IRON CHLORIDE PRECIPITATION OF VIRUSES FROM SEAWATER A simple and efficient method for concentration of ocean viruses by chemical

flocculation. Seth G John, Carolina B Mendez, Li Deng, Bonnie Poulos, Anne-Kathryn Kauffman, Suzanne Kern, Jennifer Brum, Martin F Polz, Ed A Boyle, Matthew B Sullivan Environ Microbiol Rep. 2011 3(2):195-202. 1. Pre-filter 20L seawater using a 150mm Whatman GF/A filter followed by a 0.22um, 142mm Millipore Express Plus filter and attaching the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi). This requires the use of two 142mm filtering towers to be efficient. (see note # 4 below for alternative pre-filtration method) Treat the virus fraction (0.22m filtrate) with FeCl3 to precipitate the viruses. Add 2ml of 10g/L Fe stock solution to each 20 L of filtrate. Shake vigorously for 1 min after addition of each 1ml FeCl3 and repeat shaking several times. Let sit for 1 hr at RT. Filter the FeCl3-treated filtrate using a 1.0m, 142mm, polycarbonate (PC) membrane filter on top of a 0.8m, 142mm, Supor support filter and attaching the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi). The FeCl3 precipitate will be captured on the PC filter; the Supor is just for support in the 142mm stainless steel filtration apparatus. Although the 20L can be collected on a single 142mm PC membrane, it is faster to change the PC membrane one or two times during the filtration; normally 3 PC membranes and 1 Supor membrane are used per 20L seawater. Place all of the polycarbonate filters from the 20L into a 50ml centrifuge tube being careful not to scrape off any of the FeCl3 on the edge of the tube (having precipitate facing out aids in dissolving the precipitate). The filters can be stored in the 50ml tubes at 4oC until ready to resuspend the precipitated viruses. Be sure the caps are on securely so that the filters do not dry out. Prepare fresh 0.2M Ascorbate-0.1M EDTA-Mg buffer pH6-7 and add 1ml buffer for each 1mg Fe, which is 20ml of 1x buffer for the 20L seawater precipitate or, alternatively, 10ml of a 2x concentrated buffer. Shake the tube vigorously. Incubate at 4oC, shaking when you can for the next day or so. You will see the solution change colors a number of times that is okay. Leave this stored at 4oC until ready to analyze or further purify the viral fraction.

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Reagents 10g/L Fe Stock Solution FeCl3-6H2O (FW=270.3) 4.83g Q-water 100ml qs Store at room temperature or 4oC. 0.2M Ascorbate-0.1M EDTA-0.2M Mg EDTA-Mg2 (FW=336.82) 3.37g Ascorbic acid (FW=176.12) 3.52g 5N NaOH 4ml
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Q-water 100ml qs Prepare fresh (w/in 48 hr of use). Dissolve EDTA-Mg2 first in 90ml water. Once in solution, add ascorbic acid and NaOH. EDTA may precipitate out initially, but should go back into solution after addition of NaOH. pH should be between 6-7. QS to 100ml with water. Store dark at 4oC. This can be made as a 2x solution if less volume is desired to redissolve precipitate.

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Notes: 1. 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 FeCl3 per liter SW or to 4.83mg FeCl3-6H2O per liter SW. The filters needed for this procedure are as follows: Whatman glass fiber filter GF/A (cat.no. 1820-150; 1.6m retention; 150mm diameter). Millipore Express Plus filter (GPWP 14250; 142mm, 0.22m) Pall Supor-800 filter (cat.no.60114; 142mm, 0.8um). GE Water Systems Polycarbonate membrane (cat.no.K10CP14220; 142mm, 1.0m) there is also a model number associated with this membrane (1216611). This is not easy to find but should be available for online ordering through Midland Scientific Inc. If only working with a small volume of seawater (1-2 liters) you can use 47mm or 90mm filters. Carboys and collection bottles should be washed with 1N HCl and rinsed with Q-water before use. Wear gloves and use forceps for handling filters. Checking tubing before use and replace if worn. An alternative method for preparing seawater is to pre-filter the seawater through a Whatman GF/D glass fiber filter (cat.no. 1823-150; 2.7m retention; 142mm diameter). to remove large particles. The filtrate is filtered again using a Millipore Steripak GP10 or GP20 (cat.no. SPGPM10RJ or SPGPM20RJ; 0.22m). The GP 10 works for <10L volumes and GP20 works for <20L volumes. Larger capsule filters will easily filter 100200 liters: Pall Acropak 200, 500, 1000 and 1500 ranging in price from $83-165 each. The cellular fraction is retained on the filter while the virus fraction is in the filtrate. Filtration should be done at a maximum pressure of 15 psi. A peristaltic pump ($3000) applies the pressure and a stainless steel Millipore filter rig (PN YY3014236, $2000) holds filters. For less expense, use an acrylic or polycarbonate 142 mm filter holder (Geotech Environmental Equipment, $445 acrylic; $320 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. Chemicals needed: a. Ferric Chloride hexahydrate (FeCl36 H2O): Mallinckrodt 5029-04 500g $88.75 OR Merck 1039430250 250 g (no price given) b. Ascorbic Acid (C6H8O6): Mallinckrodt 1852-57 100g $68.30 c. Sodium hydroxide (NaOH): JT Baker 3728-04 125g $46.05 OR Merck 1064950250 (no price given). d. EDTA, Magnesium derivative, Magnesium salt (C10H12Mg2N2O8): JT Baker L701-05 100g $235.95 (this is the one that is difficult to find from other manufacturers).

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Infectivity of viruses may be improved used the following buffer: 0.2M Mg2EDTA, 0.25M Tris HCl, and 0.25M oxalic acid. If Mg2EDTA is difficult to obtain, an alternative is a buffer containing 0.1M Na2EDTA, 0.2M MgCl2, 0.125M Tris, and 0.125M reductant (ascorbate or oxalate), adjusted to pH ~6 with NaOH

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