Materials:
1. Sodium pyrophosphate 10mM and EDTA 5mM
2. Plastic zip-close bags
3. Multi-tube vortex mixer capable of holding 50 ml centrifuge tubes
4. 0.22 µm Sterivex (Millipore, Corp.) filters
5. 15 and 50 ml conical sterile polypropylene centrifuge tubes
6. 10 ml luer-loc syringes
7. Low speed swing-out bench-top centrifuge
Methods:
1. Sediment samples can be processed immediately or stored frozen at –20°C if they are to be used for viral analysis only.
2. Although not necessary prior to extraction of viruses from sediment, pore water can be selectively removed from the sample by low speed centrifugation. If the sediment is too thick to separate pore water by direct centrifugation, then the apparatus shown in Fig. 2 can be used to remove pore water.
3. Place a 2 cc sediment sample in a 50 ml conical centrifuge tube and add 8 ml of 0.02µm –filtered 10mM sodium pyrophosphate and 8ul of 5 mM EDTA to each tube. Samples that do or do not contain pore water can also be used for viral extraction.
4. Place tube(s) inside two plastic zip-style bags, place horizontally on a vortex mixer and vortex on the highest setting for 20 minutes.
5. Centrifuge tubes at 2000 g, 20oC, for 25 minutes. Remove tubes from centrifuge carefully to prevent resuspension of the pellet.
6. Carefully decant the supernatant into a 10ml luer-loc syringe to which a 0.22µm Sterivex (Millipore Corp.) filter has been attached. Replace plunger and gently push sample through the filter into a clean sterile 15 ml tube. Sample storage conditions or addition of biological fixatives depend on subsequent use of the sample. Typically, snap freezing in liquid N2 followed by storage at – 80°C provides the best preservation of virus particles (Wen et al. 2004; Helton et al. 2006).
