virus particles as compared with the ultracentrifugation procedure. The lower cost of this procedure is welcomed; however, we have found that postconcentration procedures can have a dramatic effect on the quality of PFGE virioplankton fingerprints. In contrast to the reported improvement in fingerprints after PEG precipitation (Colombet et al. 2007), we found that postconcentration of viral concentrates by ultrafiltration consistently produces the best resolved PFGE fingerprints (<a href="http://www.aslo.org/books/mave/MAVE_110.pdf" target="_blank">Fig. 3</a>). The source of loss in PFGE band clarity and sharpness for samples processed by PEG precipitation or spin filtration is not known and did not appear to be attributable to sample loading as the PEG and Centricon samples were loaded with 2-fold less and 3-fold more viruses, respectively, than the ultracentrifuged sample.<br /><br /><br />

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