Dead-end Ultrafiltration

_Part I

Equipment and Supplies

• Geopump™ Peristaltic Pump Series II Package (includes easy-load II pump head and a portable battery), Geotech, Cat No. 91352123
• Pump Assembly (or equivalent assembly):
  • DIN adapter (for End Port), Molded Products, Cat. No. MPC855 NS.375
  • Blood port (End) cap, Molded Products, Cat. No. MPC-40
  • Dialysate Port (Side) cap, Molded Products, Cat. No. MPC-60D
  • L/S 36 tubing (9.7mm ID), Cole Parmer, Cat. No. EW-96410-36 or EW-06434-36
  • SNP-8 hose clamps (for DIN adapter), Cole Parmer, Cat. No. EW-06832-08
  • SNP-12 hose clamps (for side port), Cole Parmer, Cat. No. EW-06832-12 (optional)
• Hollow fiber ultrafilter Rexeed-25S (Asahi Kasei Medical Co.)
• 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.
• 500 ml plastic bottles for turbidity measurement samples and sodium thiosulfate solution.
• Flow meter
• Scissors (required to cut tubing)
• Pliers (to close the hose clamps)
• Autoclavable biohazard waste bags
• Appropriate Personal protective equipment (PPE) such as hand sanitizer, gloves, protective eyewear, and lab coats as needed.

Reagents

• Sodium thiosulfate, Fisher Cat. No S446 (Only needed for chlorinated water)

Recovery and Detection of Cyclospora cayetanensis from Ultrafilters

_Part II

Laboratory Facilities

• A laboratory bench for the ultrafilter backflushing and concentration procedures.
• A laboratory bench or hood for the DNA extraction procedure.
• Separate workstations for each step of the qPCR protocol to allow an efficient workflow and eliminate the potential for positive results due to laboratory contamination.

Equipment and Supplies

• Geopump™ Peristaltic Pump Series II Package (includes easy-load II pump head and batteries), Geotech, Cat No. 91352123
• Turbidity Meter 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.
• Pump Assembly (or equivalent assembly):
  • DIN adapter (for End Port), Molded Products, Cat. No. MPC855 NS.375
  • Blood port (End) cap, Molded Products, Cat. No. MPC-40
  • Dialysate Port (Side) cap, Molded Products, Cat. No. MPC-60D
  • L/S 36 tubing (9.7mm ID), Cole Parmer, Cat. No. EW-96410-36 or EW-06434-36
  • SNP-8 hose clamps (for DIN adapter), Cole Parmer, Cat. No. EW-06832-08
  • SNP-12 hose clamps (for side port), Cole Parmer, Cat. No. EW-06832-12
• Laboratory lift stand for pump (optional)
• Laboratory ring stand and clamps to support filter during backflush procedure
• 1 L glass beaker for collecting backflushed sample, Fisher, Cat. No. NC9370942
• 1-L Nalgene bottles, Fisher, Cat. No. 02-893D
• 500-ml Nalgene bottles, Fisher, Cat No. 02-893C
• Centrifuge (or equivalent assembly) capable of spinning 15 ml conical tubes and 175 ml centrifuge bottles at 4000 x g:
  • Thermo Scientific™ Sorvall™ Legend™ XTR Centrifuge Package, Fisher, Cat. No.75-217-420. Package must include:
    • Thermo Scientific™ Sorvall™ Legend™ TX-1000 high capacity rotor
    • 15 ml Conical Tube Adapters, set of 4
  • 250/175ml bottle adapters for TX-1000 Swinging Bucket Rotor, sold as set of 4, Fisher, Cat. No. 75005392. Not included in above centrifuge package, must be ordered separately.
• Vacuum aspiration system (or equivalent assembly):
  • Foxx Life Sciences, Vactrap™, PP (Autoclavable), 4L, Red Bin, SKU: 305-4001-FLS
  • Vacuum pump or house vacuum
  • Glass Pasteur pipettes or equivalent disposable plastic pipettes for vacuum aspiration of supernatants
• Autoclavable Polypropylene Centrifuge Tubes, 225 ml, Fisher, Cat. No. 05-538-61
• Conical Sterile Polypropylene Centrifuge Tubes, 15 ml, Fisher, Cat. No. 339650
• FastPrep®-24 Instrument (bead beater), 5G (SKU 116005500) or Classic (SKU 116004500), MP Biomedicals
• Benchtop microcentrifuge (24 tube)
• Heat block with core for 2.0 ml microcentrifuge tubes
• Benchtop Vortex mixer
• Eppendorf Repeater Repeating Pipette and assorted Combitips (optional)
• Sterile DNase-free polypropylene microcentrifuge tubes, 1.5 ml
• Sterile DNase-free polypropylene microcentrifuge tubes, 2.0 ml
• Eppendorf Tubes® with snap cap, 5.0 ml Biopur, Fisher, Cat. No. 14-282-303 (optional)
• Sterile applicator Sticks, Fisher, Cat. No. 22-029-641
• Applied Biosystems 7500 Fast Real-Time PCR System with Software versions 1.4, 2.0, or 2.3 or newer, ThermoFisher Scientific
• VWR Mini Centrifuge (or comparable), VWR, Cat. No. 76269-064
• Applied Biosystems MicroAmp® Fast 8-Tube Strip, 0.1 ml and MicroAmp® Optical 8-Cap Strips, Cat Nos. 4358293 and 4323032, (ThermoFisher Scientific) or equivalent, OR Applied Biosystems MicroAmp® Fast Optical 96-Well Reaction Plates, 0.1 ml, and MicroAmp® Optical Adhesive Film, Cat Nos. 4346907 and 4311971, (ThermoFisher Scientific) or equivalent
• Benchtop centrifuge capable of spinning 96-Well reaction plates OR a mini-centrifuge capable of spinning 0.1 ml tube strips
• Analytical Balance (0.01g resolution)
• Magnetic Stir Plate
• Teflon coated stir bar
• Digital laboratory timer
• Scalpel or scissors (required to cut tubing)
• Pliers (to close the hose clamps)
• Pipet-Aid
• 1 ml serological pipettes
• 2 ml serological pipettes
• 5 ml serological pipettes
• 10 ml serological pipettes
• Micropipettors
• 2 µL aerosol resistant pipette tips
• 20 µL aerosol resistant pipette tips
• 200 µL aerosol resistant pipette tips
• 1000 µL aerosol resistant pipette tips
• Transfer pipettes
• 15 ml conical tubes
• Appropriate personal protective equipment (PPE) such as hand sanitizer, protective eyewear, gloves and lab coats as needed.

Reagents

• Sodium polyphosphate (NaPP), Sigma, Cat. No. 305553-25G
• Tween 80, Fisher, Cat. No. T164-500 ml
• Antifoam Y-30 emulsion, Sigma, Cat. No. A5758-250 ml
• Dulbecco’s Phosphate Buffered Saline (PBS), no calcium, no magnesium, Thermo Fisher Scientific, Cat. No. 14190250, (or equivalent)
• FastDNA SPIN Kit for Soil, 50 preps, MP Biomedicals, Fisher, Cat. No. MP116560200
• QIAquick® PCR Purification Kit (50), Qiagen, Cat. No. 28106
• 100% Ethanol
• 3 M Sodium Acetate Solution, pH 5.2, Fisher Scientific, Cat. No. FERR1181
• Sterile DNase-free TE buffer, pH 7.5, (commercially prepared or refer to FDA BAM Chapter 19B, Appendix 2)
• DNA AWAY Surface Decontaminant, Fisher, Cat. No. 21-236-28 OR 10% bleach
• Real-time PCR reagents (refer to FDA BAM Chapter 19B for additional details and ordering instructions)
  • QuantiFast Multiplex PCR kit (400), Qiagen, Cat. No. 204654
  • Negative control (Water, included in the QuantiFast Multiplex PCR Kit above)
  • Real-time PCR primers for Cyclospora and IAC amplification
  • Real-time PCR probes for Cyclospora and IAC detection
  • IAC Target (HMultra130-synIAC)
  • Positive control DNA (HMgBlock135m)