Materials and Equipment

• BagPage®+ filter bags (400 ml) and Bag Clips (Interscience, St Nom, France)
• Envirochek™ sampling capsule, 1 µm nominal (Pall Gelman Laboratory)
• Rocker platform
• Rotary shaker
• Rotating wheel
• 150 ml analytical filter units (Nalgene, Cat No. 130-4045)
• 25 mm disposable filter funnels (Whatman Biosciences)
• Dynal L10 tubes Prod. No. 740-03)
• Dynal MPC®-1 (Prod. No 120.01)
• Dynal MPC®-S (Prod. No 120.20)
• Vacuum manifold (Vac-Man® Laboratory Vacuum Manifold 20-sample capacity; Promega)
• FTA Filters, Classic Card of 960circle pre-printed card formats (Whatman Biosciences)
• Photo laminating sheets (Scotch®)
• Conical 250 ml centrifuge tubes
• Sorvall RT7 Plus refrigerated centrifuge or equivalent (to centrifuge 250 ml conical centrifuge tubes)
• Epi-illuminated Fluorescence Microscope-equipped with the following: UV 1A filter block (excitation Filter, EX 365/10; Dichroic mirror, DM 440; Barrier Filter, BA-400; or equivalent. Optics for differential interference contrast (DIC). For Cryptosporidium spp.: appropriate filter for viewing fluorescein isothiocyanate (FITC) conjugated oocysts. (Table 1)
• Glass microscope slides and cover slips
• Blotting paper
• Heating block for incubation at 56°C
• Single punch (6 mm diameter) hole puncher
• Thin-walled 0.65 ml PCR tube (PGC)
• ART pipette tips (Molecular BioProducts)
• PTC-200 DNA Engine (MJ Research) or comparable thermal cycler.
• Horizontal gel electrophoresis apparatus and power supply.
• Polaroid camera or digital imaging system to capture ethidium bromide-stained gels
• Polaroid Type 667 film
• UV Transilluminator

Reagents

• Water
  • Deionized water (for washing produce [dH2O])
  • Sterile deionized water (for PCR procedure)
• Envirochek™ elution buffer (0.01M Tris, pH 7.4 ; 0.001 M EDTA; 1% SDS)
• Silicone vacuum grease
• Albumin, bovine (BSA) (Sigma, A-7030)
• Celite (Sigma, C-8656)
• Polyvinyl polypyrrolidone (PVPP) (Sigma, P-6755)
• NET buffer: 0.1 M Tris, pH 8.0, 0.15M NaCl, 0.001M EDTA
• NET-BSA buffer-NET buffer containing 1% (w/v) BSA
• 20% celite in NET-BSA buffer (w/v)
• 10% PVPP (w/v) in dH2O
• Dynabeads anti-Cryptosporidium Kit (Dynal Biotech Inc, USA)
• Hydrofluor Combo Detection Kit for Cryptosporidium and Giardia (Strategic Diagnostics, Inc.)
• 0.1 N HCl
• 0.1 N NaOH
• Immersion oil
• Clear fingernail polish, slide compound, paraffin wax or equivalent
• FTA Purification Buffer (Whatman Biosciences)
• FTA filter wash buffer: 0.01 M Tris, Ph 8.0; 0.1 mM EDTA
• DNA Primers-See Table 1 in PCR Section
• HotStartTaq™ Master Mix Kit (Qiagen)
• 0.5x Tris-acetate-EDTA buffer (0.5x TAE)
• Molecular biology-grade agarose (BioRad)
• Ethidium bromide
• 6x gel loading solution
• 100 bp and 25 bp DNA ladders (Invitrogen)
• Vsp I restriction endonuclease (Promega)
• Dra II restriction endonuclease (Hoffman-La Roche)
• NuSieve® 3:1 agarose (Biowhitaker Molecular Applications)

Optional Reagents, Supplies, and Equipment

• LightCycler®-FastStart DNA Master SYBR Green I kit (Roche Diagnostics)
• LightCycler® Capillaries (Roche Diagnostics)
• LightCycler® System (Roche Diagnostics)