<IP_ADDRESS> Setup <IP_ADDRESS>.1 Assemble the laboratory shaker with the clamps aligned vertically so that the filters will be aligned horizontally. Extend the clamp arms to their maximum distance from the horizontal shaker rods to maximize the shaking action. <IP_ADDRESS>.2 Prepare sufficient elution buffer so that all samples to be eluted that day can be eluted with the same batch of buffer. Elution may require up to 275 mL of buffer per sample. <IP_ADDRESS>.3 Designate at least one 250 mL conical centrifuge tube for each sample and label with the sample number. <IP_ADDRESS> Elution <IP_ADDRESS>.1 Record the elution date and time on the bench sheet. Using a ring stand or other means, clamp each capsule in a vertical position with the inlet end up. Remove the inlet cap and allow the liquid level to stabilize. Method 1623  Cryptosporidium and Giardia June 2003 37 <IP_ADDRESS>.2 Pour elution buffer through the inlet fitting. Remove the inlet cap and allow the liquid level to stabilize. Sufficient elution buffer must be added to cover the pleated white membrane with buffer solution. Replace the inlet cap and clamp the cap in place. <IP_ADDRESS>.3 Securely clamp the capsule in one of the clamps on the laboratory shaker with the bleed valve positioned at the top on a vertical axis ( in the 12 o'clock position). Turn on the shaker and set the speed to maximum ( approximately 900 rpm). Agitate the capsule for approximately 5 minutes. Time the agitation using a lab timer, rather than the timer on the shaker to ensure accurate time measurement. <IP_ADDRESS>.4 Remove the filter from the shaker, remove the inlet cap, and pour the contents of the capsule into the 250 mL conical centrifuge tube. <IP_ADDRESS>.5 Clamp the capsule vertically with the inlet end up and add sufficient volume of elution buffer through the inlet fitting to cover the pleated membrane. Replace the inlet cap. <IP_ADDRESS>.6 Return the capsule to the shaker with the bleed valve positioned at the 4 o'clock position. Turn on the shaker and agitate the capsule for approximately 5 minutes. <IP_ADDRESS>.7 Remove the filter from the shaker, but leave the elution buffer in the capsule. Re clamp the capsule to the shaker at the 8 o'clock position. Turn on the shaker and agitate the capsule for a final 5 minutes. <IP_ADDRESS>.8 Remove the filter from the shaker and pour the contents into the 250 mL centrifuge tube. Rinse down the inside of the capsule filter walls with reagent water or elution buffer using a squirt bottle inserted in the inlet end of the capsule. Invert the capsule filter over the centrifuge tube and ensure that as much of the eluate as possible has been transferred. 12.2.7 Proceed to Section 13.0 for concentration and separation ( purification). 12.3 Sample filtration using the Filta Max   foam filter. This procedure was validated using 50 L sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water and source water samples ( Section 9.1.2).

NOTE: The filtration procedures specified in Sections <IP_ADDRESS>  <IP_ADDRESS>.3 are specific to laboratory filtration of a bulk sample. These procedures may require modification if samples will be filtered in the field.

12.3.1 Filtration <IP_ADDRESS> Flow rate adjustment <IP_ADDRESS>.1 Connect the sampling system, minus the filter housing, to a carboy filled with reagent water. <IP_ADDRESS>.2 Place the peristaltic pump upstream of the filter housing. Method 1623  Cryptosporidium and Giardia June 2003 38 <IP_ADDRESS>.3 Turn on the pump and adjust the flow rate to 1 to 4 L per minute.

NOTE: A head pressure of 0.5 bar ( 7.5 psi) is required to create flow through the filter, and the recommended pressure of 5 bar ( 75 psi) should produce the flow rate of 3 to 4 L per minute. The maximum operating pressure of 8 bar ( 120 psi) should not be exceeded.

<IP_ADDRESS>.4 Allow 2 to 10 L of reagent water to flush the system. Adjust the pump speed as necessary during this period. Turn off the pump when the flow rate has been adjusted. <IP_ADDRESS> Place filter module into the filter housing and secure lid, hand tighten housings, apply gentle pressure to create the seal between the module and the ` O' rings in the base and the lid of the housing. Excessive tightening is not necessary, and may shorten the life of the ` O' rings. Tools may be used to tighten housing to the alignment marks ( refer to manufacturer's instructions). ` O' rings should be lightly greased before use ( refer to manufacturer's instructions). <IP_ADDRESS> Install the filter housing in the line, securing the inlet and outlet ends with the appropriate clamps/ fittings. Verify that the filter housing is installed so that the end closest to the screw top cap is the inlet and the opposite end is the outlet. <IP_ADDRESS> Record the sample number, sample turbidity ( if not provided with the field sample), and the name of the analyst filtering the sample on a bench sheet. <IP_ADDRESS> Filtration <IP_ADDRESS>.1 Connect the sampling system to the field carboy of sample water, or transfer the sample water to the laboratory carboy used in Section <IP_ADDRESS>.1. If the sample will be filtered from a field carboy, a spigot can be used with the carboy to facilitate sample filtration.

NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory carboy must be cleaned and disinfected before it is used with another field sample.

<IP_ADDRESS>.2 Place the drain end of the sampling system tubing into an empty graduated container with a capacity greater than or equal to the volume to be filtered. This container will be used to determine the sample volume filtered. Alternately, connect a flow meter downstream of the filter, and record the initial meter reading. <IP_ADDRESS>.3 Allow the carboy discharge tube and filter housing to fill with sample water. Turn on the pump to start water flowing through the filter. Verify that the flow rate is between 1 and 4 L per min. <IP_ADDRESS>.4 After all of the sample has passed through the filter, turn off the pump. Allow the pressure to decrease until flow stops. <IP_ADDRESS> Disassembly <IP_ADDRESS>.1 Disconnect the inlet end of the filter housing assembly while maintaining the level of the inlet fitting above the level of the outlet fitting to prevent backwashing and the loss of oocysts and cysts from the filter. Restart the Method 1623  Cryptosporidium and Giardia June 2003 39 pump and allow as much water to drain as possible. Turn off the pump. <IP_ADDRESS>.2 Based on the water level in the graduated container or the meter reading, record the volume filtered on a bench sheet to the nearest quarter liter. <IP_ADDRESS>.3 Loosen the outlet fitting, the filter housing should be sealed with rubber plugs.

NOTE: Filters should be prevented from drying out, as this can impair their ability to expand when decompressed.

12.3.2 Elution <IP_ADDRESS> The filter is eluted to wash the oocysts from the filter. This can be accomplished using the Filta Max   wash station, which moves a plunger up and down a tube containing the filter and eluting solution ( Section <IP_ADDRESS>), or a stomacher, which uses paddles to agitate the stomacher bag containing the foam filter in the eluting solution ( Section <IP_ADDRESS>). If the Filta MaxTM automatic wash station is used please see the manufacturer's operator's guide for instructions on its use. <IP_ADDRESS> Filta Max   wash station elution procedure <IP_ADDRESS>.1 First wash ( a) Detach the removable plunger head using the tool provided, and remove the splash guard. ( b) Place the filter membrane flat in the concentrator base with the rough side up. Locate the concentrator base in the jaws of the wash station and screw on the concentrator tube ( the longer of the two tubes), creating a tight seal at the membrane. Take the assembled concentrator out of the jaws and place on the bench. ( c) Replace the splash guard and temporarily secure it at least 15 cm above the end of the rack. Secure the plunger head with the tool provided ensuring that the lever is fully locked down. ( d) Remove the filter module from the filter housing or transportation container. Pour excess liquid into the assembled concentrator, then rinse the housing or container with PBST and add the rinse to the concentrator tube. Screw the filter module onto the base of the plunger. Locate the elution tube base in the jaws of the wash station and screw the elution tube ( the shorter of the two tubes) firmly in place. ( e) Pull the plunger down until the filter module sits at the bottom of the elution tube; the locking pin ( at the top left of the wash station) should " click" to lock the plunger in position. ( f) Remove the filter module bolt by turning the adapted allen key ( provided) in a clockwise direction ( as seen from above). Attach the steel tube to the elution tube base. Method 1623  Cryptosporidium and Giardia June 2003 40 ( g) Add 600 mL of PBST to the assembled concentrator. If more than 50 mL of liquid has been recovered from the shipped filter module, reduce the volume of PBST accordingly. Screw the concentrator tube onto the base beneath the elution tube. Release the locking pin.

NOTE: Gentle pressure on the lever, coupled with a pulling action on the locking pin should enable the pin to be easily released.

( h) Wash the foam disks by moving the plunger up and down 20 times. Gentle movements of the plunger are recommended to avoid generating excess foam.

NOTE: The plunger has an upper movement limit during the wash process to prevent it popping out of the top of the chamber.

( i) Detach the concentrator and hold it such that the stainless steel tube is just above the level of the liquid. Purge the remaining liquid from the elution tube by moving the plunger up and down 5 times, then lock the plunger in place. To prevent drips, place the plug provided in the end of the steel tube. ( j) Prior to the second wash the eluate from the first wash can be concentrated using the Filta Max   apparatus according to Section <IP_ADDRESS>.1 or the eluate can be decanted into a 2 L pooling beaker and set aside. <IP_ADDRESS>.2 Second wash ( a) Add an additional 600 mL of PBST to the concentrator module, remove the plug from the end of the steel tube and screw the concentrator tube back onto the elution module base. Release the locking pin. ( b) Wash the foam disks by moving the plunger up and down 10 times. Gentle movements of the plunger are recommended to avoid generating excess foam. ( c) The eluate can be concentrated using the Filta Max   apparatus according to Section <IP_ADDRESS>.2 or the eluate can be decanted into the 2 L pooling beaker containing the eluate from the first wash and concentrated using centrifugation, as described in Section <IP_ADDRESS>. <IP_ADDRESS> Stomacher elution procedure <IP_ADDRESS>.1 First wash ( a) Place the filter module in the stomacher bag then use the allen key to remove the bolt from the filter module, allowing the rings to expand. Remove the end caps from the stomacher bag and rinse with PBST into the stomacher bag. Method 1623  Cryptosporidium and Giardia June 2003 41 ( b) Add 600 mL of PBST to stomacher bag containing the filter pads. Place bag in stomacher and wash for 5 minutes on a normal setting. ( c) Remove the bag from the stomacher and decant the eluate into a 2 L pooling beaker. <IP_ADDRESS>.2 Second wash ( a) Add a second 600 mL aliquot of PBST to the stomacher bag. Place bag in stomacher and wash for 5 minutes on a normal setting. Remove the bag from the stomacher and decant the eluate from the stomacher bag into the 2 L pooling beaker. Wring the stomacher bag by hand to remove eluate from the foam filter and add to the pooling beaker. Remove the foam filter from the bag and using a squirt bottle, rinse the stomacher bag with reagent water and add the rinse to the pooling beaker. ( b) Proceed to concentration ( Section 12.3.3). 12.3.3 Concentration <IP_ADDRESS> The eluate can be concentrated using the Filta Max   concentrator apparatus, which pulls most of the eluate through a membrane filter leaving the oocysts concentrated in a small volume of the remaining eluting solution ( Section 12.3.. 2), or by directly centrifuging all of the eluting solution used to wash the filter ( Section <IP_ADDRESS>). <IP_ADDRESS> The Filta Max   concentrator procedure <IP_ADDRESS>.1 Concentration of first wash ( a) If the stomacher was used to elute the sample ( Section <IP_ADDRESS>), transfer 600 mL of eluate from the pooling beaker to the concentrator tube. Otherwise proceed to Step ( b). ( b) Stand the concentrator tube on a magnetic stirring plate and attach the lid ( with magnetic stirrer bar). Connect the waste bottle trap and hand or electric vacuum pump to the valve on the concentrator base. Begin stirring and open the tap. Increase the vacuum using the hand pump.

NOTE: The force of the vacuum should not exceed 30 cmHg.

( c) Allow the liquid to drain until it is approximately level with the middle of the stirrer bar then close the valve. Remove the magnetic stirrer, and rinse it with PBST or distilled water to recover all oocysts. Decant the concentrate into a 50 mL tube, then rinse the sides of the concentration tube and add the rinsate to the 50 mL tube. <IP_ADDRESS>.2 Concentration of second wash ( a) If the stomacher was used to elute the sample ( Section <IP_ADDRESS>), transfer the remaining 600 mL of eluate from the pooling beaker to the concentrator tube. Otherwise proceed to Step ( b). Method 1623  Cryptosporidium and Giardia June 2003 42 ( b) Add the concentrate, in the 50 mL tube, retained from the first concentration ( Section <IP_ADDRESS>.1 ( c)) to the 600 mL of eluate from the second wash, then repeat concentration steps from Sections <IP_ADDRESS>.1 ( b) and <IP_ADDRESS>.1 ( c). The final sample can be poured into the same 50 mL tube used to retain the first concentrate. Rinse the sides of the concentrator tube with PBST and add the rinse to the 50 mL tube. ( c) Remove the magnetic stirrer. Insert the empty concentrator module into the jaws of the wash station and twist off the concentrator tube. ( d) Transfer the membrane from the concentrator base to the bag provided using membrane forceps. <IP_ADDRESS>.3 Membrane elution. The membrane can be washed manually or using a stomacher: ° Manual wash. Add 5 mL of PBST to the bag containing the membrane. Rub the surface of the membrane through the bag until the membrane appears clean. Using a pipette, transfer the eluate to a 50 mL tube. Repeat the membrane wash with another 5 mL of PBST and transfer the eluate to the 50 mL tube. ( Optional: Perform a third wash using another 5 mL of PBST, by hand kneading an additional minute or placing the bag on a flat headed vortexer and vortexing for one minute. Transfer the eluate to the 50 mL tube.)

NOTE: Mark the bag with an " X" to note which side of the membrane has the oocysts to encourage the hand kneading to focus on the appropriate side of the membrane.

° Stomacher wash. Add 5 mL of PBST to the bag containing the membrane. Place the bag containing the membrane into a small stomacher and stomach for 3 minutes. Using a pipette transfer the eluate to a 50 mL tube. Repeat the wash two times using the stomacher and 5 mL aliquots of PBST. ( Optional: Perform a fourth wash using another 5 mL of PBST, by hand kneading an additional minute or placing the bag on a flat headed vortexer and vortexing for one minute. Transfer the eluate to the 50 mL tube.) <IP_ADDRESS>.4 If the membrane filter clogs before concentration is complete, there are two possible options for completion of concentration. One option is replacing the membrane as often as necessary. Filter membranes may be placed smooth side up during the second concentration step. Another option is concentrating the remaining eluate using centrifugation. Both options are provided below. ° Using multiple membranes. Disassemble the concentrator tube and pour any remaining eluate back into the pooling beaker. Remove the membrane using membrane forceps, placing it in the bag provided. Place a new membrane in the concentrator Method 1623  Cryptosporidium and Giardia June 2003 43 tube smooth side up, reassemble, return the eluate to the concentrator tube, rinse the pooling beaker and add rinse to the eluate, and continue the concentration. Replace the membrane as often as necessary. ° Centrifuging remaining volume. Decant the remaining eluate into a 2 L pooling beaker. Rinse the sides of the concentrator tube and add to the pooling beaker. Remove the filter membrane and place it in the bag provided. Wash the membrane as described in Section <IP_ADDRESS>.3, then concentrate the sample as described in Section <IP_ADDRESS>.1. <IP_ADDRESS> If the Filta Max   concentrator is not used for sample concentration, or if the membrane filter clogs before sample concentration is complete, then the procedures described in Section <IP_ADDRESS>.1 should be used to concentrate the sample. If less than 50 mL of concentrate has been generated, the sample can be further concentrated, as described in Section <IP_ADDRESS>.2, to reduce the volume of sample to be processed through IMS.

NOTE: The volume must not be reduced to less than 5 mL above the packed pellet. The maximum amount of pellet that should be processed through IMS is 0.5 mL. If the packed pellet is greater than 0.5 mL then the pellet may be subsampled as described in Section 13.2.4.

<IP_ADDRESS>.1 Centrifugation of greater than 50 mL of eluate ( a) Decant the eluate from the 2 L pooling beaker into 250 mL conical centrifuge tubes. Make sure that the centrifuge tubes are balanced.

( b) Centrifuge the 250 mL centrifuge tubes containing the eluate at 1500 × G for 15 minutes. Allow the centrifuge to coast to a stop.

( c) Using a Pasteur pipette, carefully aspirate off the supernatant to 5 mL above the pellet. If the sample is reagent water ( e. g. initial or ongoing precision and recovery sample) extra care must be taken to avoid aspirating oocysts and cysts during this step.

( d) Vortex each 250 mL tube vigorously until pellet is completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after vortexing. Combine the contents of each 250 mL centrifuge tube into a 50 mL centrifuge tube. Rinse each of the 250 mL centrifuge tubes with PBST and add the rinse to the 50 mL tube.

( e) Proceed to Section <IP_ADDRESS>.2. <IP_ADDRESS>.2 Centrifugation of less than 50 mL of eluate ( a) Centrifuge the 50 mL centrifuge tube containing the combined concentrate at 1500 x G for 15 minutes. Allow the centrifuge to coast to a stop. Record the initial pellet volume ( volume of solids) and the date Method 1623  Cryptosporidium and Giardia June 2003 44 and time that concentration was completed on a bench sheet. ( b) Proceed to Section 13.0 for concentration and separation ( purification). 12.3.4 Maintenance and cleaning <IP_ADDRESS> Maintenance of O rings <IP_ADDRESS>.1 Check all rubber O rings for wear or deterioration prior to each use and replace as necessary. <IP_ADDRESS>.2 Lubricate the plunger head O ring inside and out with silicon before each use. <IP_ADDRESS>.3 Lubricate all other O rings ( concentrator tube set, filter housing) regularly in order to preserve their condition. <IP_ADDRESS> Cleaning <IP_ADDRESS>.1 All components of the Filta MaxTM system can be cleaned using warm water and laboratory detergent. After washing, rinse all components with oocyst and cyst free reagent water and dry them. All O rings should be re lubricated. Alternatively a mild ( 40 ° C) dishwasher cycle without bleach or rinse aid can be used. <IP_ADDRESS>.2 To wash the detachable plunger head slide the locking pin out and wash the plunger head and locking pin in warm water and laboratory detergent. Rinse the plunger head and locking pin with oocyst and cyst free reagent water and dry. Lightly lubricate the locking pin and reassemble the plunger head.

13.0 Sample Concentration and Separation ( Purification) 13.1 During concentration and separation, the filter eluate is concentrated through centrifugation, and the oocysts and cysts in the sample are separated from other particulates through immunomagnetic separation ( IMS). Alternate procedures and products may be used if the laboratory first demonstrates equivalent or superior performance as per Section 9.1.2. 13.2 Adjustment of pellet volume 13.2.1 Centrifuge the 250 mL centrifuge tube containing the capsule filter eluate at 1500 × G for 15 minutes. Allow the centrifuge to coast to a stop   do not use the brake. Record the pellet volume ( volume of solids) on the bench sheet.

NOTE: Recoveries may be improved if centrifugation force is increased to 2000 × G. However, do not use this higher force if the sample contains sand or other gritty material that may degrade the condition of any oocysts and/ or cysts in the sample.

13.2.2 Using a Pasteur pipette, carefully aspirate the supernatant to 5 mL above the pellet. Extra care must be taken to avoid aspirating oocysts and cysts during this step, particularly if the sample is reagent water ( e. g. initial or ongoing precision and recovery sample). 13.2.3 If the packed pellet volume is # 0.5 mL, vortex the tube vigorously until pellet is completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after vortexing. Record the resuspended pellet volume on the bench sheet. Proceed to Section 13.3. Method 1623  Cryptosporidium and Giardia June 2003 45 NOTE: Extra care must be taken with samples containing sand or other gritty material when vortexing to ensure that the condition of any oocysts and/ or cysts in the sample is not compromised.

13.2.4 If the packed pellet volume is > 0.5 mL, the concentrate needs to must be separated into multiple subsamples ( a subsample is equivalent to no greater than 0.5 mL of packed pellet material, the recommended maximum amount of particulate material to process through the subsequent purification and examination steps in the method). Use the following formula to determine the total volume required in the centrifuge tube before separating the concentrate into two or more subsamples:

total volume ( mL) required = pellet volume x 5 mL 0.5 mL ( For example, if the packed pellet volume is 1.2 mL, the total volume required is 12 mL.) Add reagent water to the centrifuge tube to bring the total volume to the level calculated above. Vortex the tube vigorously for 10 to 15 seconds to completely resuspend the pellet. Record the resuspended pellet volume on the bench sheet.

NOTE: Extra care must be taken with samples containing sand or other gritty material when vortexing to ensure that the condition of any oocysts in the sample is not compromised.

<IP_ADDRESS> Analysis of entire sample. If analysis of the entire sample is required, determine the number of subsamples to be processed independently through the remainder of the method: <IP_ADDRESS>.1 Calculate number of subsamples: Divide the total volume in the centrifuge tube by 5 mL and round up to the nearest integer ( for example, if the resuspended volume in Section 13.2.4 is 12 mL, then the number of subsamples would be 12 mL / 5 mL = 2.4, rounded = 3 subsamples). <IP_ADDRESS>.2 Determine volume of resuspended concentrate per subsample. Divide the total volume in the centrifuge tube by the calculated number of subsamples ( for example, if the resuspended volume in Section 13.2.4 is 12 mL, then the volume to use for each subsample = 12 mL / 3 subsamples = 4 mL). <IP_ADDRESS>.3 Process subsamples through IMS. Vortex the tube vigorously for 10 to 15 seconds to completely resuspend the pellet. Record the resuspended pellet volume on the bench sheet. Proceed immediately to Section 13.3, and transfer aliquots of the resuspended concentrate equivalent to the volume in the previous step to multiple, flat sided sample tubes in Section <IP_ADDRESS>. Process the sample as multiple, independent subsamples from Section 13.3 onward, including the preparation and examination of separate slides for each aliquot. Record the volume of resuspended concentrate transferred to IMS on the bench sheet ( this will be equal to the volume recorded in Section 13.2.4). Also record the number of Method 1623  Cryptosporidium and Giardia June 2003 46 subsamples processed independently through the method on the bench sheet. <IP_ADDRESS> Analysis of partial sample. If not all of the concentrate will be examined, vortex the tube vigorously for 10 to 15 seconds to completely resuspend the pellet. Record the resuspended pellet volume on the bench sheet. Proceed immediately to Section 13.3, and transfer one or more 5 mL aliquots of the resuspended concentrate to one or more flat sided sample tubes in Section <IP_ADDRESS>. Record the volume of resuspended concentrate transferred to IMS on the bench sheet. To determine the volume analyzed, calculate the percent of the concentrate examined using the following formula:

percent examined = total volume of resuspended concentrate transferred to IMS x 100% total volume of resuspended concentrate in Section 13.2.4 Then multiply the volume filtered ( Section <IP_ADDRESS>) by this percentage to determine the volume analyzed. 13.3 IMS procedure ( adapted from Reference 20.11)

NOTE: The IMS procedure should be performed on a bench top with all materials at room temperature, ranging from 15 ° C to 25 ° C.

13.3.1 Preparation and addition of reagents <IP_ADDRESS> Prepare a 1X dilution of SL buffer A from the 10X SL buffer A ( clear, colorless solution) supplied. Use reagent water ( demineralized; Section 7.3) as the diluent. For every 1 mL of 1X SL buffer A required, take 100 µ L of 10X SL buffer A and make up to 1 mL with the diluent water. A volume of 1.5 mL of 1X SL buffer A will be required per sample or subsample on which the Dynal IMS procedure is performed. <IP_ADDRESS> For each sample or subsample ( Section 13.2) to be processed through IMS, add 1 mL of the 10X SL buffer A ( supplied   not the diluted 1X SL buffer A) to a flat sided tube ( Section 6.5.4). <IP_ADDRESS> For each subsample, add 1 mL of the 10X SL buffer B ( supplied   magenta solution) to the flat sided tube containing the 10X SL buffer A. 13.3.2 Oocyst and cyst capture <IP_ADDRESS> Use a graduated, 10 mL pipette that has been pre rinsed with elution buffer to transfer the water sample concentrate from Section 13.2 to the flat sided tube( s) containing the SL buffers. If all of the concentrate is used, rinse the centrifuge tube twice with reagent water and add the rinsate to the flat sided tube containing the concentrate ( or to the tube containing the first subsample, if multiple subsamples will be processed). Each of the two rinses should be half the volume needed to bring the volume in the flat sided sample tube to 10 mL ( including the buffers added in Sections <IP_ADDRESS> and <IP_ADDRESS>). ( For example, if the tube contained 1 mL of SL buffer A and 1 mL of SL buffer B, and 5 mL of sample was transferred after resuspension of the pellet, for a total of 7 mL, the centrifuge tube would be rinsed twice with 1.5 mL of reagent water to bring the total volume in the flat sided tube to 10 mL.) Visually inspect the centrifuge tube after completing the transfer to ensure that no concentrate remains. If multiple subsamples will be processed, bring the Method 1623  Cryptosporidium and Giardia June 2003 47 volume in the remaining flat sided tubes to 10 mL with reagent water. Label the flat sided tube( s) with the sample number ( and subsample letters). <IP_ADDRESS> Vortex the Dynabeads ® Crypto Combo vial from the IMS kit for approximately 10 seconds to suspend the beads. Ensure that the beads are fully resuspended by inverting the sample tube and making sure that there is no residual pellet at the bottom. <IP_ADDRESS> Add 100 µ L of the resuspended Dynabeads ® Crypto Combo ( Section <IP_ADDRESS>) to the sample tube( s) containing the water sample concentrate and SL buffer. <IP_ADDRESS> Vortex the Dynabeads ® Giardia Combo vial from the IMS kit for approximately 10 seconds to suspend the beads. Ensure that the beads are fully resuspended by inverting the tube and making sure that there is no residual pellet at the bottom. <IP_ADDRESS> Add 100 µ L of the resuspended Dynabeads ® Giardia Combo ( Section <IP_ADDRESS>) to the sample tube( s) containing the water sample concentrate, Dynabeads ® Crypto Combo, and SL buffer. <IP_ADDRESS> Affix the sample tube( s) to a rotating mixer and rotate at approximately 18 rpm for 1 hour at room temperature. <IP_ADDRESS> After rotating for 1 hour, remove each sample tube from the mixer and place the tube in the magnetic particle concentrator ( MPC 1) with flat side of the tube toward the magnet. <IP_ADDRESS> Without removing the sample tube from the MPC 1, place the magnet side of the MPC 1 downwards, so the tube is horizontal and the flat side of the tube is facing down. <IP_ADDRESS> Gently rock the sample tube by hand end to end through approximately 90 ° , tilting the cap end and base end of the tube up and down in turn. Continue the tilting action for 2 minutes with approximately one tilt per second. <IP_ADDRESS> Ensure that the tilting action is continued throughout this period to prevent binding of low mass, magnetic or magnetizable material. If the sample in the MPC 1 is allowed to stand motionless for more than 10 seconds, remove the flat sided tube from the MPC 1, shake the tube to resuspend all material, replace the sample tube in the MPC 1 and repeat Section <IP_ADDRESS> before continuing to Section <IP_ADDRESS>. <IP_ADDRESS> Return the MPC 1 to the upright position, sample tube vertical, with cap at top. Immediately remove the cap and, keeping the flat side of the tube on top, pour off all of the supernatant from the tube held in the MPC 1 into a suitable container. Do not shake the tube and do not remove the tube from MPC 1 during this step. Allow more supernatant to settle; aspirate additional supernatant with pipette. <IP_ADDRESS> Remove the sample tube from the MPC 1 and resuspend the sample in 1 mL 0.5 mL 1X SL buffer A ( prepared from 10X SL buffer A stock   supplied). Mix very gently to resuspend all material in the tube. Do not vortex. <IP_ADDRESS> Quantitatively transfer ( transfer followed by two rinses) all the liquid from the sample tube to a labeled, 1.5 mL microcentrifuge tube. Use 1 mL 0.5 mL of 1X SL buffer A to perform the first rinse and 0.5 mL of reagent water for the second rinse. Liberally rinse down the sides of the Leighton tube before transferring and transfer the rinsate to the Method 1623  Cryptosporidium and Giardia June 2003 48 microcentrifuge tube. Allow the flat sided sample tube to sit for a minimum of 1 minute after transfer of the second rinse volume, then use a pipette to collect any residual volume that drips down to the bottom of the tube to ensure that as much sample volume is recovered as possible. Ensure that all of the liquid and beads are transferred. <IP_ADDRESS> Place the microcentrifuge tube into the second magnetic particle concentrator ( MPC M), with its magnetic strip in place. <IP_ADDRESS> Without removing the microcentrifuge tube from MPC M, gently rock/ roll the tube through 180 ° by hand. Continue for approximately 1 minute with approximately one 180 ° roll/ rock per second. At the end of this step, the beads should produce a distinct brown dot at the back of the tube. <IP_ADDRESS> Immediately aspirate the supernatant from the tube and cap held in the MPC M. If more than one sample is being processed, conduct three 90 ° rock/ roll actions before removing the supernatant from each tube. Take care not to disturb the material attached to the wall of the tube adjacent to the magnet. Do not shake the tube. Do not remove the tube from MPC M while conducting these steps. 13.3.3 Dissociation of beads/ oocyst/ cyst complex NOTE: Two acid dissociations are required.

<IP_ADDRESS> Remove the magnetic strip from the MPC M. <IP_ADDRESS> Add 50 µ L of 0.1 N HCl, then vortex at the highest setting for approximately 50 seconds.

NOTE: The laboratory should must use 0.1 N standards purchased directly from a vendor, rather than adjusting the normality in house.

<IP_ADDRESS> Place the tube in the MPC M without the magnetic strip in place and allow to stand in a vertical position for at least 10 minutes at room temperature. <IP_ADDRESS> Vortex vigorously for approximately 30 seconds. <IP_ADDRESS> Ensure that all of the sample is at the base of the tube. Place the microcentrifuge tube in the MPC M. <IP_ADDRESS> Replace magnetic strip in MPC M and allow the tube to stand undisturbed for a minimum of 10 seconds. <IP_ADDRESS> Prepare a well slide for sample screening and label the slide. <IP_ADDRESS> Add 5 µ L of 1.0 N NaOH to the sample wells of two well slides ( add 10 µ L to the sample well of one well slide if the volume from the two required dissociations will be added to the same slide).

NOTE: The laboratory should must use 1.0 N standards purchased directly from a vendor rather than adjusting the normality in house.

<IP_ADDRESS> Without removing the microcentrifuge tube from the MPC M, transfer all of the sample from the microcentrifuge tube in the MPC M to the sample well with the NaOH. Do not disturb the beads at the back wall of the tube. Ensure that all of the fluid is transferred. <IP_ADDRESS> Do not discard the beads or microcentrifuge tube after transferring the volume from the first acid dissociation to the well slide. Perform the steps in Sections <IP_ADDRESS> through <IP_ADDRESS> a second time. The volume Method 1623  Cryptosporidium and Giardia June 2003 49 from the second dissociation can be added to the slide containing the volume from the first dissociation, or can be applied to a second slide.

NOTE: If one slide is used, exert extra care when using The wells on Dynal Spot On slides are likely to be too small to accommodate the volumes from both dissociations.

<IP_ADDRESS> Record the date and time the purified sample was applied to the slide( s). <IP_ADDRESS> Air dry the sample on the well slide( s). Because temperature and humidity varyies from laboratory to laboratory, no minimum time is specified. However, the laboratory must take care to ensure that the sample has dried completely before staining to prevent losses during the rinse steps. A slide warmer set at 35 ° C to 42 ° C also can be used. 13.3.4 Tips for minimizing carry over of debris onto microscope slides after IMS ° Make sure the resuspended pellet is fully homogenized before placing the tube in the MPC 1 or MPC M to avoid trapping " clumps" or a dirty layer between the beads and the side of the tube.

° When using the MPC 1 magnet, make sure that the tube is snugged flat against the magnet. Push the tube flat if necessary. Sometimes the magnet is not flush with the outside of the holder and, therefore, the attraction between the beads and the magnet is not as strong as it should be. However, it can be difficult to determine this if you do not have more than one MPC 1 to make comparisons.

° After the supernatant has been poured off at Section <IP_ADDRESS>, leave the tube in the MPC 1 and allow time for any supernatant remaining in the tube to settle down to the bottom. Then aspirate the settled supernatant and associated particles from the bottom of the tube. The same can be done at Section <IP_ADDRESS> with the microcentrifuge tube.

° An additional rinse can also be performed at Section <IP_ADDRESS>. After the supernatant has been poured off and any settled material is aspirated off the bottom, leave the tube in the MPC 1 and add an additional 10 mL of reagent water or PBS to the tube and repeat Sections <IP_ADDRESS> and <IP_ADDRESS>. Although labs have reported successfully using this technique to reduce carryover, because the attraction between the MPC 1 and the beads is not as great as the attraction between the MPC M and the beads, the chances would be greater for loss of cysts and oocysts doing the rinse at this step instead of at Section <IP_ADDRESS>.

° After the supernatant has been aspirated from the tube at Section <IP_ADDRESS>, add 0.1 mL of PBS, remove the tube from the MPC M, and resuspend. Repeat Sections <IP_ADDRESS> and <IP_ADDRESS>.

° Use a slide with the largest diameter well available to spread out the sample as much as possible.

14.0 Sample Staining NOTE: The sample must be stained within 72 hours of application of the purified sample to the slide.

14.1 Prepare positive and negative controls. 14.1.1 For the positive control, pipette 10 µ L of positive antigen or 200 to 400 intact oocysts and 200 to 400 cysts to the center of a well. 14.1.2 For the negative control, pipette 50 µ L of 150 mM PBS ( Section 7.6.4) into the center of a well and spread it over the well area with a pipette tip. 14.1.3 Air dry the control slides ( see Section <IP_ADDRESS> for guidance). Method 1623  Cryptosporidium and Giardia June 2003 50 NOTE: If the laboratory has a large batch of slides that will be examined over several days, and is concerned that a single positive control may fade, due to multiple examinations, the laboratory should prepare multiple control slides with the batch of field slides and alternate between the positive controls when performing the positive control check.

14.2 Apply 50 µ L of absolute methanol to each well containing the dried sample and allow to air dry for 3 to 5 minutes. 14.2 Follow manufacturer's instructions in applying stain to slides. 14.3 Place the slides in a humid chamber in the dark and incubate at room temperature for approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container containing damp paper towels on top of which the slides are placed. 14.4 Remove slides from humid chamber and allow condensation to evaporate, if present. 14.5 Apply one drop of wash buffer ( prepared according to the manufacturer's instructions [ Section 7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the excess detection reagent from below the well using a clean Pasteur pipette or absorb with paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.

NOTE: If using the Merifluor stain ( Section 7.6.1), do not allow slides to dry completely.

14.6 Apply 50 µ L of 4', 6 diamidino 2 phenylindole ( DAPI) staining solution ( Section 7.7.2) to each well. Allow to stand at room temperature for a minimum of 1 minute. ( The solution concentration may be increased up to 1 µ g/ mL if fading/ diffusion of DAPI staining is encountered, but the staining solution must be tested first on expendable environmental samples to confirm that staining intensity is appropriate.) 14.7 Apply one drop of wash buffer ( prepared according to the manufacturer's instructions [ Section 7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the excess DAPI staining solution from below the well using a clean Pasteur pipette or absorb with paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.

NOTE: If using the Merifluor stain ( Section 7.6.1), do not allow slides to dry completely.

14.8 Add mounting medium ( Section 7.8) to each well. 14.9 Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the coverslip. Seal the edges of the coverslip onto the slide using clear nail polish. 14.10 Record the date and time that staining was completed on the bench sheet. If slides will not be read immediately, store in a humid chamber in the dark at 0 ° C to < 8 ° C ( but not frozen) until ready for examination.

15.0 Examination NOTE: Although immunofluorescence assay ( FA) and 4', 6 diamidino 2 phenylindole ( DAPI) and differential interference contrast ( DIC) microscopy examination and confirmation should be performed immediately after staining is complete, laboratories have up to 7 days from completion of sample staining to complete the examination and confirmation of samples. However, if fading/ diffusion of FITC or DAPI staining is noticed, the laboratory must reduce this holding time. In addition the laboratory may adjust the concentration of the DAPI staining solution ( Sections 7.7.2) so that fading/ diffusion does not occur.

15.1 Scanning technique: Scan each well in a systematic fashion. An up and down or a side to side scanning pattern may be used ( Figure 4). Method 1623  Cryptosporidium and Giardia June 2003 51 15.2 Examination using immunofluorescence assay ( FA), 4', 6 diamidino 2 phenylindole ( DAPI) staining characteristics, and differential interference contrast ( DIC) microscopy. The minimum magnification requirements for each type of examination are noted below.

NOTE: All shape characterization and size measurements must be determined using 1000X magnification and reported to the nearest 0.5 µ m.

Record examination results for Cryptosporidium oocysts on a Cryptosporidium report examination results form; record examination results for Giardia cysts on a Giardia report examination results form. All oocysts and cysts that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical organisms specifically identified as non target organisms by DIC or DAPI ( e. g. possessing spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc), must be reported. 15.2.1 Positive and negative staining control. Positive and negative staining controls must be acceptable. <IP_ADDRESS> Each analyst must characterize a minimum of three Cryptosporidium oocysts and three Giardia cysts on the positive staining control slide before examining field sample slides. This characterization must be performed by each analyst during each microscope examination session. FITC examination must be conducted at a minimum of 200X total magnification, DAPI examination must be conducted at a minimum of 400X, and DIC examination and size measurements must be conducted at a minimum of 1000X. Size, shape, and DIC and DAPI characteristics of the three Cryptosporidium oocysts and Giardia cysts must be recorded by the analyst on a microscope log. The analyst also must indicate on each sample report examination results form whether the positive staining control was acceptable. <IP_ADDRESS> Examine the negative staining control to confirm that it does not contain any oocysts or cysts ( Section 14.1). Indicate on each sample report examination results form whether the negative staining control was acceptable. <IP_ADDRESS> If the positive staining control contains oocysts and cysts within the expected range and at the appropriate fluorescence for both FA and DAPI, and the negative staining control does not contain any oocysts or cysts ( Section 14.1), proceed to Sections 15.2.2 and 15.2.3. 15.2.2 Sample examination   Cryptosporidium <IP_ADDRESS> FITC examination ( the analyst must use a minimum of 200X total magnification). Use epifluorescence to scan the entire well for applegreen fluorescence of oocyst and cyst shapes. When brilliant apple green fluorescing ovoid or spherical objects 4 to 6 µ m in diameter are observed with brightly highlighted edges, increase magnification to 400X and switch the microscope to the UV filter block for DAPI ( Section <IP_ADDRESS>), then to DIC ( Section <IP_ADDRESS>) at 1000X. <IP_ADDRESS> DAPI examination ( the analyst must use a minimum of 400X total magnification). Using the UV filter block for DAPI, the object will exhibit one of the following characteristics: ( a) Light blue internal staining ( no distinct nuclei) with a green rim ( b) Intense blue internal staining ( c) Up to four distinct, sky blue nuclei Record oocysts in category ( a) as DAPI negative; record oocysts in categories ( b) and ( c) as DAPI positive. Method 1623  Cryptosporidium and Giardia June 2003 52 <IP_ADDRESS> DIC examination ( the analyst must use a minimum of 1000X total magnification [ oil immersion lens]). Using DIC, look for external or internal morphological characteristics atypical of Cryptosporidium oocysts ( e. g., spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.) ( adapted from Reference 20.7). If atypical structures are not observed, then categorize each apple green fluorescing object as: ( a) An empty Cryptosporidium oocyst ( b) A Cryptosporidium oocyst with amorphous structure ( c) A Cryptosporidium oocyst with internal structure ( one to four sporozoites/ oocyst) Using 1000X total magnification, record the shape, measurements ( to the nearest 0.5 µ m), and number of sporozoites ( if applicable) for each apple green fluorescing object meeting the size and shape characteristics. Although not a defining characteristic, surface oocyst folds may be observed in some specimens.

NOTE: All measurements must be made at 1000X magnification.

15.2.3 Sample examination   Giardia <IP_ADDRESS> FITC examination ( the analyst must use a minimum of 200X total magnification). When brilliant apple green fluorescing round to oval ovoid objects ( 8  18 µ m long by 5  15 µ m wide) are observed, increase magnification to 400X and switch the microscope to the UV filter block for DAPI ( Section <IP_ADDRESS>) then to DIC ( Section <IP_ADDRESS>) at 1000X. <IP_ADDRESS> DAPI examination ( the analyst must use a minimum of 400X total magnification). Using the UV filter block for DAPI, the object will exhibit one or more of the following characteristics: ( a) Light blue internal staining ( no distinct nuclei) and a green rim ( b) Intense blue internal staining ( c) Two to four sky blue nuclei Record cysts in category ( a) as DAPI negative; record cysts in categories ( b) and ( c) as DAPI positive. <IP_ADDRESS> DIC examination ( the analyst must use a minimum of 1000X total magnification [ oil immersion lens]). Using DIC, look for external or internal morphological characteristics atypical of Giardia cysts ( e. g., spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.) ( adapted from Reference 20.7). If atypical structures are not observed, then categorize each object meeting the criteria specified in Sections <IP_ADDRESS>  <IP_ADDRESS> as one of the following, based on DIC examination: ( a) An empty Giardia cyst ( b) A Giardia cyst with amorphous structure ( c) A Giardia cyst with one type of internal structure ( nuclei, median body, or axonemes), or ( d) A Giardia cyst with more than one type of internal structure Using 1000X total magnification, record the shape, measurements ( to the nearest 0.5 µ m), and number of nuclei and presence of median body or axonemes ( if applicable) for each apple green fluorescing object meeting the size and shape characteristics.

NOTE: All measurements must be made at 1000X magnification. Method 1623  Cryptosporidium and Giardia June 2003 53 15.2.4 Record the date and time that sample examination was completed on the report examination results form. 15.2.5 Report Cryptosporidium and Giardia concentrations as oocysts/ L and cysts/ L. 15.2.6 Record analyst name 16.0 Analysis of Complex Samples 16.1 Some samples may contain high levels (> 1000/ L) of oocysts and cysts and/ or interfering organisms, substances, or materials. Some samples may clog the filter ( Section 12.0); others will not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain materials that preclude or confuse microscopic examination. 16.2 If the sample holding time has not been exceeded and a full volume sample cannot be filtered, dilute an aliquot of sample with reagent water and filter this smaller aliquot ( Section 12.0). This dilution must be recorded and reported with the results. 16.3 If the holding times for the sample and for microscopic examination of the cleaned up retentate/ eluate have been exceeded, the site should be re sampled. If this is not possible, the results should be qualified accordingly.

17.0 Method Performance 17.1 Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The initial and ongoing precision and recovery criteria are based on the results of spiked reagent water samples analyzed during the Information Collection Rule Supplemental Surveys ( Reference 20.8). The matrix spike and matrix spike duplicate criteria are based on spiked source water data generated during the interlaboratory validation study of Method 1623 involving 11 laboratories and 11 raw surface water matrices across the U. S. ( Reference 20.10).

NOTE: Some sample matrices may prevent the MS acceptance criteria in Tables 3 and 4 to be met. An assessment of the distribution of MS recoveries across 430 MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5.

18.0 Pollution Prevention 18.1 The solutions and reagents used in this method pose little threat to the environment when recycled and managed properly. 18.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the volume of expired materials that need to be discarded to be disposed.

19.0 Waste Management 19.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the biohazard and hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance with all sewage discharge permits and regulations is also required. An overview of these requirements can be found in the Environmental Management Guide for Small Laboratories ( EPA 233 B 98 001). 19.2 Samples, reference materials, and equipment known or suspected to have viable oocysts or cysts attached or contained must be sterilized prior to disposal. 19.3 For further information on waste management, consult The Waste Management Manual for Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste Reduction, both available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N. W., Washington, D. C. 20036. Method 1623  Cryptosporidium and Giardia June 2003 54 20.0 References 20.1 Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and Environmental Microbiology 61 ( 10), 3759 3763. 20.2 Fleming, Diane O., et al.( eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995. ASM Press, Washington, DC 20.3 " Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77 206, ( 1977). 20.4 " OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 ( 1976). 20.5 " Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety ( 1979). 20.6 USEPA. Manual for the Certification of Laboratories Analyzing Drinking Water: Criteria and Procedures; Quality Assurance; Fourth Edition, EPA 815 B 97 001, Office of Ground Water and Drinking Water, U. S. Environmental Protection Agency, 26 West Martin Luther King Drive, Cincinnati, OH 45268 ( 1997). 20.7 ICR Microbial Laboratory Manual, EPA/ 600/ R 95/ 178, National Exposure Research Laboratory, Office of Research and Development, U. S. Environmental Protection Agency, 26 Martin Luther King Drive, Cincinnati, OH 45268 ( 1996). 20.8 USEPA. EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA 821 D 96 004. Office of Water, Engineering and Analysis Division, Washington, DC 20460 ( 1996). 20.8 Connell, K., C. C. Rodgers, H. L. Shank Givens, J Scheller, M. L Pope, and K. Miller, 2000. Building a Better Protozoa Data Set. Journal AWWA, 92: 10: 30. 20.9 " Envirochek   Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann Arbor, MI 48103 9019 ( 1996). 20.10 USEPA. Results of the Interlaboratory Method Validation Study for Determination of Cryptosporidium and Giardia Using USEPA Method 1623, EPA 821 R 01 028. Office of Water, Office of Science and Technology, Engineering and Analysis Division, Washington, DC ( 2001). 20.11 " Dynabeads ® GC Combo," Dynal Microbiology R& D, P. O. Box 8146 Dep., 0212 Oslo, Norway ( September 1998, Revision no. 01). 20.12 USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys. EPA 815 R 01 003. Office of Water, Office of Ground Water and Drinking Water, Standards and Risk Management Division, Washington, DC ( 2001). 20.13 Connell, K., J. Scheller, K. Miller, and C. C. Rodgers, 2000. Performance of Methods 1622 and 1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water Quality Technology Conference, November 5  9, 2000, Salt Lake City, UT. Method 1623  Cryptosporidium and Giardia June 2003 55 21.0 Tables and Figures Table 1. Method Holding Times ( See Section 8.2 for details)

Sample Processing Step Maximum Allowable Time between Breaks ( Samples should be processed as soon as possible)

Collection Filtration ' Up to 96 hours are permitted between sample collection ( if shipped to the laboratory as a bulk sample) or filtration ( if filtered in the field) and initiation of elution Elution These steps must be completed in 1 working day Concentration Purification Application of purified sample to slide Drying of sample ' Up to 72 hours are permitted from application of the purified sample to the slide to staining Staining ' Up to 7 days are permitted between sample staining and examination Examination Method 1623  Cryptosporidium and Giardia June 2003 56 Table 2. Tier 1 and Tier 2 Validation/ Equivalency Demonstration Requirements Test Description Tier 1 modification( 1) Tier 2 modification( 2)

IPR ( Section 9.4) 4 replicates of spiked reagent water Required. Must be accompanied by a method blank. Required per laboratory Method blank ( Section 9.6) Unspiked reagent water Required Required per laboratory MS ( Section 9.5.1) Spiked matrix water Required on each water to which the modification will be applied and on every 20th sample of that water thereafter. Must be accompanied by an unspiked field sample collected at the same time as the MS sample Not required MS/ MSD ( Section 9.5) 2 replicates of spiked matrix water Recommended, but not required. Must be accompanied by an unspiked field sample collected at the same time as the MS sample Required per laboratory. Each laboratory must analyze a different water. ( 1) If a modification will be used only in one laboratory, these tests must be performed and the results must meet all of the QC acceptance criteria in the method ( these tests also are required the first time a laboratory uses the validated version of the method) ( 2) If nationwide approval of a modification is sought for one type of water matrix ( such as surface water), a minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all QC acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the laboratories must meet all QC acceptance criteria.

NOTE: The initial precision and recovery and ongoing precision and recovery ( OPR) acceptance criteria listed in Tables 3 and 4 are based on results from 293 Cryptosporidium OPR samples and 186 Giardia OPR samples analyzed by six laboratories during the Information Collection Rule Supplemental Surveys ( Reference 20.13). The matrix spike acceptance criteria are based on data generated through interlaboratory validation of Method 1623 ( Reference 20.11). Method 1623  Cryptosporidium and Giardia June 2003 57 Table 3. Quality Control Acceptance Criteria for Cryptosporidium Performance test Section Acceptance criteria Initial precision and recovery Mean recovery ( percent)

Precision ( as maximum relative standard deviation) 9.4 9.4.2 24  100 9.4.2 55 Ongoing precision and recovery ( percent) 9.7 11  100 Matrix spike/ matrix spike duplicate ( for method modifications) 9.5 Mean recovery1, 2 ( as percent) 9.5.2 13  111 Precision ( as maximum relative percent difference) 9.5.2 61 ( 1) The acceptance criteria for mean MS/ MSD recovery serves as the acceptance criteria for MS recovery during routine use of the method ( Section 9.5.1). ( 2) Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5.

Table 4. Quality Control Acceptance Criteria for Giardia Performance test Section Acceptance criteria Initial precision and recovery Mean recovery ( percent)

Precision ( as maximum relative standard deviation) 9.4 9.4.2 24  100 9.4.2 49 Ongoing precision and recovery ( percent) 9.7 14  100 Matrix spike/ matrix spike duplicate ( for method modifications) 9.5 Mean recovery* ( as percent) 9.5.2 15  118 Precision ( as maximum relative percent difference) 9.5.2 30 ( 1) The acceptance criteria for mean MS/ MSD recovery serves as the acceptance criteria for MS recovery during routine use of the method ( Section 9.5.1). ( 2) Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of MS recoveries across multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5. Method 1623  Cryptosporidium and Giardia June 2003 58 Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 Source Waters During the ICR Supplemental Surveys ( Adapted from Reference 20.14)

MS Recovery Range Percent of 430 Cryptosporidium MS Samples in Recovery Range Percent of 270 Giardia MS Samples in Recovery Range < 10% 6.7% 5.2%

> 10%  20% 6.3% 4.8%

> 20%  30% 14.9% 7.0%

> 30%  40% 14.2% 8.5%

> 40%  50% 18.4% 17.4%

> 50%  60% 17.4% 16.3%

> 60%  70% 11.2% 16.7%

> 70%  80% 8.4% 14.1%

> 80%  90% 2.3% 6.3%

> 90% 0.2% 3.7% Method 1623  Cryptosporidium and Giardia June 2003 59 A B E D C 1.

3. 4. 2. 1 mm 1/ 5 mm Figure 1. Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4 are used to count stock suspensions of Cryptosporidium oocysts and Giardia cysts ( after Miale, 1967) Method 1623  Cryptosporidium and Giardia June 2003 60 Figure 2. Manner of Counting Oocysts and Cysts in 1 Square mm. Dark organisms are counted and light organisms are omitted ( after Miale, 1967). Method 1623  Cryptosporidium and Giardia June 2003 61 Figure 3. Laboratory Filtration System Method 1623  Cryptosporidium and Giardia June 2003 62 Figure 4. Methods for Scanning a Well Slide Method 1623  Cryptosporidium and Giardia June 2003 63 22.0 Glossary of Definitions and Purposes These definitions and purposes are specific to this method but have been conformed to common usage as much as possible.

22.1 Units of weight and measure and their abbreviations 22.1.1 Symbols ° C degrees Celsius µ L microliter < less than > greater than % percent 22.1.2 Alphabetical characters cm centimeter g gram G acceleration due to gravity hr hour ID inside diameter in. inch L liter m meter MCS microscope cleaning solution mg milligram mL milliliter mm millimeter mM millimolar N normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of solution RSD relative standard deviation sr standard deviation of recovery X average percent recovery 22.2 Definitions, acronyms, and abbreviations ( in alphabetical order)

Analyst   The analyst should have at least 2 years of college in microbiology or equivalent or closely related field. The analyst also should have a minimum of 6 months of continuous bench experience with Cryptosporidium and IFA microscopy. The analyst shouldhave a minimum of 3 months experience using EPA Method 1622 and/ or EPA Method 1623 and should have successfully analyzed a minimum of 50 samples using EPA Method 1622 and/ or EPA Method 1623. The analyst must have at least 2 years of college lecture and laboratory course work in microbiology or a closely related field. The analyst also must have at least 6 months of continuous bench experience with environmental protozoa detection techniques and IFA microscopy, and must have successfully analyzed at least 50 water and/ or wastewater samples for Cryptosporidium and Giardia. Six months of additional experience in the above areas may be substituted for two years of college.

Analyte   A protozoan parasite tested for by this method. The analytes in this method are Cryptosporidium and Giardia.

Axoneme   An internal flagellar structure that occurs in some protozoa, such as Giardia, Spironucleous, and Trichonmonas. Method 1623  Cryptosporidium and Giardia June 2003 64 Cyst   A phase or a form of an organism produced either in response to environmental conditions or as a normal part of the life cycle of the organism. It is characterized by a thick and environmentally resistant cell wall.

Flow cytometer   A particle sorting instrument capable of counting protozoa.

Immunomagnetic separation ( IMS)   A purification procedure that uses microscopic, magnetically responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid stream. Pathogens are selectively removed from other debris using a magnetic field.

Initial precision and recovery ( IPR)   Four aliquots of spiking suspension analyzed to establish the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first time this method is used and any time the method or instrumentation is modified.

Laboratory blank   See Method blank Laboratory control sample ( LCS)   See Ongoing precision and recovery ( OPR) standard Matrix spike ( MS)   A sample prepared by adding a known quantity of organisms to a specified amount of sample matrix for which an independent estimate of target analyte concentration is available. A matrix spike is used to determine the effect of the matrix on a method's recovery efficiency.

May   This action, activity, or procedural step is neither required nor prohibited.

May not   This action, activity, or procedural step is prohibited.

Median bodies   Prominent, dark staining, paired organelles consisting of microtubules and found in the posterior half of Giardia. In G. intestinalis ( from humans), these structures often have a claw hammer shape, while in G. muris ( from mice), the median bodies are round.

Method blank   An aliquot of reagent water that is treated exactly as a sample, including exposure to all glassware, equipment, solvents, and procedures that are used with samples. The method blank is used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the apparatus.

Must   This action, activity, or procedural step is required.

Negative control   See Method blank Nucleus   A membrane bound organelle containing genetic material. Nuclei are a prominent internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium oocysts, there is one nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts.

Oocyst   The encysted zygote of some sporozoa; e. g., Cryptosporidium. The oocyst is a phase or form of the organism produced as a normal part of the life cycle of the organism. It is characterized by a thick and environmentally resistant outer wall.

Ongoing precision and recovery ( OPR) standard   A method blank spiked with known quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified in this method for precision and recovery. Method 1623  Cryptosporidium and Giardia June 2003 65 Oocyst and cyst spiking suspension   See Spiking suspension Oocyst and cyst stock suspension   See Stock suspension Positive control   See Ongoing precision and recovery standard Principle analyst   The principle analyst ( may not be applicable to all monitoring programs) should have a BS/ BA in microbiology or closely related field and a minimum of 1 year of continuous bench experience with Cryptosporidium and IFA microscopy. The principle analyst also should have a minimum of 6 months experience using EPA Method 1622 and/ or EPA Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622 and/ or EPA Method 1623.

PTFE   Polytetrafluoroethylene Quantitative transfer   The process of transferring a solution from one container to another using a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of the source container with a small volume of rinsing solution ( e. g., reagent water, buffer, etc.), followed by transfer of the rinsing solution, followed by a second rinse and transfer.

Reagent water   Water demonstrated to be free from the analytes of interest and potentially interfering substances at the method detection limit for the analyte.

Reagent water blank   see Method blank Relative standard deviation ( RSD)   The standard deviation divided by the mean times 100.

RSD   See Relative standard deviation Should   This action, activity, or procedural step is suggested but not required.

Spiking suspension   Diluted stock suspension containing the organism( s) of interest at a concentration appropriate for spiking samples.

Sporozoite   A motile, infective stage of certain protozoans; e. g., Cryptosporidium. There are four sporozoites in each Cryptosporidium oocyst, and they are generally banana shaped.

Stock suspension   A concentrated suspension containing the organism( s) of interest that is obtained from a source that will attest to the host source, purity, authenticity, and viability of the organism( s).


[Federal Register: December 27, 2005 (Volume 70, Number 247)]
[Notices]
[Page 76475-76477]
[DOCID:fr27de05-71]

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SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-52983; File No. SR-ISE-2005-047]

Self-Regulatory Organizations; International Securities Exchange,
Inc.; Notice of Filing and Immediate Effectiveness of Proposed Rule
Change and Amendment No. 1 Thereto To Adopt a Flat Execution Fee For
Public Customer Orders in ``Premium Products'' and Firm Proprietary
Orders, and To Incorporate the Current Facilitation Mechanism Fee Into
the Flat Execution Fee For Firm Proprietary Orders

December 20, 2005.
    Pursuant to section 19(b)(1) of the Securities Exchange Act of 1934
(``Act''),\1\ and Rule 19b-4 thereunder,\2\ notice is hereby given that
on December 1, 2005, the International Securities Exchange, Inc.
(``ISE'' or ``Exchange'') filed with the Securities and Exchange
Commission (``Commission'') the proposed rule change as described in
Items I, II and III below, which Items have been prepared by the ISE.
On December 7, 2005, the Exchange filed Amendment No. 1 to the proposed
rule change.\3\ The ISE filed the proposal as a ``non-controversial''
proposed rule change pursuant to section 19(b)(3)(A)(iii) of the Act
\4\ and Rule 19b-4(f)(2) thereunder,\5\ which renders the proposal
effective upon filing with the Commission. The Commission is publishing
this notice to solicit comments on the proposed rule change, as
amended, from interested persons.
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    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 17 CFR 240.19b-4.
    \3\ In Amendment No. 1, ISE explained further its basis for the
proposed flat execution fee and its reason for eliminating the
Facilitation Mechanism fee. Amendment No. 1 also corrected several
minor errors. For purposes of calculating the 60-day period within
which the Commission may summarily abrogate the proposed rule change
the Commission considers the period to commence on December 7, 2005,
the date on which the ISE filed Amendment No. 1. See 15 U.S.C.
78s(b)(3)(C).
    \4\ 15 U.S.C. 78s(b)(3)(A)(iii).
    \5\ 17 CFR 240.19b-4(f)(2).
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I. Self-Regulatory Organization's Statement of the Terms of Substance
of the Proposed Rule Change

    The ISE proposes to amend its Schedule of Fees to (i) adopt a flat
execution fee for Public Customer Orders \6\ in ``Premium Products''
(as defined in the Schedule of Fees) and firm proprietary orders; and
(ii)

incorporate the current Facilitation Mechanism fee into the flat
execution fee for firm proprietary orders. The text of the proposed
rule change is available at the Exchange and at the Commission's Public
Reference Room, and at the Exchange's Web site (http://www.iseoptions.com/legal/proposed_rule_changes.asp
).

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    \6\ A Public Customer Order is defined in ISE Rule 100(a)(33) as
an order for the account of a Public Customer. A Public Customer is
defined in ISE Rule 100(a)(32) as a person that is not a broker or
dealer in securities.
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II. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

    In its filing with the Commission, the ISE included statements
concerning the purpose of, and basis for, the proposed rule change and
discussed any comments it received on the proposal. The text of these
statements may be examined at the places specified in Item IV below.
The Exchange has prepared summaries, set forth in sections A, B, and C
below, of the most significant aspects of such statements.

A. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

1. Purpose
    The Exchange's Schedule of Fees currently contains a formula that
determines the amount of the execution fee to be charged to all order
types, i.e., Public Customer (for certain index-based products), firm
proprietary and market maker, based on the Exchange's average daily
volume during the previous month. Under the current formula, the
execution fee can range anywhere between $0.12 per contract to $0.21
per contract, depending on the Exchange's average daily volume during
the previous month. The Exchange proposes to change this structure so
that Public Customer Orders in ``Premium Products'' \7\ and firm
proprietary orders pay a flat execution fee of $0.15 per contract.\8\
Public Customer Orders in products other than Premium Products will
continue to be subject to a flat execution fee of $0.05, although that
fee is currently waived until June 30, 2006. ISE market makers will
continue to be charged the variable fee under the current formula. A
$0.03 per contract comparison fee shall continue to apply to all these
order types, unless specifically waived in the Schedule of Fees.
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    \7\ The ISE proposes to define the term ``Premium Products'' in
the Schedule of Fees as options on DIA, IBB, IEF, IJH, IJR, IWM,
IWN, IWO, NYC, NY, OEF, OIH, SHY, SMH, SPY, TLT, XLB, XLE, XLF, XLI,
XLU, BYT, HSX, HVY, IXK, IXX, IXZ, JLO, MID, MNX, MSH, NDX, OOG,
RMN, RND, RUF, RUI, RUT, SIN, and SML.
    \8\ These fees will be charged only to Exchange members.
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    Additionally, the Exchange proposes to delete the separate
Facilitation Mechanism fee line item. Previously, there was a separate
line item in the Schedule of Fees for orders executed in that
mechanism: Orders executed in the Facilitation Mechanism were charged
the lower of the standard execution fee or $0.15 per contract.
Historically, fees charged for trades executed in the Facilitation
Mechanism were carved out on the Schedule of Fees as a separate line
item because, as a matter of inducement for members to transact in the
Facilitation Mechanism, these fees were lower than the standard
execution fees. Over time, as the standard execution fees decreased, as
a result of increased volume, the two fees became identical.
Accordingly, the Exchange now proposes to eliminate the separate line
item for orders executed in the Facilitation Mechanism because it is no
longer necessary when there is a flat $0.15 fee for firm proprietary
order and public customer orders in Premium Products.
    As a matter of ``housekeeping,'' in addition to using ``Premium
Products'' as a defined word throughout the Schedule of Fees, the
Exchange proposes to identify each product that appears on the Schedule
of Fees by its ticker symbol alone rather than by its name and ticker
symbol.
2. Statutory Basis
    The Exchange believes that the proposed rule change is consistent
with the requirements of section 6(b)(4) of the Act,\9\ which requires
that an exchange have an equitable allocation of reasonable dues, fees
and other charges among its members and other persons using its
facilities. The Exchange believes the proposed fees are reasonable
because they closely correlate to the variable execution fees charged
to market maker and firm proprietary orders over the course of the last
12 months. For example, for nine months during 2005, the variable
execution fees charged to market maker and firm proprietary orders were
$0.15 per contract, and for 3 months during 2005, these same fees were
$0.14 per contract. The Exchange further believes that the proposed fee
changes will enable the ISE to continue offering competitively priced
products and services.
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    \9\ 15 U.S.C. 78f(b)(4).
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B. Self-Regulatory Organization's Statement on Burden on Competition

    The Exchange believes that the proposed rule change will not impose
any burden on competition that is not necessary or appropriate in
furtherance of the purposes of the Act.

C. Self-Regulatory Organization's Statement on Comments on the Proposed
Rule Change Received From Members, Participants, or Others

    The Exchange has neither solicited nor received comments on the
proposed rule change. The ISE has not received any unsolicited written
comments from members or other interested parties.

III. Date of Effectiveness of the Proposed Rule Change and Timing for
Commission Action

    The foregoing proposed rule change has become effective pursuant to
section 19(b)(3)(A)(ii) of the Act,\10\ and paragraph (f)(2) of Rule
19b-4 thereunder \11\ because it establishes or changes a due, fee, or
other charge. At any time within 60-days of the filing of the proposed
rule change, the Commission may summarily abrogate such rule change if
it appears to the Commission that such action is necessary or
appropriate in the public interest, for the protection of investors, or
otherwise in furtherance of the purposes of the Act.\12\
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    \10\ 15 U.S.C. 78s(b)(3)(A)(ii).
    \11\ 17 CFR 240.19b-4(f)(2).
    \12\ See footnote 3, supra.
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IV. Solicitation of Comments

    Interested persons are invited to submit written data, views, and
arguments concerning the foregoing, including whether the proposed rule
change is consistent with the Act. Comments may be submitted by any of
the following methods:

Electronic Comments

     Use the Commission's Internet comment form (http://www.sec.gov/rules/sro.shtml.
); or     Send an e-mail to<EMAIL_ADDRESS>Please include

File Number SR-ISE-2005-047 on the subject line.

Paper Comments

     Send paper comments in triplicate to Jonathan G. Katz,
Secretary, Securities and Exchange Commission, 100 F Street, NE.,
Washington, DC 20549-9303.

All submissions should refer to File Number SR-ISE-2005-047. This file
number should be included on the subject line if e-mail is used. To
help the

Commission process and review your comments more efficiently, please
use only one method. The Commission will post all comments on the
Commission's Internet Web site (http://www.sec.gov/rules/sro.shtml).

Copies of the submission, all subsequent amendments, all written
statements with respect to the proposed rule change that are filed with
the Commission, and all written communications relating to the proposed
rule change between the Commission and any person, other than those
that may be withheld from the public in accordance with the provisions
of 5 U.S.C. 552, will be available for inspection and copying in the
Commission's Public Reference Room. Copies of such filing also will be
available for inspection and copying at the ISE. All comments received
will be posted without change; the Commission does not edit personal
identifying information from submissions. You should submit only
information that you wish to make available publicly. All submissions
should refer to File Number SR-ISE-2005-047 and should be submitted on
or before January 17, 2006.

    For the Commission, by the Division of Market Regulation,
pursuant to delegated authority.\13\
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    \13\ 17 CFR 200.30-3(a)(12).
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Jonathan G. Katz,
Secretary.
[FR Doc. E5-7844 Filed 12-23-05; 8:45 am]

BILLING CODE 8010-01-P