Materials:
1. Small 2 land 500 ml polycarbonate bottles with caps (Nalgene)
2. 6.4mm diameter PharMed peristaltic pump tubing (Cole-Parmer)
3. 6.4mm diameter Tygon tubing for plumbing of water sample between filters, pumps and reservoirs (Nalgene)
4. Peristaltic pump capable of providing sufficient cross-flow for a 0.1 m2 ultrafiltration membrane (Cole Parmer).
5. 0.1 m2 Prep-Scale molecular weight cut-off (MWCO) ultrafiltration cartridge (Millipore, Corp. Bedford, MA)
6. Stopwatch timer
7. 2 L graduated cylinders
Methods:
1. If processing a small water sample remove bacteria and small particulates by filtration through a 0.22 µm filter. Prefiltration can be avoided with viral concentrates from the Helicon filter, as this sample is already free of bacteria and particulates.
2. Because the flow direction of the Prep-Scale filter is fixed, plumb the filter similarly to that of the Helicon filter. Fully open the permeate valve, and slowly increase the pump speed to expel air from the filter. Once the pump speed has reached 1 L min-1 slowly close the permeate valve until a retentate:permeate ratio of 3:1 is achieved with a steady drip out the permeate line.
3. Collect 100 ml of virus-free permeate in a 250 ml or 500 ml polycarbonate bottle.
4. Once the retentate volume is close to the minimum hold up volume of the Prep-Scale filter and lines( ~ 100 ml), open the permeate control valve, and slow the pump down to a full stop. Unclamp the feed line from the peristaltic pump head and drain the system. This is the viral concentrate sample.
5. Run the feed, retentate, and permeate lines from the polycarbonate bottle containing virus-free permeate. Prime and then rinse the Prep-Scale filter by recirculating virus-free permeate for about 5 minutes. Avoid excessive bubbling of air through the sample.
6. Drain the filter and pool the rinse water with the viral concentrate from step #4. The final volume should be ca. 300 ml.
7. To insure sterility of the viral concentrate, use a 0.22 µm syringe filter to remove any unintended bacterial contamination
8. Wash the Prep-Scale filter by flushing with ca. 2 L of virus-free permeate followed by 4 L of ultrapure water.
9. If the cartridges are not to be used for 2 days or longer, filter membranes should be cleaned following manufacturer’s instructions. Care must be taken to ensure that cleaning and storage solutions are compatible with the filtration membrane. Integrity test filter according to manufacturer’s directions after every 5-10 uses.
