TITLE: Backflush of Dead-end Ultrafilter
AUTHORS: Andrea    Ottesen, Brandon   Kocurek

[DESCRIPTION]
The backflush procedure pushes out all the cells and particles that were captured during the pumping procedure. It is most easily carried out in the lab with established  (pump holding equipment) or ad hoc installation of pump, flushing material , filter and a >500 sterile vessel to collect the backflush.

[BEFORE_START]
Make a fresh backflush solution:
Add 2.5 µL of Tween 80 to 500 mL  sterile distilled water. Swirl slowly to dissolve the 0.5% Tween 80. 


[GUIDELINES]
Ultrafilter is backflushed with a 500 mL  solution of 0.5% Tween 80


[STEPS]
SECTION: Backflush Station Set Up
4. Open the side port of the "top" of the ultrafilter by removing the cap. Push the freshly cut tubing on the side port all the way to where the port meets the ultrafilter. Feed the tubing through the peristatic pump and place the end of the tubing into the backflush solution.
SECTION: Backflush Station Set Up
6. Secure all of the connection points with zip ties or clamps as needed. Attach the tubing and feed the tube into the sterile vessel for collection of backflush solution that has flowed through the ultrafilter.

SECTION: Prepare backflushing solution
1. A 500 mL volume is required per ultrafilter to be backflushed. Make a fresh backflush solution as follows:

Add 2.5 µL of Tween 80 to 500 mL  sterile distilled water. Swirl slowly to dissolve the 0.5% Tween 80.

SECTION: Backflush the filter
8. Determine the desired direction of water flow and toggle the switch for flow direction. Turn the pump on in the direction that pulls backflush solution towards the ultrafilter and then into the sterile vessel. 
SECTION: Backflush Station Set Up
2. Assemble the filtration system by coordinating the position of the peristaltic pump, ultrafilter and backflush solution. 

Tubing will flow from backflushing solution, through peristaltic pump, into the "top" of the ultrafilter. Backflush will then flow from the ultrafilter to a sterile beaker or bottle.
SECTION: Prepare backflushing solution
1.1. Be patient, the Tween 80 can take a while to dissolve. Attempt to limit the amount of foam produced, but some foam is inevitable.
SECTION: Backflush Station Set Up
3. Cut enough fresh tubing (L/S 36) to run from the 500 mL vessel containing backflush solution, through the pump, into the side of the "top" of the ultrafilter.

SECTION: Backflush Station Set Up
7. Once the tubing, pump, ultrafiltration cartridge, and sterile vessel have been set up, plug the appropriate power cord into the outlet in the back of the pump and the other end of the power cord into a battery source or outlet within the laboratory.
SECTION: Backflush Station Set Up
5. Cut another piece of fresh tubing (L/S 36) to run from the colored "bottom" part of the ultrafilter into a sterile vessel. Attach a DIN adapter and screw into the bottom of the ultrafilter which will feed into the sterile vessel. 
SECTION: Backflush Station Set Up
6.1. Ensure the 2 remaining caps on the ultrafilter are secured tightly so as to prevent leaks.
SECTION: Backflush Station Set Up
4.1. Secure all of the connection points with zip ties or clamps as needed.
SECTION: Backflush the filter
9. Continue pumping until no backflush solution remains in the 500 mL  bottle and the flow out of the ultrafilter has slowed to a trickle. 

SECTION: Backflush the filter
9.1. It will be necessary to briefly pump air after the backflush solution is used up in order to get all the liquid out of the filter.
SECTION: Backflush the filter
10. Turn off the pump and replace the lid on the backflush solution that has passed through the ultrafilter.
SECTION: Backflush the filter
10.1. This is your sample. Place at 4 °C  until further analysis. Ensure this vessel is properly labeled.

SECTION: Backflush the filter
11. Disassemble backflush station and ensure all used materials are placed in the appropriate biohazard waste containers.
SECTION: Backflush the filter
11.1. The ultrafilter may be stored at 4 °C  if needed. Ensure all open ports (side and end) are capped to prevent any possible leaks)