TITLE: Seawater virome concentration with Vivaflow 
AUTHORS: Natascha Varona, Cynthia Silveira

[DESCRIPTION]
Concentration of viral particles from seawater samples using a Vivaflow.

[GUIDELINES]
This protocol is written for one sample only, however, with additional pumpheads and vivaflows you can run up to 4 viromes at the same time

[STEPS]
SECTION: Virome collection
1. Collect 500mL - 2L of seawater (depending on microbial density) in a sterile container.
SECTION: Virome collection
2. Wipe down outside of all masterflex tubing with 70% ethanol.
SECTION: Virome collection
2.1. Insert one end of the tubing into the sample and feed it through the Masterflex pump.
SECTION: Virome collection
2.2. Attach 0.22 µm (or 0.45 µm depending on the viral size fraction you want to concentrate) Sterivex to the other end of the tube using a luer lock adapter. 
SECTION: Virome collection
3. Turn the Masterflex on a low setting (between 2-3).
SECTION: Virome collection
4. Keep the flowthrough for virome concentration. 
SECTION: Virome concentration
5.  Set up Vivaflow per instruction according to the image above.

SECTION: Virome concentration
5.1. Before running the sample, rinse Vivaflow with 500 mL of DI water by inserting a sample tube into a container with DI water and placing the filtrate and return tubes in a waste container.

SECTION: Virome concentration
5.2. Pump water at a rate of 200-400 mL/min. 
SECTION: Virome concentration
5.3. After 400 mL have passed into the waste, stop the pump, and check for leakage at connections.

SECTION: Virome concentration
5.4. Tighten if needed. Drain system. It is now ready for use. 
SECTION: Virome concentration
6. Replace the DI water with the sample and collect the filtrate in a sterile container (this filtrate will be used for backflush later).
SECTION: Virome concentration
6.1. Move the return tubing to a waste container to rinse out residual DI-water before recirculating.
SECTION: Virome concentration
6.2. Pump liquid at a rate of 200-400 mL/min for a few seconds to ensure the DI water is removed and stop the pump. 
SECTION: Virome concentration
6.3. Move the return tube to the sample container (note that the feed tube and return tube are now in the sample, this is necessary to recirculate and concentrate the sample). 
SECTION: Virome concentration
7. Pump liquid at a rate of 200-400 mL/min. (If using a pressure indicator it should be approximately 2.5 bar).
SECTION: Virome concentration
8. When the sample container is nearly empty, remove the feed line, and collect the sample in a 50 mL conical tube. This usually contains about 30 mL of the sample. 

SECTION: Virome concentration
9. For a more complete recovery, preform a backflush.
SECTION: Virome concentration
9.1. Remove and screw off filtrate line.
SECTION: Virome concentration
9.2. Take about 20 mL aliquot of the filtrate, and insert the feed tube into the filtrate.  

SECTION: Virome concentration
9.3. Run the Vivaflow in reverse until the sample reaches about 50 mL .

SECTION: Virome concentration
10. Add chloroform to your virome sample for a final concentration of 0.1 % (v/v). 
Example: 50 µL Chloroform for 50 mL of sample. 

SECTION: Virome concentration
11. Samples can be stored at 4 °C for later extraction. 

SECTION: Vivaflow clean up
12. Reattach the filtrate line and flush the system with 200 mL of DI water with the filtrate going to the waste.

SECTION: Vivaflow clean up
13. Place the feed, return and filtrate lines into a container with a cleaning solution of 250 mL of 0.5 millimolar (mM) NaOCl and 0.5 Molarity (m) NaOH. (250 mL  DI-water + 133 µL  NaOCl + 5 g NaOH) 

SECTION: Vivaflow clean up
14. Recirculate at 50-100 mL/min for 30 minutes. 
SECTION: Vivaflow clean up
15. Drain the system and recirculate 250 mL of DI-water through the system for 5-10 minutes. 

SECTION: Vivaflow clean up
16. Drain and rinse system with a further 500 mL of DI-water.

SECTION: Vivaflow clean up
17. The system is now ready for the next sample, or for long term storage, fill the module with 10% ethanol and (in DI-water) store at 4 °C .