A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

The overall goal of this procedure is to detect and visualize waterborne, cryptosporidium, and giardia species. This is accomplished by first concentrating the water sample using tangential flow, hollow fiber, ultra filtration. The second step is to elute the organisms and trap debris from the filter following a centrifugation step.

Immuno magnetic separation allows for selective separation of the parasites from the concentrated debris. Magnetic beads are detached from the parasites by heat dissociation and removed using a magnet. Cysts and O cysts are then placed on a slide, and the sample is stained for visualization by immunofluorescence microscopy.

The main advantage of hofi filtration over existing techniques is that the ultra filter is significantly less expensive and captures multiple microorganisms in addition to Cryptosporidium and Giardia like viruses and bacteria. Generally, individuals new to immuno separation will be challenged by the amount and type of debris associated with different samples from very climate conditions. Start by assembling the ultra filtration apparatus inside a biosafety cabinet.

Once assembled the apparatus functions as follows, the water sample enters into the system by passing through the black T connector and moves through the pump head the pressure gauge and into the hollow fiber ultra filter, the filtered water passes out of the side of the filter into the waste container. The retained water containing the organisms are forced into the retent tape bottle and recirculated through the system until the volume of water is reduced. To begin the procedure, remove the vent cap and open all clamps except for pinch clamp two, which should be closed to allow the reten ate bottle to fill with the water, set the pump direction such that the water sample will flow into the filter.

Then adjust the pump speed to 25%and turn on the pump when the renate bottle is two thirds full open. Pinch clamp two and quickly seal the bottle tightly using the vent cap. Check all the lines and fittings to ensure that there are no leaks.

Then slowly increase the pump speed to about 1.5 liters per minute using a graduated cylinder and timer or flow meter, verify that the filtrate rate the rate of the water exiting from the effluent line is indeed 1.5 liters per minute. Monitor the filtration process ensuring that the reten bottle never empties, although the volume may increase or decrease slightly. If the volume drops below one third, remove the vent cap and close pinch clamp two.

Again, allow the volume in the bottle to increase to two thirds. Then replace the vent cap tightly and open clamp two again. Once the sample container is empty, close pinch clamp one immediately reduce the pump speed to 20%Remove the vent cap from the retent ate bottle and close the ramp clamp connecting the filter column effluent tube to the waste tank.

Tighten or loosen the ramp clamp to adjust the volume in the retent ate bottle to about 200 milliliters. Then tighten the ramp clamp for the elucian to elute the sample. First, add 500 milliliters of elucian solution to the sample container, rinsing the inside of the container in the process.

Then place the 10 milliliter pipette connected to the sample uptake line into the elucian solution container. Open pinch clamp one and close pinch clamp two momentarily to draw up the elucian solution. Once all the solution has been loaded, close pinch clamp one, open pinch clamp two and allow the solution to circulate for five minutes with a pump speed of 20%tighten or loosen the ramp clamp to adjust the volume of sample in the renate bottle to about 100 milliliters.

Then tighten the ramp clamp completely and allow the sample to recirculate for one minute. At this point, reverse the direction of the pump for 20 seconds. This will result in the accumulation of about 225 milliliters of sample in the tate bottle.

Then turn off the pump. Remove the tubing from the pump head and tubing exiting from the filter and hold the tubing over the tate bottle to force any remaining sample into the bottle. Then disconnect all tubing from the bottle and replace the venting cap with a non venting cap.

Transfer the concentrated sample to a 250 milliliter conical centrifuge tube. Rinsing the renate bottle twice with 10 milliliters of reagent water and transferring the rinse fluid to the centrifuge tube. Centrifuge the sample at a minimum of 1500 times G for 15 minutes.

Do not use a break to stop the centrifugation. Following the centrifugation, carefully aspirate the supernatant to five milliliters above the pellet. Extra care should be taken to remain at the top of the liquid level and avoid aspirating O cysts and cysts that are within the pellet.

Vortex the tube to resuspend the pellet thoroughly. Then transfer the sample to a flat sided dyal L 10 tube containing one milliliter each of 10 X SL buffer A and buffer B.Rinse the centrifuge tube twice with 2.5 milliliters of reagent water and add the rinse solution to the dyal L 10 tube. Bringing the final volume to 12 milliliters, add 100 microliters each of magnetic bead conjugated, anti cryptosporidium and anti giardia antibodies to the tube.

Then rotate the tube at 18 RPM for one hour at room temperature on a rotating mixer after rotation. Place the flat side of the tube against the MP six magnet and gently rock the tube for two minutes without removing the tube from the magnet. Keep the flat side up and decant the supernatant.

Being careful not to disturb the beads retained on the flat side of the tube near the magnet. Remove the sample tube from the magnet and rinse the flat side of the tube three times with 500 microliters of buffer A avoiding the debris on the round side of the tube. Transfer each rinse volume to a 1.5 milliliter micro centrifuge tube held inside an MPCS magnet.

With the magnet in the vertical position. Close the tube and gently rock it for one minute. Then with the magnet in place, aspirate off the supernatant using a pasture pipette positioned at the bottom of the tube gently add one milliliter of PBS to the tube.

Take care not to disturb the bead pellet. Attach to the wall of the tube adjacent to the magnet. Remove the magnetic strip and rock the tube gently until the beads are resuspended.

Reinsert the magnetic strip into the vertical position. Rock the tube gently for a minute and aspirate the supernatant, removing as much debris as possible without disturbing the beads. After this wash, remove the magnet and add 50 microliters of reagent water directly onto the bead palette.

Vortex the tube at full speed for 50 seconds to resuspend the beads. Then incubate the sample at 80 degrees Celsius for 10 minutes, allowing the Giardia cysts and cryptosporidium o cysts to dissociate from the beads After the incubation, vortex the sample for 30 seconds. Then place the magnetic strip into the MPCS using the slanted position to separate the beads from the cysts and O cysts in suspension.

Transfer the supernatant containing cysts and O cysts to a well slide. Repeat the water addition vortex and heat dissociation steps a second time adding the supernatant to the same slide. Well then place the slide on a 37 degrees Celsius slide warmer to dry the suspension.

Start by applying 50 microliters of methanol to the slide to fix the sample and allow it to dry at room temperature. Next, add 50 microliters of DPI solution and incubate the slide for two minutes at room temperature. To stain the nuclei, use a Kim wipe to wick the dappy from the well being careful not to touch the well.

Then add 50 microliters of easy stain to stain the cell wall of the Giardia cysts and cryptosporidium o cysts. Incubate the sample at 35 degrees Celsius for 30 minutes. Remove the stain with the Kim wipe as before.

Then slowly add 300 microliters of cold easy stain fixing buffer by carefully placing the pipette tip outside of the well and allowing the liquid to move into the, well. Incubate the slide for two minutes at room temperature. Then remove the buffer using a kim wipe and apply 10 microliters of mounting medium gently place a cover slip on the sample, removing any bubbles, and seal the cover slip with clear nail polish Using a fluorescent microscope, examine the slide at a 200 x to 1000 x magnification and a fse then DPI filter at a 200 x magnification cysts and O cysts appear as brilliant apple green fluorescent ovoid structures with brightly highlighted edges.

Cryptosporidium O cysts are round and range in size from four to six microns. Giardia cysts are larger with a width of five to 15 microns and eight to 18 microns in length and can be round or ovoid at a 1000 x magnification. And using the DPI filter, the internal structure of the cysts and CYS can be better appreciated.

Typically, up to four sporozoites, which appear as sky blue DPI stain nuclei are visible within the Cryptosporidium O cys and Giardia Cys.DIC. Microscopy can also aid in the proper identification of parasites as additional morphological characteristics may be appreciated. Cryptosporidium sporozoites may be visible by DIC Giardia cysts often exhibit one or more discernible internal structures such as visible nuclei, crescent shaped organelles called median bodies, and internal flagellar structures called axons.

It is important to note that the holo fiber ultra filtration procedure has the ability to capture multiple microorganisms. In addition, other detection assays like QPCR and genotyping can be applied to the concentrated samples, which makes this method more flexible and more adaptable to your applications. Thank you for watching and let us know if you have any questions.