Aug 22, 2024

Public workspaceStandard operating procedure for dead-end ultrafiltration water sampling in the field for bacterial pathogens using REXEED 25S replacement filters

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8nydl5r/v1
  • 1US FDA;
  • 2Goldbelt C6, LLC
Ai Kataoka
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.j8nlk8nydl5r/v1
Protocol Citation: Julie Ann Kase, Jose Roberto Guzman, Jennifer Wolny, Tina Pfefer, Andrew Battin, Ai Kataoka 2024. Standard operating procedure for dead-end ultrafiltration water sampling in the field for bacterial pathogens using REXEED 25S replacement filters. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8nydl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2024
Last Modified: October 08, 2024
Protocol Integer ID: 104073
Keywords: Ultrafilters, DEUF, Water Sampling, FDA, Backflush, Bacterial Pathogens, Dead-end ultrafiltrations
Abstract
This protocol details the standard operating procedure for dead-end ultrafiltration water sampling in the field for bacterial pathogens using REXEED 25S replacement filters.
Guidelines
Appendix A
Pictures of Hose Clamp, Blood and Dialysate Port Cap and DIN Adapters





Appendix B
Fig 8. Ultrafilters with designated ports and outlets.


Appendix C
List of supplies in ultrafiltration kit

Each kit contains: 2 bench protectors; one clean/unused 50 mL conical tube with one DIN adapter, 2 rubber storage caps and 2 blood port caps; one SNP-04 hose clamp to secure the MPC-855 DIN adapter or SNP-8 hose clamp/zip tie for MPC-855NS.375 DIN adapter; 2 SNP-12 Plastic hose clamp or zip tie to secure the two rubber storage caps; 2 zip ties to secure the tubing at the telescoping pole; one roll of tubing; one ultrafilter; and one 2.5-gallon sealable bag (for storage of the ultrafilter after sample collection).

Fig 9. Original caps removed and replaced with storage caps.


Appendix D
Illustrations of tubing connection to different ultrafilter brands
Fig 10. Ultrafilters tubing connection configuration.

1. Tubing connected onto DIN adapter and secured with plastic hose clamp SNP-8 and needle nose pliers.
2. Dialysate port storage cap secured with a plastic hose clamp SNP-12 and needle nose pliers.
3. Tubing connected to the dialysate fluid outlet and effluent directed away from water sample intake. No clamps are needed.
4. Blood inlet port secured with the storage blood port caps.



Appendix E.
Ultrafilter priming and flow meter configuration
Fig 11. Set up for ultrafilter priming.



Fig 12. Appropriate configuration of effluent tubing and water flow meter



Appendix F
Water sample collection
Fig 13. Example of dead-end ultrafiltration set up for field sampling.



Fig 14. Dead end ultrafiltration field arrangement diagram.



Appendix G
Backflush procedure.
Fig 15. Ultrafilter upon receipt.




Fig. 16. The ultrafilter is secured vertically using the stand clamps with the sample inlet port pointing downwards, and the effluent side port oriented towards the pump.


Fig. 17. During the ultrafilter set up, the sample inlet port points downwards, and the effluent side port faces the pump. The sample receiving bottle is placed under blood return port without touching the bottle surfaces. The tubing is attached to the dialysate outlet port by pushing onto the port and is secured with a SNP-12 clamp or a zip tie.












Materials
EQUIPMENT AND SUPPLIES FOR DEAD-END ULTRAFILTRATION

Disclaimer: Names of vendors or manufacturers are provided as examples of suitable product sources; inclusion does not imply endorsement by the US Food and Drug Administration or the Department of Health and Human Services. Alternative products may be used if they provide equivalent performance to the items cited in this SOP.

Note
Ultrafilter sampling kits can be made ahead of time to facilitate field activities. One kit is prepared per ultrafilter. See Appendix C for details.

1. Filtrate port tubing (Masterflex® Platinum-cured Silicone Tubing L/S 36 (sold in 25-foot rolls; Avantor, Item # MFLX96410-36)
ReagentMasterflex® L/S® High-Performance Precision Pump Tubing, Platinum-Cured Silicone, Avantor®VWR InternationalCatalog #MFLX96410-36

Note
This tubing is not sold pre-sterilized, although the likelihood of pathogen presence is infinitesimal. If concern over sterility exists, contact manufacturer for autoclaving instructions or consider using an approximately 3-foot piece of tubing from same lot as a control sample that could be flushed with sterile water that is subsequently tested accordingly.

a. Alternative tubing (less expensive but doesn’t appear to be as durable) – Silicone Tubing, Size 36 (sold in 25-foot rolls; Geotech Environmental Equipment, Item # 77050011, http://www.geotechenv.com )
b. Alternative tubing (needs to be used with the smaller DIN adapter and clamp) Masterflex® Platinum-cured Silicone Tubing L/S 24 (Avantor, Item # MFLX96410- 24); Small Plastic hose clamp - (Grainger, Item #1ENJ3, Avantor Item # MFLX06832-02, or equivalent (clamping diameter 0.36in – 0.39in)ReagentMasterflex® L/S® High-Performance Precision Pump Tubing, Platinum-Cured Silicone, L/S 24; 25 ftVWR InternationalCatalog #MFLX96410-24
ReagentMasterflex® Hose Clamps, Acetal Copolymer, Avantor®VWR InternationalCatalog #MFLX06832-02
2. Kidney dialysis ultrafilters: Medtronic Clearum H22 (Item # 1428580) and Nipro Medical Elisio 25H (Item # 9120016) can be purchased from Henry Schein Corporation (1-800-472-4346);
B. Braun Xevonta Hi23 (Item # 7204405) can be purchased from Optimal Golden Max LLC (1-800-778-4718)ReagentXEVONTA DIALYZER HI 23 US, GAMMAB. Braun SECatalog #7204405
3. SNP-8 plastic hose clamps (for DIN adapter), (Avantor, Item # MFLX06832-08 or equivalent; clamping diameter = 0.513in x 0.586in). See Fig. 4, Appendix AReagentMasterflex® Hose Clamps, Acetal Copolymer, Avantor®VWR InternationalCatalog #MFLX06832-08
4. SNP-12 plastic hose clamps (for side port), (Avantor, Item # MFLX06832-12 or equivalent; clamping diameter = 0.616in x 0.707in). See Fig. 7, Appendix AReagentMasterflex® Hose Clamps, Acetal Copolymer, Avantor®VWR InternationalCatalog #MFLX06832-12
5. Zip ties (Fisher Scientific, Item # NC9269761 or similar)ReagentCole-Parmer 40 Pound Solid Nylon Cable/Zip Ties, 5.5 L, White; 1000/PkFisher ScientificCatalog #NC9269761
6. SNP-4 Plastic hose clamp, (Grainger, Item # 1ENP8 or equivalent (clamping diameter of 0.36in – 0.41in) for DIN adapter MPC-855). See Fig. 1, Appendix AReagentHose Clamp: Nylon, Black, Single Tooth, 0.36 in – 0.41 in Clamping Dia, 0.23 in Band WdGraingerCatalog #1ENP8
7. DIN adapter (Molded Products, Item # MPC-855, sold by the set, sterile, 2 pieces per set; https://moldedproducts.com); See Fig. 2, Appendix A. Alternative DIN Adapter – larger version, not pre-sterilized, only used with the L/S 36 tubing (Molded Products, Item # MPC- 855NS.375). See Fig.3, Appendix A.ReagentDIN AdapterMOLDED PRODUCTS, INC.Catalog # MPC-855NS.375

Note
Zip ties can be used instead of clamps.
8. Blood port caps (Molded Products, Item # MA-40 or equivalent); See Fig. 5 in Appendix AReagentBlood Port Kidney Storage CapMOLDED PRODUCTS, INC.Catalog # MPC-40
9. Dialysate port storage ap (Molded Products, Item # MPC-60D or equivalent) See Fig. 6 in Appendix AReagentBlood Port Kidney Storage CapMOLDED PRODUCTS, INC.Catalog # MPC-60D
10. Two 20L-cubitainers or other container with demarcations for measuring total volume (liters) passed through filter if not using a flow meter (having two containers will allow for water measurements to continue while one container is being emptied).
11. Mosquito/insect fiberglass mesh (sterile) for sampling waters with substantial, visible debris.
12. Permanent marker
13. Sealable, watertight plastic bags (e.g., Ziploc® 2.5 gallon) for storing ultrafilter after sampling.
14. Spray bottle of 70% ethanol (to field-disinfect scissors, pliers, and gloved hands)
15. Needle nose pliers (to close and remove the hose clamps)
16. Cutting pliers to cut the zip ties, if used
17. Cooler box or other suitable container for keeping filters chilled after collection and during transport to laboratory.
18. Heavy duty tape (Gorilla Tape or equivalent since will be used to seal cooler for shipment and will need to withstand rough treatment)
19. Ice / freezer packs
20. Bench protector paper (Fisher Scientific, Item # 14-206-65, or similar)ReagentAbsorbent UnderpadsThermo FisherCatalog #1420665
21. UPS or FedEx Shipping label(s) for Next Day Delivery, if shipping
22. Trash bag or biohazard bag depending on individual laboratory guidelines.
23. Nitrile or latex disposable gloves
24. Scissors (capable of cutting tubing)
25. 50 mL conical tubes or other clean, sterile container for storing clamps and caps.
26. Umbrella (golf-type or other large size, if bench protector paper not available) if sampling in sunny conditions to protect ultrafilter during collection and prevent pump from overheating.
27. Sodium thiosulfate, (Fisher Scientific, Item # S446) (Only needed for chlorinated water)ReagentSodium Thiosulfate Anhydrous (Certified), Fisher Chemical™Fisher ScientificCatalog #S446-500
28. Peristaltic pump (Geotech Environmental Equipment; http://www.geotechenv.com/ Geopump™ Peristaltic Pump Series II Package, Geotech Environmental Equipment, Item # 91352123 or equivalent) with one battery (recommend purchasing extra batteries)ReagentGeopump™ Peristaltic PumpsGeotech Environmental EquipmentCatalog #91352123
29. Extra batteries for field sampling pump (Geotech Environmental Equipment, Item # 77250000, Grainger Item # 54XH38, or equivalent)ReagentAPPROVED VENDOR Sealed Lead Acid Battery: 12V DC, 8 Ah Capacity, 3.7 in Ht, 2.56 in Wd, 5.94 in DpGraingerCatalog #54XH38
30. DC to AC converter for pump operation (Geotech Environmental Equipment, Item # 81400127 or equivalent) – this piece of equipment has alligator clips (to attach to a car battery) and a place to plug in the pump. It provides a way to power the pump in the field if the battery is depleted.
31. Flat head and #1 Phillips head screwdrivers (if the easy-load II pump head on Geopump needs to be moved).
32. Turbidity meter (optional) with a resolution of 0.01 NTU, a range up to at least 200 NTU, and compliant with EPA 180.1 method or ISO 7027 standard.
33. Flow meter (optional and can be used instead of containers for measuring pump output). Recommend – Model # 01N12LM, Part# 113255-6, GPImeters.com ReagentFlow meterGPImetersCatalog #01N12LM
34. Sterile deionized (D.I.) water for priming filter (1L per ultrafilter)

EQUIPMENT AND SUPPLIES FOR BACKFLUSH PROCEDURE

1. Silicone Tubing L/S 36 Masterflex® (Avantor, Item # MFLX96410-36) or Silicone Tubing size 36 (Geotech Environmental Equipment, http://www.geotechenv.com/ Item # 77050011)ReagentMasterflex® L/S® High-Performance Precision Pump Tubing, Platinum-Cured Silicone, Avantor®VWR InternationalCatalog #MFLX96410-36
2. Plastic hose clamp, SNP-12 (Avantor, Item # MFLX06832-12 or equivalent; clamping diameter = 0.616in x 0.707in); See Fig. 7 in Appendix A. ReagentMasterflex® Hose Clamps, Acetal Copolymer, Avantor®VWR InternationalCatalog #MFLX06832-12
3. Zip ties, optional, in place of hose clamp (Fisher Scientific, Item # NC9269761 or similar). See Fig. 7 in Appendix A.ReagentCole-Parmer 40 Pound Solid Nylon Cable/Zip Ties, 5.5 L, White; 1000/PkFisher ScientificCatalog #NC9269761
4. Paper towels
5. Scissors
6. Needle nose pliers (to close the hose clamps)
7. Bench protector paper (Fisher Scientific, Item # 14-206-65, or similar)ReagentAbsorbent UnderpadsThermo FisherCatalog #1420665
8. Face shield, optional (Fisher Scientific, Item # 17-310, or similar). ReagentSplash Shield™ North™ Protecto-Shield Face Shields Fisher ScientificCatalog #17-310

Note
Please consult safety protocols of individual laboratories.

9. Nitrile or latex disposable gloves
10. 10% bleach solution
11. 70% ethanol
12. Tween® 80 (Fisher Scientific Item # ICN10317080 or equivalent)ReagentTween 80, MP BiomedicalsFisher ScientificCatalog #ICN10317080
13. Sodium polyphosphate (NaPP), (Fisher Scientific, Item # 390930010 or equivalent)ReagentSodium polyphosphate, pure, Thermo Scientific ChemicalsFisher ScientificCatalog #AC390930010
14. Antifoam Emulsion. Prepare solution by mixing three parts of antifoam A concentrate (MilliporeSigma, Item # A6582-100G) in seven parts of propylene glycol (MilliporeSigma, Item# 398039).ReagentAntifoam A concentrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6582-100G Reagent1,2-PropanediolMerck MilliporeSigma (Sigma-Aldrich)Catalog #398039-25ML
15. Sterile stir bars, one per sample
16. 500 mL bottle, sterile, one per sample
17. 1 Liter bottle, sterile, one per sample
18. Sterile D.I. water
19. 5-10 mL serological pipette
20. Bench scale with 0.1 g sensitivity
21. Peristaltic pump (Geotech Environmental Equipment; http://www.geotechenv.com/ Geopump™ Peristaltic Pump Series II Package, Geotech Environmental Equipment, Item # 91352123 or equivalent)
22. Construction of pump platform: Adjustable height jack, (e.g., Synthware® Item # J112028; Fisher Scientific, Item # 31-502-290), or a rods and clamps assembly to elevate pump for ease in backflushing filters.ReagentSynthware™ Aluminum Support JacksFisher ScientificCatalog #31-502-290
a. Eisco Aluminum Rod 1/2 in. Diameter x 12 in. Length - Round Bar (0.50 in. x 12 in.); (Fisher Scientific, Item # S24256) (need four per set up)ReagentEisco™ Aluminum Rod 1/2 in. Diameter x 12 in. Length - Round Bar (0.50 in. x 12 in.)Fisher ScientificCatalog #S24256
b. Troemner™ Stainless Steel Support Rod, Length: 22.76 inches, Diameter: 0.51 inches; (Fisher Scientific, Item # 02-300-253) (need four per set up)ReagentTroemner™ Stainless Steel Support RodFisher ScientificCatalog #02-300-253
c. Troemner™ Two-Prong Single Adjust Swivel Clamp; (Fisher Scientific, Item # 02-216-212) (need two per set up)ReagentTroemner™ Two-Prong Single Adjust Swivel ClampFisher ScientificCatalog #02-216-212
d. Troemner™ Talboys™ Labjaws™ Regular Clamp Holder; (Fisher Scientific, Item # 02-300-201) (need eight per set up)ReagentTroemner™ Talboys™ Labjaws™ Regular Clamp HolderFisher ScientificCatalog #02-300-201
e. Bel-Art™ SP Scienceware™ Compact Support Stand, 21 x 16cm L x W footprint; (Fisher Scientific, Item # 12-947-976) (need one per set up)ReagentBel-Art™ SP Scienceware™ Compact Support StandFisher ScientificCatalog #12-947-976


SCOPE AND APPLICABILITY
SCOPE AND APPLICABILITY
This standard operating procedure (SOP) will be used to filter up to Amount100 L of water using an ultrafilter at a field sampling site to collect bacteria for pathogen analyses in the laboratory.
Included are instructions on how to properly backflush the ultrafilters in a laboratory setting to retrieve any bacteria captured.
SUMMARY OF METHOD
SUMMARY OF METHOD
Using a field-portable peristaltic pump, volumes up to Amount100 L of water are passed continuously through a Clearum H22, Xevonta Hi23, Elisio 25H or equivalent dialysis ultrafilter having a pore size small enough to capture any bacteria and other microorganisms present in the site water.
These ultrafilters are verified for their equivalent performance compared to REXEED 25S ultrafilter, which is no longer being distributed for sale in the United States.
Parameters such as time, date, and Sample ID are recorded on the filter using permanent marker.
The ultrafilter is kept at Temperature4 °C (refrigerated or on ice packs) after sample collection.
Temperature
Following sampling, a backflush procedure is performed in the laboratory with a Amount500 mL solution of 0.5% Tween 80, 0.01% sodium polyphosphate, and 0.001% Antifoam Emulsion.
HEALTH AND SAFETY WARNINGS
HEALTH AND SAFETY WARNINGS
Disposable gloves are recommended to be worn during all steps of this sample collection procedure to prevent possible exposure to waterborne pathogens and contamination of the filtration components.
Gloves are mandatory when removing caps from the ultrafilter, attaching tubing to the filter ports or otherwise touching exposed parts of the ultrafilter.
For the backflush procedure, disposable gloves should be worn and changed appropriately during all steps to prevent possible exposure to waterborne pathogens and to prevent cross-contamination of samples.
Since the filtration system is under pressure, a face shield is recommended to protect eyes and mouth.
Please consult safety protocols developed for your specific laboratory for exact requirements in terms of precautions and personal protection equipment (PPE).
FIELD FILTRATION AND SHIPPING PROCEDURES
FIELD FILTRATION AND SHIPPING PROCEDURES
30s
30s
Please see Figs. 1-7 in Appendix A and Fig. 8 in Appendix B for ultrafilter components and port locations for the various filters.





Fig 8. Ultrafilters with designated ports and outlets.

1. Blood outlet port
2. Dialysate inlet port
3. Dialysate outlet port
4. Blood inlet port

Note
Since these ultrafilters need to be primed with D.I. water prior to use, please read through the instructions below before beginning the filtration process and review the figures (11 - 14) in Appendix E and F. If chlorination of sampling water or the presence of free chlorine is suspected, ultrafilters should be treated with a 1% sodium thiosulfate solution post sample collection. See 6.19 below.

Fig 11. Set up for ultrafilter priming.

Fig 12. Appropriate configuration of effluent tubing and water flow meter.

Fig 13. Example of dead-end ultrafiltration set up for field sampling.

Fig 14. Dead end ultrafiltration field arrangement diagram.

Put on gloves.
Place clean bench protector(s) on the area(s) where sampling will be performed.
Remove ultrafilter from packaging.

Note
Clearum H22 and Xevonta Hi23 come without dialysates port caps, (Fig. 8, Appendix B). Dialysate storage caps must be attached and secured before starting the procedure (See Fig. 9, Appendix C). Secure the storage dialysate caps with a plastic hose clamp SNP-12 using needle nose pliers.

Fig 8. Ultrafilters with designated ports and outlets.

Fig 9. Original caps removed and replaced with storage caps.

Write the date, time of collection, your initials, and sample number and place ultrafilter on the bench protector.

Note
Ultrafilters should be primed before the sampling procedure with 1L of sterile D.I. water if the turbidity of the sampling water is unknown, measured turbidity is > 7.0 NTU, or if sampling volumes are < 50L. It is preferred that priming take place immediately before sampling is initiated however.

Disinfect scissors with 70% ethanol and allow to dry for Duration00:00:30 on a paper towel. Then, after locating one end of the tubing inside the bag, cut open the bag and cut approximately 2 ft of tubing for use in priming. Place it on the bench protector. All remaining tubing should stay in packaging to the greatest extent possible.
30s
Remove the ultrafilter blood outlet port cap, screw in the DIN adapter and connect one end of priming tubing.
Secure the DIN adapter with the SNP-8 plastic hose clamps when DIN adapter MPC-855NS.375 is used or SNP-04 plastic hose clamp if small DIN adapter MPC-855 is used.

Note
Zip ties can be used alternatively for both DIN adapters.

Place the other tubing end into the bottle of priming liquid and align the center portion of the tubing into the peristaltic water pump head. See Fig. 11, Appendix E. Make sure the pump flow direction “Forward/Reverse” setting is from liquid to ultrafilter, remove the blood inlet port cap and turn on the peristaltic pump at low speed (200-300 rpm) until the liquid emerges from the blood inlet port.

Fig 11. Set up for ultrafilter priming.

Secure the blood inlet port with the storage blood port cap, then carefully detach and discard the tubing from the DIN adapter, positioning the ultrafilter such that the DIN adapter won’t become contaminated while sitting on the bench paper.
Using a permanent marker, indicate by writing an arrow on the filter exterior, the direction of water filtration as confirmation. Water should be entering through the blood outlet port. See Appendix D Fig. 10. Priming step is complete.
Fig 10. Ultrafilters tubing connection configuration.

Attach a new tubing end to a sampling telescoping pole with a zip tie. Leave around three inches off the telescoping pole and approach your water source leaving the remainder of the tubing in the packaging. Place influent tubing into the body of water and ensure the end of the tubing will stay below the surface of the water and away from plant material or other large debris. Be cautious not to disturb the area (especially any sediment) near the filtration site.

If debris or material that may cause premature filter clogging is present, a sterilized mosquito/insect mesh can be attached to the tubbing end with a rubber band or zip tie to avoid clogging the tubing and ultrafilter with debris.
Using disinfected scissors, cut the tubing remaining in the packaging to the desired length.
Insert the tubing into the DIN adapter and secure it with SNP-8 plastic hose clamps for DIN adapter MPC-855NS.375 or SNP-4 for small DIN adapter, MPC-855.
Connect the remaining tubing from the original packaging to the dialysate fluid outlet and direct the effluent downstream from sample intake.

If a flow meter will be used to measure the volume of water filtered, cut the effluent tubing in half after it is connected into the dialysate fluid outlet and screw into the 3/4" GHT water flow meter adapter onto each end. (See Fig. 12 Appendix E). Ensure directional flow of the meter is correct. No clamps are needed.

Fig 12. Appropriate configuration of effluent tubing and water flow meter.

If flow meter is not used, place the tubing into a 20-L cubitainer or other container equivalent to measure the total volume that has passed through the filter. Return the measured water downstream far from the influent water source to avoid disturbing the water near intake. Do this 5 times when a 20-L cubitainer is used to measure 100L water sample. Adjust accordingly for containers of different volumes.
Verify set up is correct before staring the pump. See Appendix F. Make sure the tubing is passing through the water pump head without pinching the tubing and review the flow direction on the “Forward/Reverse” setting is correct.
Start the pump by flipping the switch to ON and turning the knob gradually to maximum speed of 600 rpm.

Note
With the Geotech peristaltic pump, the easy-load II pump head can be in one of two positions (Series #1 Drive or #2 Drive) and either will work fine for the purposes of this method. However, the position to the right (Series #2) will result in faster pumping speeds and thus, shorter sampling times. A #1 Phillips head screwdriver is needed to move the easy-load II pump head from one position to another.

The pump will begin pumping the water through the influent port tubing into the filter and out the side port through the filtrate port tubing. Make sure no ports on the ultrafilter are leaking water.
If sampling outdoors and the ultrafilter and pump is in the sun, cover the ultrafilter and pump with a clean bench protector, large umbrella, or other device to shield it from the sun and prevent pump overheating.
When sampling is completed and the pump is stopped, slowly open the pump head to release any pressure and remove the filtration tubing from the pump head.

If chlorination of sampling water or the presence of free chlorine is suspected, ultrafilters should be treated with a 1% sodium thiosulfate solution post sample collection.

Mix Amount10 g sodium thiosulfate and Amount1 L sterile, D.I. water, shake to dissolve. Place the influent tubing into the 1% sodium thiosulfate solution and pump the entire volume through the filter without pumping air into the ultrafilter.
Unplug the pump from the battery. Remove all tubing from the ultrafilter and place end caps and storage caps on the ports, being careful not to allow water in the ultrafilter to escape.

Note
*IMPORTANT* Be sure endcaps are attached as snug as possible as they tend to leak if not secure. See Fig. 9, Appendix C. Be sure that water does not exit the filter as you are removing the tubing and securing the caps.

Fig 9. Original caps removed and replaced with storage caps.

Used blood port caps, DIN adapters, and rubber storage caps can be returned to laboratory for cleaning and disinfecting before reuse or dispose of material in appropriate trash or biohazard bag.
Tubing that came in contact with the water source should be disposed of and not reused to collect another sample.
Dispose of trash (tubing, gloves, bench protectors, paper towels, etc.) in trash or biohazard bag as per individual laboratory or onsite guidelines.
Label a sealable 2.5 gallon bag (e.g., Ziploc® bag) with the Sample ID, date, time, and your initials.
Place the ultrafilter inside the bag, seal the bag, and gently place in a chilled cooler (wet ice / freezer packs) or in a refrigerator until shipment or processing.
TROUBLESHOOTING
TROUBLESHOOTING
Pulling air instead of water through tubing.

Symptom: Pump is struggling to pull water from source.

Remedy: Stop pump, slowly release pressure in tubing by lifting the pump head clamp and resume pumping after checking to be sure intake tubing end is submerged under the water.
Clogging of tubing or filter

Symptom: Debris is observed in tubing near connection with filter inlet or flow of water out of filter is visibly decreasing.

Remedy: Stop pump, consider if area where debris is can be simply cut out and remainder of tubing reattached; if filter is clogging, it may be a hard stop and end to sampling with current ultrafilter. Note the volume of water filtered on the ultrafilter and on any field datasheets. If debris is noticed before it reaches the filter, the tubing should be disconnected at the DIN adapter and tubing flushed with source water until the debris is removed. Then, reconnect everything and proceed with filtration.
Split tubing

Symptom: Water is leaking where tubing is threaded thru pump head due to crack in tubing.

Remedy: Stop pump, remove tubing from pump head and replace influent tubing piece, clear out any visible sand or debris that might be causing friction in pump head and resume pumping.
BACKFLUSH OF ULTRAFILTERS FOR BACTERIAL PATHOGENS PROCEDURE - Since there exists limited experimental information in terms of viability of bacterial cells following long-term storage of the ultrafilter, it is recommended that filters be processed upon receipt in the laboratory.
BACKFLUSH OF ULTRAFILTERS FOR BACTERIAL PATHOGENS PROCEDURE - Since there exists limited experimental information in terms of viability of bacterial cells following long-term storage of the ultrafilter, it is recommended that filters be processed upon receipt in the laboratory.
2d
2d
Create backflush solution. A Amount500 mL volume is required per ultrafilter to be backflushed.

Note
The backflush solution (0.5% Tween® 80/0.01% NaPP/0.001% Antifoam emulsion) can be made ahead of time but should be used within Duration48:00:00 .

Make Amount10 mL of a 10% NaPP/1% Antifoam emulsion stock solution in a 15 mL conical tube. Note that the use of a larger volume tube facilitates the easier mixing process. It is fine to prepare this stock solution ahead of time and store it for up to one week at Temperature4 °C .

  1. Add Amount1 g NaPP to Amount10 mL sterile D.I. water. Shake vigorously to dissolve the NaPP.
  2. Add Amount100 µL of Antifoam emulsion to the solution and gently mix by inverting repeatedly.

Mix
Temperature
Add Amount500 mL of sterile D.I. water to a 500 mL or 1 L bottle.
Add Amount2.5 mL of Tween® 80 with a 5-10 mL serological pipette.
Pipetting
Stir with a sterile stir bar until the Tween® 80 is dissolved. If stir bar is not used, swirl vigorously (don’t shake) to dissolve the Tween® 80.

Note
Be patient, the Tween® 80 can take a while to dissolve. Attempt to limit the amount of foam produced, but some foam production is inevitable.

Mix
Add Amount500 µL of the 10% NaPP/1% Antifoam emulsion stock solution to the water.
Pipetting
Set aside for later use or store at Temperature4 °C for no more than Duration48:00:00 .
2d
Temperature
If filters have been held at Temperature4 °C or TemperatureOn ice packs from shipping, allow the filters to warm to TemperatureRoom temperature on bench for approximately Duration01:00:00 .
1h
Temperature
Record all pertinent details of the ultrafilter sample on your 1-Liter sample collection bottle.
Place clean bench protectors on the lab bench where backflushing will be performed.
Position the peristaltic pump on a level surface or on a lift at least 20” above the bench surface.
Secure filter vertically using the stand clamps pointing the sample inlet port downwards, and the sampling effluent side port facing through the water pump as shown in Fig. 16 and 17 in Appendix G.

Fig. 16. The ultrafilter is secured vertically using the stand clamps with the sample inlet port pointing downwards, and the effluent side port oriented towards the pump.

Fig. 17. During the ultrafilter set up, the sample inlet port points downwards, and the effluent side port faces the pump. The sample receiving bottle is placed under blood return port without touching the bottle surfaces. The tubing is attached to the dialysate outlet port by pushing onto the port and is secured with a SNP-12 clamp or a zip tie.

Note
Confirm the filtration direction by locating an arrow written on the filter surface by the collector. The water flow should have occurred through the blood outlet port. See Appendix B Fig. 8. If filtration happened instead through the blood inlet port, the backflush process should occur with this port facing downwards (i.e., backflush should always occur in the reverse direction from sample filtration).

Fig 8. Ultrafilters with designated ports and outlets.

Position a 1-L bottle directly under the end port, making sure the ultrafilter doesn’t touch the opening of the bottle.
Unscrew and remove the cap from the filter end port facing downwards (blood outlet port). Backflush solution will exit this port and into the 1-Liter bottle upon backflushing.
Disinfect the scissors with 70% ethanol and cut around 3ft section of tubing. Remove the cap from the top side port (dialysate outlet port). Push one end of the tubing fully onto the port as much as possible.
Feed the tubing through the peristaltic pump head and place the other end into the Amount500 mL backflush solution.
Secure the tubing attached on the upper side port (dialysate outlet port) with either a hose clamp SNP-12 or zip-tie. This tubing can slip off the side port when under pressure and must be secured.
The blood inlet port and dialysate inlet port (top port and bottom side port, respectively) must remain secured. Check to ensure they are finger tightened and ensure that a SNP-12 hose clamp, or a zip tie was applied to the bottom side port (dialysate inlet port) adequately to prevent the rubber cap from leaking or coming off.
Put on face shield if part of laboratory safety requirements.
Close the pump head lever.
Ensure pump flow direction is set from backflush solution to ultrafilter. Pump backflush solution through the ultrafilter at 650 mL/min (or 250-300 rpms), capturing backflush solution into the bottle. If the pump doesn’t have a digital display set the dial to a slow pumping rate.
Continue pumping until no backflush solution remains in the Amount500 mL bottle and the flow out of the ultrafilter has slowed to a trickle. It will be necessary to briefly pump air after the backflush solution is depleted to get as much liquid out of the filter as possible. Do not pump air into the filter for more than approximately Duration00:00:10 .
10s
Turn off the pump.
Release the tubing from the pump head slowly, with one hand on the pump and the other hand releasing the lever gradually. BE VERY CAREFUL - THE SYSTEM IS UNDER PRESSURE.
Cap the 1-Liter bottle and ensure the bottle is properly labeled. This is your backflush sample. Refrigerate until analysis. Measure and record backflush sample volume, if necessary. Backflush sample volume is typically between Amount500 mL -Amount700 mL .
Remove the tubing from the ultrafilter by releasing the SNP-12 hose clamp with needle nose pliers.
After backflushing, the ultrafilter may be stored if required by individual laboratory protocols for sample remains. However, backflushing the ultrafilter a second time is not likely to result in the additional recovery of bacterial pathogens. Be sure to cap all open ports (side and end) to prevent leaks.
Discard bench protector. Disinfect area with 10% bleach followed by 70% ethanol.
Proceed to pathogen-specific detection method or desired secondary concentration method.