General protocol for the concentration of virioplankton from a large volume (>20 L) water sample
Prefilter ambient water sample with a 25-µm wound polypropylene sediment filter before any concentration using a peristaltic or diaphragm pump.
Rinse the concentration container three times with prefiltered water before filling with prefiltered sample water.
Once the concentration container is full of 25 µm prefiltered ambient water, arrange the tubing for tangential flow filtration (TFF).
Attach the 0.22-µm tangential flow filtration (TFF) filter feed and retentate tubing to the TFF filter housing inlet and outlet ports, respectively.
Attach the permeate tubing to the permeate port of the TFF filter housing and direct into a separate container to catch virus-containing water.
Run the feed tubing through a large peristaltic pump head.
If the peristaltic pump head has an occlusion setting, set it to maximal occlusion to help prime the 0.22-µm TFF filter and remove all air.
Turn the peristatic pump on and increase the peristaltic pump speed to 20% to prime the tubing and remove excess air.
Once the tubing and filter are completely filled with water, adjust the occlusion knob to a looser setting to prevent excessive tubing wear.
Slowly increase the pump speed to 45%.
Once the system is running smoothly, partially open the permeate valve on the 0.22-µm TFF filter to the second tick mark (~20° open).
Collect the <0.22 µm permeate into an appropriately sized carboy.
When the permeate carboy is more than half full with <0.22 µm permeate, prepare the 30-kD TFF filter for viral concentration.
Slow the large peristaltic pump speed to 10%.
Attach the feed and retentate tubing to the appropriate ports on the 30-kD TFF.
Direct the permeate tubing from the 30-kD TFF filter into a clean carboy to collect ultrafiltrate (UF; virus-free water).
Carefully work the feed tubing into the second pump head of the large peristaltic pump.
Increase the large peristaltic pump speed to 20%.
Again, tighten the occlusion knob on the pump head to prime the 30-kD TFF filter and remove all air.
Once the filter and lines are fully primed loosen the pump head occlusion knob.
Slowly increase the pump speed to 45%.
Slowly close the backpressure knob on the 30-kD TFF filter until permeate begins to flow.
Monitor the level of <0.22 µm water in the <0.22 µm permeate carboy.
The level should be maintained at half full until the pre filtered ambient water is nearly gone.
Collect 1 L of ultra filtrate (30 kD TFF permeate) in a 2-L bottle for rinsing of the 30-kD TFF filter.
When ~5 L of 25 µm filtered ambient water remains in the large carboy, release the pump head and remove the 0.22-µm TFF filter feed tubing from the large peristaltic pump.
Continue to run the 30-kD TFF filter until ~1 L of <0.22 µm water remains in the carboy.
When 1L or less of <0.22 µm water remains, stop the large peristaltic pump and drain the 30-kD TFF filter into the <0.22 µm water carboy.
Slowly prime the 30-kD TFF filter from the 1 L of collected UF from step 22 and then recirculate (feed, retentate and permeate lines of the 30-kD filter in the 1L of UF) the UF at 30% pump speed for 5 min.
Stop the pump, release the pump head, and drain all tubing into the 2-L UF bottle.
Pool the recirculated UF with the ~1 L of retentate from the primary viral concentration through the 30-kD TFF filter.
The volume of the ~2 L viral concentrate can be further reduced by using a small-scale TFF ultrafilter.
To avoid excessive degradation of viruses, it is advisable to store the first stage VC at 4°C and perform a second stage small-scale concentration a soon as possible (i.e., within no more than 1 d).
Insure that the permeate port is closed, then begin priming the system as follows.
