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 .
