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Introduction
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Welcome to the Environmental Protection Agency on-line training module detailing the immunomagnetic separation, IMS, procedure used in Method 1623. IMS is designed to separate Cryptosporidium oocysts and
Giardia cysts from extraneous debris in a water sample. This module is designed to assist you in maximizing recoveries of the target organisms using Section 13 of Method 1623.
Narration:
Welcome to the EPA on-line training module detailing the immunomagnetic separation, or IMS, procedure used in Method 1623. IMS is designed to separate Cryptosporidium oocysts and Giardia cysts from extraneous debris in a water sample. This module is designed to assist you in maximizing recoveries of the target organisms.
Lesson Information
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Time to complete this module is approximately 1 to 1.5 hours.
Narration, photographs, and video are used to explain the key processing steps to help analysts refine their lab technique to maximize recovery and precision between samples, analysts and laboratories.
Knowledge Reviews at the end of each section help highlight and reinforce key learning points.
Computer requirements:
- 800 x 600 screen resolution, minimum
- Macromedia Flash 8, required for interactive graphics and video segments
- Computer audio on to hear narration
- Pop-up blockers disabled
Module features:
- The capital T icon in the menu bar displays narration.
- Use the Print icon on the toolbar to print the complete module or print photographs through individual icons.
- Use the document icon on the toolbar to access the IMS Supplement.
Narration:
Narration, photographs, and video are used to explain the key processing steps to help all analysts refine their lab technique to maximize recovery and precision between samples, analysts and laboratories. Time to complete this module is approximately 1 to 1.5 hours.
Lesson Objectives
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Upon completion of the module, you should be able to summarize and apply the techniques demonstrated to achieve the best recovery and precision. Specifically, you should know how to:
- Efficiently transfer the sample through successive steps in the method.
- Optimize the performance of the magnets.
- Improve the recovery from difficult matrices.
- Troubleshoot the IMS section of Method 1623.
Narration:
Upon completion of the module, you should be able to summarize and apply the techniques used to optimize and troubleshoot the IMS procedure for the best recovery and precision.
Definition and Purpose
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Immunomagnetic separation (IMS) uses magnetic beads coated with antibodies that selectively attach to Cryptosporidium and Giardia. The
bead-organism complexes are separated from the rest of the sample using a magnet. The extraneous debris is discarded, resulting in a small volume of purified sample which contains the organisms.
Because of the variation in amount and type of debris in different samples, IMS can be a challenging step in Method 1623.
Narration:
Immunomagnetic separation, or IMS, uses magnetic beads coated with antibodies that selectively attach to Cryptosporidium and Giardia.
Technology Overview (1 of 3)
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The beads used in IMS contain iron and are coated with anti-Cryptosporidium and anti-Giardia monoclonal antibodies which are genus specific but not species specific. For example, oocysts of Cryptosporidium parvum, C. hominis, and other species of Cryptosporidium will be recovered. The antibodies attached to the beads selectively bind to the
epitope of the specific
antigen in the cell wall of the (oo)cysts forming an antigen-antibody complex. Monoclonal antibodies are used because they reduce cross reaction with non-target organisms.
The beads commonly used by laboratories performing Method 1623 exhibit the following properties:
- Magnetism only when exposed to a magnetic field (beads will not clump or attract particles outside a magnetic field)
- Uniform size (approx. 5 µm), spherical shape and surface area
Narration:
The beads used in IMS contain iron and are coated with antibodies that are genus specific but not species specific. The antibodies attached to the beads selectively bind to the specific antigen in the cell wall of the oocysts/cysts to form an antigen-antibody complex.
Technology Overview (2 of 3)
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The beads and buffering solutions are added to a water sample concentrate. The buffer helps maintain a pH conducive to bead attachment with the (oo)cysts.
The sample is mixed allowing the bead-organism complexes to form.
After mixing, the sample is exposed to a magnet which holds the bead-organism complexes so the extraneous suspension can be poured off.
Narration:
IMS beads are mixed with a water sample concentrate and bead-organism complexes are separated from the suspension using a magnet.
Technology Overview (3 of 3)
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The bead-organism complex is resuspended in hydrochloric acid. The new suspension is vigorously vortexed which breaks the bonds of the complexes, freeing the organisms. The acidic solution prevents the bead-organism complexes from reforming.
The beads are separated from the suspension containing the (oo)cysts with the magnet. The suspension is carefully removed from the microcentrifuge tube with a pipette leaving behind the beads. The beads have been dissociated from the organisms to prevent their interference with microscopy.
Narration:
The bead-organism complex is resuspended in hydrochloric acid and the suspension is vigorously vortexed. The vortex action breaks the bonds of the bead-organism complexes, freeing the organisms. The acidic solution prevents the bead-organism complexes from reforming.
The beads are removed using the magnet, leaving behind a solution containing the organisms which is then placed on a well slide.
Pellets
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A packed centrifugate pellet is formed after processing the water sample through Method 1623 filtration, elution and concentration. Most of the supernatant is then aspirated from above the concentrated pellet, and Cryptosporidium oocysts and Giardia cysts now can be targeted with IMS.
After completing this section of the module, you should be able to describe:
- Estimation of packed pellet size.
- Proper aspiration technique.
- Volume of suspension fluid to remain after aspiration.
- Pellet resuspension.
Narration:
The previous screens described an overview of the IMS process. The next sections describe details of Section 13 in Method 1623.
After sample processing, it is important to understand how to estimate the size of the packed pellet and the proper steps for aspiration and pellet resuspension.
Pellet Measurement
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- Centrifuge the filter eluate using one appropriately sized tube or bottle depending on the volume of eluting solution.
- Use a set of prepared pellet standards to compare and estimate the volume of the packed pellet.
- Record pellet volume on the Bench Sheet.
Prepared Pellet Standards
Tips and Tricks
Narration:
Centrifuge the filter eluate from the water sample using an appropriately sized bottle based on the type of filtration and elution used. Use a set of prepared pellet standards to estimate the volume of packed pellet and record on the bench sheet.
Tips and Tricks
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- Centrifuge for the specific time and speed in the laboratory standard operating procedure.
- Don't use the brake during deceleration of the centrifuge.
- Balance weight of centrifuge bottles to within 0.5 g and use a centrifuge with a self-balancing rotor to correct imbalances.
- Start timing when the centrifuge achieves the required speed.
- Use a lab timer.
- Locate the aspiration equipment beside the centrifuge to avoid carrying samples. Eliminate any disruption of the pellet with vibration from other equipment on the same bench.
- Perform annual maintenance and calibration of the centrifuge to keep it operating properly.
Note: Method 1623 states centrifugation is to be performed at 1500 x G. Increasing the centrifugation to 2000 x G may improve recoveries in water matrices without sand or other debris; however, abrasive particulates may result in (oo)cyst losses if the centrifuge speed is increased.
Prepared Pellet Standards
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-
Pellet standards should range from:- 0.1 mL to 0.5 mL, in 0.1 mL increments
- 0.5 mL to 3 mL in 0.5 mL increments
-
Prepare pellet standards based on the pellet sizes typically observed. -
Pellet standards may be prepared for long-term usage by measuring appropriate amounts of colored glycerol, sand, or colored floral arranging gel.- Colored glycerol and sand have the advantage of being "fluid" so the standard may be viewed at an angle similar to the packed pellet.
- Floral arranging gel is available as Wonder Water®, Liquid Illusion, or equivalent.
Aspiration of the Supernatant
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Aspirate the supernatant from the centrifuge tube leaving 5 mL of fluid for every 0.5 mL of pellet.
- Aspirate from the center of the water column.
- Aspirate at the air/liquid interface not below the water surface, maintaining an air suction sound.
- Aspirate gently and steadily with a low vacuum pressure (<5 in. Hg vacuum).
- Lower the vacuum pressure when 30 mL remains.
- Use standard Pasteur pipettes with inner diameter of 0.8 - 1.2 mm.
- Record the volume of fluid left in the tube (≥5 mL).
View the videos demonstrating correct and incorrect aspiration; listen to the difference.
Narration:
Aspirate the supernatant of the water sample concentrate leaving 5 mL of fluid for every 0.5 mL of pellet to a minimum of 5 mL. Aspiration must be performed carefully to reduce the loss of organisms. Aspirate gently and steadily at the air/liquid interface in the center of the water column using a pipette and a low vacuum pressure.
View the videos demonstrating correct and incorrect aspiration; listen to the difference.
Correct Aspiration of the Supernatant
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Incorrect Aspiration of the Supernatant
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Pellet Resuspension
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Proper resuspension of the packed pellet ensures that any (oo)cysts present in the water sample concentrate are released from the debris, distributed throughout the liquid, and available to bind with the IMS beads.
- Vortex, or pipette mix, the pellet thoroughly until no clumps are present before transfer to the flat-sided tube(s).
- Reduce vortex speed if the pellet contains sand or other gritty material which may compromise the organisms.
- Reduce vortex speed, or pipette mix, if excess debris is splattered onto the sides of the centrifuge tube.
Narration:
Resuspension of the packed pellet releases any organisms from the debris and distributes them throughout the liquid, allowing them to bind with the IMS beads.Vigorously vortex or pipette mix the pellet before transfer to the flat-sided tube. Be cognizant of the speed of the vortex and reduce if excess debris is splattered onto the sides of the centrifuge tube.
Knowledge Review
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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)
Equipment and Reagent Preparation
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In addition to general lab equipment, reagents and equipment specific to the IMS procedure are listed in Method 1623 Sections 6.5 and 7.5. Rollover each picture to view the description.
After completing this section, you should be able to describe:
- Transfer of the pellet suspension to the flat-sided tube.
- Correct addition of IMS buffers and beads to the flat-sided tube.
- Proper use of the rotating mixer.
Tips and Tricks
Narration:
Equipment needed to perform the IMS procedure is shown on the screen. Rollover each piece of equipment for additional information.
Tips and Tricks
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Transfer to Flat-sided Tube (1 of 2)
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- Samples with ≤0.5 mL of pellet resuspended into 5 mL of fluid will require 1 flat-sided tube.
- Samples with >0.5 mL and ≤1.0 mL of pellet resuspended into 10 mL of fluid will require 2 flat-sided tubes, and so on.
- Record the number of flat-sided tubes as the number of subsamples on the bench sheet.
For samples being analyzed for compliance with the Long Term 2 Enhanced Surface Water Treatment Rule, analyze all of the pellet resuspension, or up to 2 mL of pellet resuspended in 20 mL of fluid transferred in 5 mL aliquots into 4 flat-sided tubes.
Remember to use the IMS Supplement for processing tips.
Narration:
The number of flat-sided tubes required per sample is dependent on the pellet size. Analyze the entire pellet resuspension, up to 20 mL of fluid, transferred in 5 mL aliquots to individual flat-sided tubes. Record the number of flat-sided tubes as the number of subsamples on the bench sheet.
Transfer to Flat-sided Tube (2 of 2)
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swf*
- Allow buffers and samples to reach room temperature.
- SL-buffer A often becomes crystallized during storage in the refrigerator; dissolve by mixing, hand warming, and/or brief warming at 37°C.
- Add 1 mL of 10X SL Buffer A and 1 mL of 10X SL Buffer B to each flat-sided tube.
- Pre-rinse a pipette with elution buffer.
- Draw up 5 mL of the well-mixed pellet suspension into the pipette and dispense into the labeled flat-sided tube.
- If the entire pellet suspension has been transferred, rinse the centrifuge tube twice with ≤2.5 mL of reagent water per rinse.
- Adjust the volume in the flat-sided tube to 12 mL by adding reagent water.
Tips and Tricks
Select the videos to view demonstrations.
Narration:
Allow buffers and samples to reach room temperature; Add 1 mL of 10X SL Buffer A and 1 mL of 10X SL Buffer B to each flat-sided tube. Pre-rinse a pipette with elution buffer and transfer the well mixed pellet to the labeled flat-sided tube. Rinse the centrifuge tube twice with reagent water. Adjust the volume in the flat-sided tube to 12 mL by adding reagent water.
Select the videos to view demonstrations.
Tips and Tricks
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Single Tube: Prepare Pellet
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Single Tube: Transfer
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Single Tube: First Rinse
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Multi Tube: Transfer
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Addition of Beads
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- Thoroughly resuspend the beads by vortexing; visually inspect the bottom of the bottle and continue vortexing until a homogenous suspension of the beads is seen.
- Immediately add the following to each flat-sided tube containing sample and buffers:
- 100 µL anti-Cryptosporidium beads
- 100 µL anti-Giardia beads
Tips and Tricks
Narration:
To each flat-sided tube containing 2 mL of buffers and 10 mL of sample concentrate and rinses, add 100 µL of anti-Cryptosporidium beads and 100 µL of anti-Giardia beads.
Tips and Tricks
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Each laboratory should develop a standard procedure to ensure that beads and buffers are added properly and to reduce confusion and mistakes if analysts are working together or otherwise distracted. The following are some techniques currently used by labs to be methodical and ensure consistency:
- Remove all 4 reagents from the box and place the bottle back in the box once the reagent has been added to all the flat-sided tubes.
- Move each flat-sided tube to a new position in the rack after each reagent is added to the tube.
- After adding each reagent to the flat-sided tube, replace the cap; remove all caps before starting the next reagent.
Establish a pattern and perform these additions the same way every time to ensure all reagents are added to each tube.
Rotation
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Mixing allows the beads to come in contact with the (oo)cysts in the sample and form the bead-organism complexes. Locate the rotating mixer on the countertop to keep the IMS suspension at room temperature.
- Place the flat-sided tubes on the rotating mixer so that they rotate freely without interference.
- Balance the tubes on each side to ensure even, gentle rotation.
- Adjust the dial speed to achieve 18 rpm on the mixer.
- Rotate for 60 minutes and use a timer to consistently follow your standard operating procedure (SOP).
All rotating mixers do not rotate at the same speed or stay consistent over time. Calibrate the rotating mixer loaded with tubes by counting the revolutions per minute at least annually.
Narration:
The mixing step allows the beads to come in contact with the (oo)cysts in the sample and form the bead-organism complexes. Rotate for 60 minutes.
Knowledge Review
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Number the following statements in the order that they are performed in the space provided next to each choice.
Overview of Bead-Organism Separation
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After rotating for an hour, each tube is removed from the rotating mixer and rocked against the magnet in the magnetic particle concentrator.
The bead-organism complexes are attracted to the magnet and the unattached debris and fluid are poured off.
The bead-organism complexes are then transfered to a microcentrifuge tube.
Upon completion of this section, you should be able to describe:
- Rocking the sample suspension for optimum attraction to the magnet.
- Decanting the extraneous debris and fluid.
- Quantitatively transferring the (oo)cysts.
Narration:
After rotating for an hour, each flat-sided tube is removed from the rotating mixer and placed in a Magnetic Particle Concentrator®. Bead-organism complexes are attracted to the magnet; unattached debris and fluid are poured off. Bead-organism complexes are then transferred to a microcentrifuge tube.
Please note: During the next few steps it is critical that you be gentle. Do not vortex or mix the bead suspension too vigorously to ensure the organisms stay attached to the beads.
The Magnetic Particle Concentrator
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The flat-sided tube may be placed into either type of magnetic particle concentrator: MPC®-6 or MPC®-1.
- Check that the flat side of the tube is completely flush and tight against the magnet.
- With MPC®-6, rock each side through a 90° angle resulting in a complete 180° rock.
- With MPC®-1, hold the tube flat against the magnet during rocking.
- Rock the magnet and tube through a 90° angle.
- Rock gently and smoothly at ~1 second for each 90° rock for 2 minutes.
Select the videos for demonstrations.
Tips and Tricks
Narration:
Place the flat-sided tube in the MPC® and rock for 2 minutes. Rock gently and smoothly at ~1 second for each 90° rock. Remember to check that the flat side of the tube is flush and tight against the magnet.
Select the videos for demonstrations.
Tips and Tricks
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- Use an automatic rocking plate instead of hand rocking, to increase consistency and reduce repetitive stress injuries (e.g. Double Tier Rocking Platform from VWR (40000-304) or equivalent).
- The automated rocker may be used with all of the MPCs®, but only if the MPC® holds the tube flat and tight against the magnet.
- When using the MPC®-6 on the rocking plate, load the MPC® with ≤3 tubes.
- Rock at 1 rock per second using the automatic rocking plate.
Note: Some flat-sided tubes tend to bow and require the technician to manually hold the flat side of the tube level and tight against the magnet during rocking. Contact the tube and magnet manufacturers to resolve this problem.
MPC®-6 Steady Rocking
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MPC®-1 Steady Rocking
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MPC®-1 Unsteady Rocking
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Flat-sided Tube Decant
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At the end of the 2-minute rock:
- Hold the MPC® containing the flat-sided tube with the magnet side up and remove the tube cap.
- Smoothly and steadily pour off supernatant into a waste beaker.
- Rock the MPC®-6 (90°) 3 times before decanting the remaining tubes.
- With the MPC®-6, the supernatant may be decanted from a single flat-sided tube or 3 tubes on the same side of the magnet.
- After decanting, gently aspirate any additional supernatant from the bottom of the flat-sided tube using a Pasteur pipette and discard to waste.
Select the videos for demonstrations.
Tips and Tricks
Remember to use the IMS Supplement for processing tips.
Narration:
Decant the supernatant from the flat-sided tube after the 2 minute rock, being careful not to disturb the bead pellet attracted to the magnet. With practice, the transition between rocking and decanting will become smooth and steady.
Select the videos for demonstrations.
Tips and Tricks
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- Be careful not to disturb the bead pellet attracted to the magnet side of the tube.
- Keep the magnet rocking, not sitting motionless, except while removing the caps and decanting.
- Use a different lint-free tissue or towel to blot the end of the flat-sided tube after decanting; this removes more matrix debris.
MPC®-6: Single Tube Decant
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MPC®-6: Multi Tube Decant
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MPC®-1: Decant
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Transfer to Microcentrifuge Tube
ims0340 | page 22 of 46
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Remove the flat-sided tube from the magnet and quantitatively transfer the bead pellet to the microcentrifuge tube as described here:
- Pipette 0.5 mL of 1X SL-Buffer A onto the flat-side of the tube to gently wash the bead pellet to the bottom of the tube.
- Resuspend the bead pellet by gently sliding the suspension across the length of the flat-side.
- Draw-up the resuspended bead pellet and dispense into a labeled microcentrifuge tube.
- This microcentrifuge tube should be in the MPC®-S with magnet in vertical position as shown on the next screen.
- Rinse the flat-side of the tube twice using 0.5 mL of 1X SL-Buffer A.
- Pipette each rinse into the microcentrifuge tube.
- Allow the flat-sided tube to sit for at least 1 minute to allow any remaining liquid to drain to the bottom, then transfer to the microcentrifuge tube.
Tips and Tricks
Select the video to view a demonstration of resuspension of the bead pellet.
Narration:
Pipette the SL-Buffer A onto the flat-side of the tube to gently wash the bead pellet to the bottom of the tube.
Gently resuspend the bead pellet by sliding the suspension across the length of the flat-side of the tube.
Transfer the resuspended bead pellet and the two rinses to a microcentrifuge tube.
The microcentrifuge tube should be in the MPC®-S with the magnet in the vertical position.
Select the video to view a demonstration.
Tips and Tricks
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- Avoid the debris that remains on the round side of the flat-sided tube after decanting.
- Do not recap the flat-sided tube; heavy mixing or inversion is not needed when resuspending or rinsing.
- The bead pellet suspension may be pipette-mixed gently to ensure complete transfer.
- The microcentrifuge tube should be in the MPC®-S with the magnet in the vertical position to produce an immediate bead pellet which aids in separating the debris.
Resuspension of Bead Pellet
ims0340b | page 22b of 46
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MPC®-S Bead Separation
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swf*
Continue the bead pellet separation in the microcentrifuge tube with the magnet placed vertically in the MPC®-S:
- Gently rock the MPC®-S containing the tubes through a 180° rotation for 1 minute.
- Aspirate the supernatant from each tube using a Pasteur pipette and pipette aid.
- Avoid disturbing the bead pellet, and discard the supernatant to waste.
- Remove as much debris and liquid from the bottom of the tube as possible.
- Rock the tubes through three 90° rocks prior to the aspiration of each subsequent sample.
It is important to note that the MPC®-S has two magnet positions - vertical and slanted. Roll over each photo to view descriptions of the two magnet positions.
Narration:
Gently rock the tubes in the MPC®-S for 1 minute. Aspirate and discard the supernatant being careful not to disturb the pellet.
It is very important to note that the MPC®-S has two magnet positions - vertical and slanted. Roll over each photo to view descriptions of the two magnet positions.
Knowledge Review
ims0370 | page 24 of 46
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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)
Knowledge Review
ims0380 | page 25 of 46
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Number the following statements in the order that they are performed in the space provided next to each choice.
Dissociation
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This section describes dissociation using a vigorous vortex to break apart the beads from the (oo)cysts. Hydrochloric acid is added to inhibit reformation of the complexes. Dissociation is performed twice to capture organisms still attached to the beads after the first dissociation.
- Remove the magnetic strip from the MPC®-S.
- Add 50 µL of 0.1N hydrochloric acid (HCl) to each microcentrifuge tube.
- Vortex vigorously at maximum speed for 50 seconds.
- Wait 10 minutes.
- Vortex vigorously for 30 seconds.
Tips and Tricks
Select the video to view a demonstration.
Narration:
During dissociation, vigorous vortex action mechanically breaks the (oo)cysts from the beads while the reduced pH inhibits reformation of the complexes. Dissociation is performed twice to capture organisms still attached to the beads after the first dissociation.
Select the video to view a demonstration.
Tips and Tricks
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- Use commercially available 0.1N HCl and 1.0N NaOH.
- Purchase in small volumes to use before expiration date.
- Aliquot stock HCl and NaOH into microcentrifuge tubes prior to use to facilitate pipetting.
- Tubes may be vortexed in the MPC®-S without the magnet strip.
- Use a microcentrifuge tube opener to eliminate the possibility of cross-contamination.
Dissociation
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Neutralize pH
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After the tube has been appropriately vortexed:
- Ensure all liquid is at the bottom of the tube; shake down the tube if necessary.
- With the magnet in the slanted position, return the tube to the MPC®-S.
- Watch for bead pellet formation at the back of the tube while it rests for a minimum of 10 seconds.
To neutralize the pH of the sample:
- Add 5 µL of 1.0N NaOH to the well on the slide.
- Use a micropipetter to transfer the dissociated sample (supernatant) to the well.
Avoid disturbing the beads at the back wall of the tube while you remove the supernatant; this may be easier if the pipetter or the tube is tipped slightly.
Select the video to view a demonstration.
Narration:
To neutralize the pH of the sample, 5 µL of 1.0N sodium hydroxide is added for every 50 µL of hydrochloric acid used in the dissociation. After the organisms have been dissociated from the beads, add the sodium hydroxide directly to each slide well, then add the dissociated sample to the well.
Select the video to view a demonstration.
Neutralize pH
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Second Dissociation
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The second dissociation step allows the breakdown of any remaining bead-organism complexes and maximizes the number of organisms released from the beads.
- Add another 50 µL of 0.1N HCl to the microcentrifuge tube and repeat the steps of the first dissociation.
The suspension resulting from the second dissociation may be applied to the same slide well as the suspension from the first dissociation or a different slide well.
If using the same slide well:
- Add another 5 µL of 1.0N NaOH to the well either before or after applying the second dissociation to the well.
If using different slide wells:
- Add 5 µL of 1.0N NaOH to a second well.
- Add the second dissociation volume to this well.
Follow Method 1623 and manufacturer instructions from staining kit for specific drying techniques.
Narration:
Repeat the first dissociation step with another 50 µL of 0.1N HCl. The second dissociation ensures any remaining organisms are available for transfer to the slide.
Knowledge Review
ims0490 | page 29 of 46
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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)
Difficult Matrices
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Some source water matrices contain substances that interfere with processing or examination of the sample and may result in low matrix spike recoveries. Special techniques can be used with IMS to help resolve problems with:
- Low matrix spike recoveries
- Large pellet sizes
- Source waters exhibiting debris carryover problems
Some special techniques are not routinely used and not firmly established, but may be beneficial for some specific source waters.
Upon completion of this section, you should be able to describe the special techniques applicable to difficult matrices.
See IMS Supplement for special techniques.
Narration:
Every analyst will encounter source water matrices containing substances that interfere with processing or examination of the sample and may result in low matrix spike recoveries. Special techniques may be used with IMS to help resolve problems with difficult matrices.
Improving Recoveries
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Special techniques to increase recoveries from samples with various interferences include:
- Removal of interfering magnetic minerals prior to addition of beads
- Adjustment of the pH of acidic or alkaline samples to 7.0
- Removal of excess debris by rinsing the flat-sided tube
- Removal of excess debris trapped in the bead pellet by washing the pellet in the microcentrifuge tube
- Use of heat dissociation instead of acid dissociation
- Distribution of sample particulates over larger surface area on microscope slide
Detailed protocols for each technique and required quality control (QC) are available in the IMS Supplement.
Tips and Tricks
Narration:
Recoveries may be improved with the use of special techniques. Practice and test the modified technique prior to use on field samples. Performing the appropriate QC validates the use of the modification(s).
Tips and Tricks
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- Always practice and test modified techniques prior to use on field samples.
- Performing the appropriate QC, discussed later in this module, validates the use of the modification(s).
- Discuss technique modifications with other analysts.
Removing Magnetic Materials
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Some source water samples can contain high concentrations of iron and other magnetic material. Low recoveries could occur if extraneous magnetic material is present to interfere with the IMS process.
Some laboratories have determined the removal of extraneous magnetic material from the pellet, prior to the addition of beads, can improve recoveries.
The sample is processed through filtration, elution, and concentration. Extraneous magnetic material is removed from the pellet and the supernatant is carried through the remainder of the process.
See the IMS Supplement for the protocol and required quality control.
Narration:
Some source water samples may contain large amounts of magnetic material that may interfere with the IMS process. If matrix spike recoveries are low, one troubleshooting analysis could include the removal of extraneous magnetic material prior to IMS.
Adjusting pH
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Some source water samples may produce a pellet with non-neutral pH characteristics. Low recoveries could result if acidity or alkalinity of the pellet is not adequately buffered during the IMS process.
Some laboratories have determined the addition of HCl or NaOH to the sample, after buffers have been added, can neutralize the pH to improve recoveries. The pH of the sample can also be checked before buffers are added, and after the 1-hour rotation to ensure the pH is stable.
See the IMS Supplement for the protocol and required quality control.
Narration:
Some source water samples may produce a pellet with non-neutral pH characteristics. Acidity or alkalinity of the pellet can interfere with the IMS process and reduce overall recoveries. The pH of the sample can be checked to determine if the pH needs to be adjusted to pH 7.
Additional Rinse of Flat-sided Tube
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Some source water matrices produce large pellet sizes
(≥ 0.5 mL) when processed, and debris carryover may interfere with recoveries. An additional rinse removes visible debris left in the flat-sided tube after aspiration of all supernatant.
See the IMS Supplement for the protocol and required quality control.
Narration:
An additional rinse of the flat-sided tube may be used to remove excess debris left in the tube after decanting and aspirating the supernatant. Care must be taken not to disturb the bead pellet.
Bead Pellet Wash
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Recoveries for source water matrices that produce debris carryover may be improved by a bead pellet wash. Use this wash with difficult matrices that exhibit visible debris trapped in the bead pellet or remaining in the bottom of the microcentrifuge tube after aspiration of the supernatant.
Remember: Any remaining debris in the microcentrifuge tube will end up on the slide!
See the IMS Supplement for the protocol and required quality control.
Select the video to view a demonstration.
Narration:
A bead pellet wash may be used to remove excess debris carried over from the flat-sided tube. The debris may be trapped in the bead pellet or may remain in the bottom of the tube.
Select the video to view a demonstration.
Bead Pellet Wash
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Heat Dissociation
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Heat can be used instead of acid to inhibit reformation of the bead (oo)cyst complexes after dissociation by vortexing. This could potentially improve recoveries from some source water matrices where acid may drive an unfavorable reaction. Samples are processed through the method up to the acid dissociation step. Dissociation is performed in an 80° C heat block rather than with acid.
See the IMS Supplement for the protocol and required quality control.
Narration:
Heat dissociation is an alternative to acid dissociation and may improve recoveries from some source water matrices.
Modification of Sample Application to Slides
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Matrix debris can produce visual obstruction or potential loss of organisms during the staining process. Techniques to modify sample application to slides can reduce matrix debris interference.
- Use slides with larger diameter wells to spread the debris and organisms over a larger surface area. Well slide diameters range from 9 mm to 15 mm.
- Split each dissociation volume and apply to multiple wells.
See the IMS Supplement for the protocol and required quality control.
Narration:
One technique to reduce the interference of matrix debris on examination of slides is to apply the sample to a larger diameter well to spread the debris and organisms over a larger surface area. Another technique is to split the first and second dissociation volumes onto multiple wells.
Knowledge Review
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Which of the following techniques may be useful to increase the recoveries and reduce the debris interference of some difficult matrices? (Select all that apply. More than one statement may be correct.)
IMS Quality Control
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On-going Quality Control (QC) monitors the IMS process and helps resolve problems with recoveries.
- An IMS control is a 10-mL reagent water sample that is spiked with a known concentration of organisms and processed through IMS and staining to determine recoveries.
- The IMS control may be run weekly or with every 20 samples and may be used to:
- Monitor performance of new lots of IMS kits, spike organisms or staining kits.
- Troubleshoot low recoveries in On-going Precision and Recovery (OPR) and proficiency test samples.
- To help evaluate matrix effect on recoveries, an additional field sample may be processed as an IMS control by spiking organisms into the flat-sided tube.
- Track performance with IMS control charts.
- Percent recovery should be at least 70%.
- Include lot numbers, analyst names, and method modifications.
Narration:
The best technique to track the effectiveness of the IMS process is to regularly process IMS controls. An IMS control is an organism-spiked 10-mL reagent water sample, processed through IMS and staining to determine recoveries. Control charts can be used to record data and track changes.
Quality Control for Special Techniques
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The effectiveness of special techniques should be confirmed by acceptable performance of initial and on-going QC.
Special techniques discussed in this module which require QC validation are:
- Removing magnetic materials
- Adjusting the pH
- Incorporating flat-sided tube rinse or bead pellet wash
- Heat dissociation method
- Modification of sample application to slides
Initial QC:
- 4 reagent water organism-spiked samples and one method blank required.
- Matrix spike, matrix spike duplicate, and unspiked field sample analyses strongly recommended.
On-going QC:
- Process matrix spike sample(s) with the same modification as the associated field sample.
- Process the equivalent percentage of OPR samples and field samples using the same special technique.
See the Lab Guidance Manual for more information on Quality Control procedures.
Narration:
Method modifications may be made at individual laboratories as long as method criteria are met through appropriate QC, both initial and on-going.
Troubleshooting the IMS Procedure
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Use IMS control charts to determine if problems are related to IMS processing, changes in reagent lot numbers or equipment, and/or analysts. When problems with the IMS process are identified, review IMS techniques and protocols carefully.
Rollover each bar to view troubleshooting tips for processing.
Narration:
Review IMS control charts, techniques and protocols to help identify problems related to IMS.
Rollover each bar to view troubleshooting tips for processing.
Troubleshooting Low or No IMS Recovery (1 of 2)
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Review processing steps when troubleshooting low or no IMS recovery. The next two screens list specific techniques to review. Rollover each bar to view troubleshooting tips.
Narration:
You should review processing steps when troubleshooting performance problems. Specific techniques to review are included on the next two screens. Rollover each bar to view troubleshooting tips.
Troubleshooting Low or No IMS Recovery (2 of 2)
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Additional processing steps to check when troubleshooting low or no IMS recovery are listed here. Rollover each bar to view troubleshooting tips.
Narration:
Additional processing steps to review when troubleshooting performance problems are listed here. Rollover each bar to view troubleshooting tips.
Knowledge Review
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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)
Summary
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Congratulations! You have completed the EPA training module on IMS. Key points addressed in the module include:
- IMS uses antibody coated beads to bind with Cryptosporidium and Giardia.
- IMS processing separates the Cryptosporidium and Giardia from the extraneous debris.
- Special techniques may help improve recoveries from difficult matrices.
- IMS controls measure the efficiency of the IMS process.
IMS is an important and challenging procedure which, with practice, may be refined to maximize recovery and precision between samples, analysts and laboratories.
Narration:
Congratulations! You have completed the EPA training module on IMS. IMS is an important and challenging procedure which, with practice, may be refined to maximize recovery and precision between samples, analysts and laboratories.
Acknowledgments and Contacts
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Photographs used in the module were generated within EPA except where individual donations are credited. EPA wishes to thank the following entities for donating photographs for use in the module:
A | Brian Strohecker, Invitrogen Corporation |
B | George D. Di Giovanni, Texas Agricultural Experiment Station - El Paso, Texas A&M University |
For questions, contact EPA, Office of Ground Water and Drinking Water, Technical Support Center at miller.carrie@epa.gov.
This completes the module.
Narration:
EPA wishes to thank the entities listed for donating photographs. The listed entities have granted permission to EPA to use the photographs. Please request permission and credit the appropriate photographer if any of these photographs are used for any publicly distributed media.