TITLE: Protocol for Dead-End Ultrafiltration using REXEED 25S for Isolation of Salmonella from Surface Water
AUTHORS: Autumn L Kraft, Manan   Sharma, Jonathan G Frye, James  E Wells, Abasiofiok  Mark Ibekwe, NARMS EWG  

[DESCRIPTION]
Protocol describes dead-end ultrafiltration (DEUF) using REXEED 25S for the recovery and isolation of Salmonella from surface water.

[STEPS]
SECTION: Protocol
1. If needed, add control strain to water sample by gently tipping vial over the opening. Close sample bottle tightly and thoroughly mix sample before proceeding.
SECTION: Protocol
2. Prepare for filtration by setting up Geotech pump in the biosafety cabinet (BSC). Ensure there is enough room around pump to include filtrate/waste collection bottle and ringstand or clamps for holding filter (Rexeed 25S).
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3. Ensure you have different lengths of Masterflex tubing to allow for insertion into  the sample bottle, through the pump head, connecting at the top port (RED) of the ultrafilter using DIN adapter with a second piece of tubing connecting to the lower side port (BLUE end, NOT bottom port) of the Rexeed 25S filter and ending in a collection container.
SECTION: Protocol
4. Attach intake tubing to the Rexeed 25S so the sample will flow through the filter as indicated on the label (from top to bottom, ORANGE to BLUE). Attach the outgoing tubing to the opposite end of the filter with the outlet end in a waste collection bottle (Figure 1).
   

SECTION: Protocol
5. Place inlet of intake tubing into the 1 L bottle containing surface water, ensure the tubing is clamped in EZ Load pump head, and water is flowing through tubing in the appropriate direction. Then turn on pump, set pump rate to ¾ of the maximum flow rate, and adjust pump speed after pumping has initiated to reach a steady/constant flow rate through the filter.
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6. Turn off pump once filtration is complete and any remaining water in tubing should be drained into the waste collection container. 
 

SECTION: Protocol
7. Reposition tubing on the Rexeed 25S filter in the configuration shown in Figure 2. Start by removing the intake tubing and DIN adapter at the top, closing the port with the cap, and discard tubing/adapter in decontamination container. Remove tubing from lower side port and re-cap so all ports on the filter are sealed. Tubing can then be attached to upper side port (Figure 2) – remember to remove cap from side port first. Thread tubing through Geotech EZ Load pump and place into Backflush solution container.
  

SECTION: Protocol
8. Place collection bottle below the bottom port (BLUE) and remove cap on filter before backflushing.
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9. Place prepared Backflush solution (100 mL) as pictured in Figure 2 and begin pumping as described in steps 4 and 5 until the backflush solution has been filtered and liquid stops flowing from the open bottom port. 
 


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10. Collect as much backflush filtrate as possible and once flow has stopped, turn off pump and remove tubing from backflush collection. To avoid spills from water remaining in tubing, all tubing should go into a bin for decontamination via autoclave.
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11. Add appropriate volume of concentrated 2X BPW to backflush filtrate bottle to make a 1:1 dilution. Incubate enriched backflush at 37 °C for 18-24 h.