Concentration of Phage Lysate using Vivaflow Tangential/Crossflow Filtration Cassette
To filter 4-8 L of phage lysate, set up one Vivaflow50 cassette per 4 L of sample (this was the capacity successfully tested by our lab).
If two cassettes are needed for a phage sample, set these up in parallel with separate tubing lines, using the double tubing pump head.
Refer to the Sartorius user manual and online videos for tubing attachment and flow set-up.
For all tubing, keep the ends to be placed in the sample and filtrate as clean as possible once removing from the packaging.
To adapt the smaller cassette tubing to our lab’s pump head size, make sure to attach the size 25 Masterflex tubing first.
Keep clean/sterile the end to be placed in the phage sample.
If running more than one phage sample simultaneously, make sure to label all input and output tubing, cassettes and bottles or flasks with sample ID’s, and keep separate to prevent any phage cross contamination.
Per the Sartorius manual, prepare the cassette (aka: module) before use by pre-rinsing the system at full pressure to remove trace amounts of glycerine and sodium azide and check for any leaks at the tubing connections.
Place the input tubing into the 1 L bottle of 0.2 um filtered, sterile nanopure water.
Place the output return tubing in the same bottle and pump liquid through the system to purge any air pockets.
You will need ~500 ml of water per cassette, and you can set up two parallel cassettes to use the same bottle.
The recirculation rate should be in the range of 200-400 ml/min and suitable flow should exit the filtrate line.
If used, the pressure indicator should read approximately 2.5 bar.
This corresponds to a pump setting of around 60.
Allow 400 ml per cassette to pass into the filtrate collection.
Check for any leaks at connections.
Finally, drain the system and empty the filtrate flask.
To concentrate the phage sample, place the input and output lines into the initial lysate container (usually a 1L bottle).
If the container lid is off, place a piece of parafilm over the opening to minimize contamination and secure the tubing in place.
Pump the liquid through the system at the same rate and pressure as specified for the rinsing step above.
For two cassettes filtering one sample, a pump setting of around 60 corresponds to a filtrate production of about 250 ml per 14 min initially, but which may slow some as more concentrated sample is added to the recirculation.
Monitor the sample level and stop the pumping when volume reaches 100-200 ml to refill the bottle with more sample.
Also monitor the filtrate flask to prevent overflowing and discard (or keep, if desired) the filtrate down the sink (this should be free of phage!).
Stop adding phage sample after you have concentrated 4 L to between 175 and 200 ml.
Note that about 15-20 ml are left in the system.
When the desired volume has been reached, reduce the recirculation rate to 20-40 ml/min (pump setting around 10) and recirculate the concentrated sample for 1-2 minutes to maximize recovery.
Stop adding phage sample after you have concentrated 4 L to between 175 and 200 ml.
Note that about 15-20 ml are left in the system.
When the desired volume has been reached, reduce the recirculation rate to 20-40 ml/min (pump setting around 10) and recirculate the concentrated sample for 1-2 minutes to maximize recovery.
If there are > 4 L of sample to concentrate, begin the next fraction: place the input and return tubes into the next lysate sample bottle, parafilm cover, and repeat the concentration process (steps 5-8).
Then transfer the concentrated sample into a second 250 ml bottle and drain the remaining sample completely from the system.
To collect a final rinse from the filters for a more complete sample recovery, place 100 ml ASW salts into a clean 250 ml bottle.
Place the input and return tubing into the bottle, cover the top with parafilm and recirculate on lower speed until the volume is reduced to about 20 ml.
Then drain the system as before and collect the remaining rinse fraction into the bottle.
Even though the Vivaflow 50 cassettes are not reusable, they were kept until the concentration of phage sample was determined.
To store the cassettes, load them with ASW salts and keep at 4 °C.
Place 50 ml ASW salts into a sterile 50 ml tube and recirculate this through the filter and tubing to remove air bubbles.
Then either leave the open tubing ends in the media tube and cover with parafilm, or seal the tubing ends with tape.
For phage concentration analysis via SYBR slide preparation, collect 0.5 ml of each of the following samples.
Sample Fractions #1, #2 and Rinse.
Filtrate from beginning of the first 4 L concentration.
Filtrate from end of the first 4 L concentration. 
Footnotes:  1) In Feb 2016 experiments, sample fractions concentrations were determined before combining the two fractions for the next step.
The second fraction was more concentrated for both phage samples.
The rinse sample was not counted or used.
2) In all previous Vivaflow concentration trials, no particles were seen on SYBR slides for the filtrate samples, as should be expected.
