Envirochek® HV Sampling Capsule Protocol

1.1 Envirochek HV sampling capsule, one capsule per sample. The capsules are not reusable.

1.2 Hose-type tubing, 12.7 mm (0.5 in.) I.D.

1.3 Clamps

1.4 Water Meter (flow totalizer)

1.5 Flow control valve

1.6 Pump, electric or gasoline powered (if needed)

1.7 Laboratory shaker (Pall Gelman Laboratory PNs 4821, 4822)

1.8 Ice chest or cooler with cold packs

1.9 250 mL conical centrifuge tubes

1.10 50 mL polypropylene tubes

1.11 Elution Buffer:

• 1 g Laureth 12 (PN 4820)
• 10 mL 1 M Tris, pH 7.4 (see 1.11.1)
• 2 mL 0.5 M Ethylenediamine Tetraacetic Acid (EDTA), 2 Na,dihydrate pH 8.0 (see 1.11.2)
• 150 µL Antifoam A
• 1 L Deionized (DI) Water

1.11.1 Preparation of 1 M Tris, pH 7.4: Dissolve 121.1 g Tris in 700 mL DI water and adjust pH to 7.4 using 1 N HCL or NaOH. Then bring volume to 1 L using DI water. Filter-sterilize through a 0.2 µm membrane (Pall Gelman Laboratory PN 4480) into a sterile container and store at room temperature.

1.11.2 Preparation of 0.5 M EDTA, 2 Na dihydrate, pH 8.0: Dissolve 186.1 g EDTA, 2 Na dihydrate in 800 mL and adjust to pH 8.0 using 1 N HCL or NaOH. Then bring volume to 1 L using DI water.

1.11.3 Preparation of Buffer Solution: Weigh 1 g Laureth-12 in a glass beaker and add 100 mL of DI water. Heat the beaker to melt the Laureth-12 using a hot plate or microwave and transfer the solution to a 1000 mL volumetric flask. Rinse the beaker several times to ensure the transfer of the detergent to the flask. Add 10 mL Tris solution, pH 7.4; 2 mL of EDTA solution, pH 8.0; and 150 µL Antifoam A. Bring solution to a final volume of 1000 mL with DI water. The buffer will have an opaque appearance.

2.1 Retain the blue vinyl caps provided with the Envirochek HV sampling capsule for sample shipping and the elution process.

2.2 The exact installation is dependent on whether or not the water is from a pressurized water source. If the source is not pressurized, a pump is required. If a pump is required the system will appear as in Diagram 1a. If a pump is not required, the system will appear as in Diagram 1b.

2.3 Before connecting the sampling system to the pressurized system or sample source, turn the pressurized system or pump on, allowing water to purge residual debris from the water lines for 2 to 3 minutes.

2.4 Turn off the pressurized source or pump and connect the sampling system without the Envirochek® HV sampling capsule.

2.5 Turn on the pressurized source or pump and adjust the flow rate to the desired volume.

2.6 Allow approximately 76 L (20 gal) to flush the system.

2.7 Install the Envirochek HV capsule in line, securing the inlet and outlet ends with the appropriate fittings/clamps.

2.8 With a water-resistant marking pen, record the sampling location, name of the person performing the sampling, date, meter reading, and turbidity of the sample on the label provided on the filter housing.

2.9 Initiate water flow.

2.10 Vent the residual air in the capsule using the vent valve by turning it in counter clockwise direction. When the filter housing is full of water, quickly close the vent valve.

2.11 After you have collected your sample through the Envirochek HV sampling capsule, shut off your water source. Allow the pressure to decay until flow stops.

2.12 Record the stop time and final meter reading in the appropriate spaces on the label on the filter.

2.13 Disconnect the inlet end of the Envirochek HV sampling capsule, making sure not to spill any of the water remaining in the capsule. This water is part of your sample.

2.14 Seal the inlet of the capsule with the blue vinyl end cap that was saved from step 2.1.

2.15 Loosen the outlet end and allow water to drain as much as possible. Water drainage from the capsule through the outlet is acceptable since the sample has passed through the membrane.

2.16 Seal the outlet of the capsule with the blue vinyl end cap that was saved from step 2.1.

2.17 The Envirochek HV capsule should be placed on cold packs for storage or transport to the testing facility.

Note: Sample should be stored at 0 - 8 °C from time of filtration. Sample testing must be completed within 36 hours of sample collection.

3.1 Assemble the laboratory shaker with the arms at full extension and the clamps aligned in a vertical position so that the capsules will be aligned horizontally, as demonstrated in Figure 1.

3.2 Prepare sufficient elution buffer so all the samples to be eluted that day can be eluted with the same stock elution buffer solution. (see 1.11)

3.3 Designate a 250 mL conical centrifuge tube for each sample and label with the sample number.

3.4 If the upper chamber of the capsule has water remaining in it, carefully decant this water into an appropriately labeled conical centrifuge tube to be used in section 4.0.

3.5 Hold the capsule in a vertical position with the inlet end up by using a ring stand or other means. Remove the end cap.

3.6 Add elution buffer to the inlet end of the capsule and allow liquid level to stabilize. Sufficient elution buffer should be added to the capsule to ensure the covering of the pleated white filter module by approximately 13 mm (0.5 in.) (see Diagram 2). Replace the blue vinyl end cap to the inlet end of the capsule.

3.7 Securely clamp the capsule in one of the clamps on the laboratory shaker such that the vent valve is facing toward you. Make sure the vent valve is in the 12 o'clock position (see Diagram 3).

3.8 Turn on the shaker and set the speed at approximately 600 rpm and agitate for 5 minutes.

3.9 Remove the capsule from the shaker. Carefully remove the inlet end cap and decant the contents of the capsule into a 250 mL conical centrifuge tube.

3.10 Add new elution buffer to the inlet end of the capsule and allow liquid level to stabilize. Sufficient elution buffer should be added to the capsule to ensure the covering of the pleated white filter module by approximately 13 mm (0.5 in.). (See Diagram 2.) Replace the blue vinyl end cap to the inlet end of the capsule.

3.11 Again, securely clamp the capsule in one of the clamps on the laboratory shaker such that the vent valve is facing toward you. This time make sure the vent valve is in the 4 o'clock position (see Diagram 3).

3.12 Turn on the shaker and set the speed at approximately 600 rpm and agitate for 5 minutes.

3.13 Turn off the shaker and loosen the clamp but do not remove the capsule or decant the elution buffer.

3.14 Rotate the capsule until the vent valve is in the 8 o'clock position (see Diagram 3), secure the capsule in place, and agitate at 600 rpm for 5 minutes.

3.15 As in step 3.9, remove the capsule from the shaker, carefully remove the inlet end cap and decant the contents of the capsule into the same 250 mL conical centrifuge tube labeled for that sample.

4.1 Spin the sample at 1000-1100 x G for 10 to 15 minutes. Allow the centrifuge to coast to a stop. DO NOT USE THE BRAKE!

4.2 Record the volume of the pellet with date and time that the concentration was completed. Use a Pasteur pipette to carefully remove the supernatant to just above the pellet.

4.2.1 If the pellet volume (volumes of solid) is 0.5 mL or less, add reagent water to the centrifuge tube to bring the volume up to 10 mL. To resuspend the pellet, vortex the tube for 10 to 15 seconds. Proceed to step 5.0.

4.2.2 If the pellet volume is greater than 0.5 mL, use the equation below to determine how much reagent water needs to be added to the centrifuge tube to adjust the pellet to 0.5 mL:

Add reagent water to the centrifuge tube to the calculated volume above. Vortex the centrifuge tube for 10 to 15 seconds to resuspend the pellet. Record the volume. Using a Pasteur pipette, reduce the resuspended volume to 10 mL (which will contain 0.5 mL of solids). Proceed to step 5.0.

5.1 The sample is now ready for the detection stage of the process. Refer to the current approved detection method for your region.