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 .
