Immunomagnetic Separation
Immunomagnetic Separation

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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.


Opening animation



Lesson Information

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Time to complete this module is approximately 1 to 1.5 hours.

Analyst performing IMS in biosafety hood.

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:

Module features:

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:

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.


Pellets, withdrawing beads, tube in magnet



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.


Pellet, tube with beads, and Cryptosporidium with beads



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:

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.


Beads



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.


Particles in multiple test tubes and 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.


Particles in multiple test tubes next to a magnet



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:

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.


Pellets, aspiration, and pellet resuspension



Pellet Measurement

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Prepared Pellet Standards

Note IconTips 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.



Pellet standards prepared with colored glycerol, Packed pellets after centrifugation and Packed pellets being compared to prepared pellet standards


Tips and Tricks

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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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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.

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.


rollover



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.

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.


Packed pellet, partially resuspended pellet, fully resuspended pellet



Knowledge Review

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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)


A. Pellet standards should be used to compare and estimate the volume of the packed pellet.
B. The pellet is resuspended before transfer to the flat-sided tubes by inverting the centrifuge tube by hand 2 or 3 times.
C. The vortex speed should be reduced on pellet resuspension if excess debris is splattered on the sides of the centrifuge tube.
D. The weight of the centrifuge bottles should be balanced to within 0.5 grams.
E. During aspiration, 10 mL of fluid should be retained for every 0.5 mL of pellet.
F. The pellet sample is aspirated using high vacuum pressure to ensure organisms are pulled out of suspension.
G. Aspiration of the supernatant should be at the air/liquid interface not below the water surface.






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:

Note IconTips 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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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.


0.5 mL, 1 mL and 2 mL pellets



Transfer to Flat-sided Tube (2 of 2)

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swf*

Note IconTips 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.





swf

8902



Tips and Tricks

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Equipment in hood, pipette measurement and various pipettes with pipetter





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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Note IconTips 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.


Beads unmixed, vortexing, beads mixed, withdrawing beads, and transfer of 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:

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.

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.


Calibrated and evenly loaded rotating mixer



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.
Rinse the centrifuge tube twice if the complete pellet is transferred to flat-sided tubes.
Pipette 5 mL of resuspended pellet concentrate into a flat-sided tube.
Bring buffers, beads, and samples to room temperature.
Add 1 mL of SL-Buffer A and 1 mL of SL-Buffer B to each flat-sided tube.
Rotate the tubes for 60 minutes.
Add 100 µL of anti-Cryptosporidium beads and 100 µL of anti-Giardia beads to each flat-sided tube.






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:

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.


Pipettes with pipetters and magnetic particle concentrators



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.

Select the videos for demonstrations.

Note IconTips 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.


rollover



Tips and Tricks

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Automated rocking plates

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:

Select the videos for demonstrations.

Note IconTips 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.


rollover



Tips and Tricks

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Blotting 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

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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:

Note IconTips 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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Resuspension of Bead Pellet

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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:

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.


rollover


swf

8924



Knowledge Review

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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)


A. After decanting the supernatant from the flat-sided tube you can use a pipette to remove additional supernatant from the bottom of the tube.
B. Both the MPC®-6 and MPC®-1 are rocked through 180° for 2 minutes.
C. After adding buffer to the decanted flat-sided tube, you should vortex vigorously to resuspend the pellet.
D. Rinse the flat-sided tube twice with additional buffer to transfer to the microcentrifuge tube.
E. When the MPC®-S magnet is in the vertical position, the pellet forms high on the back wall of the tube.
F. Automatic rocking plates are useful to provide better consistency in rocking the flat-sided tubes.
G. The magnetic particle concentrator is used to attract the bead-organism complexes to the magnet.






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.
Rock for 1 minute
Aspirate supernatant
Rock for 2 minutes
Quantitatively transfer resuspended bead pellet to microcentrifuge tube
Decant supernatant
Place the flat-sided tube in MPC®






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.

Note IconTips 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.


rollover



Tips and Tricks

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Dissociation

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Neutralize pH

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After the tube has been appropriately vortexed:

To neutralize the pH of the sample:

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.


rollover



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.

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:

If using different slide wells:

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.



Dissociation steps


Knowledge Review

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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)


A. HCl is used to break the bond between the bead and the (oo)cyst.
B. HCl is used to inhibit the reformation of the bond between the bead and the (oo)cyst.
C. Vortexing one time at minimum speed is used to disrupt the bond between the bead and the (oo)cyst.
D. NaOH neutralizes the HCl used in the dissociation process.
E. Vortexing at maximum speed is used to disrupt the bond between the bead and the (oo)cyst.
F. The MPC®-S magnet is placed in the slanted position to remove the dissociated beads from the (oo)cysts in suspension.






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:

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.


Slides with debris, and large pellet sizes



Improving Recoveries

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Special techniques to increase recoveries from samples with various interferences include:

Detailed protocols for each technique and required quality control (QC) are available in the IMS Supplement.

Note IconTips 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).



Debris remaining in tube, heatblock, and multiple wells


Tips and Tricks

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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.


Packed pellets, magnetic material being removed, supernatant being poured



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.


Buffers, reagents and pH meter



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.


Tube with debris, 10 mL rinse, tube with debris removed



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.


rollover



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.


Tubes in heat block with graphic showing process of going to vortex then exposure to magnet. Instructions to add 50 µL of reagent water and place tube(s) in 80°C heat block for 10 minutes



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.

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.


Slides with debris



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.)


A. Removing the extraneous magnetic material from the pellet prior to adding beads.
B. Rinsing the flat-sided tube with PBS while still in the MPC®-1 or MPC®-6.
C. Applying the sample to a larger well slide to spread out the organisms and debris.
D. Adusting the pH of the sample with HCl or NaOH after the addition of the buffers.






IMS Quality Control

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IMS control

On-going Quality Control (QC) monitors the IMS process and helps resolve problems with recoveries.

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.

4 reagent water spikes and 1 method blank

Special techniques discussed in this module which require QC validation are:

Initial QC:

On-going QC:

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.


rollover



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.


rollover



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.


rollover



Knowledge Review

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Which of the following statements are correct?
(Select all that apply. More than one statement may be correct.)


A. Initial QC for Method modifications involves the analysis of 3 organism-spiked reagent water samples.
B. Percent recovery for an IMS control should be at least 70%.
C. Method modifications require validation with initial and on-going QC.
D. Excess beads on a slide can result from improper technique during the dissociation procedure.
E. An IMS control is processed through the complete Method to troubleshoot low percent recovery.
F. Low or no IMS recovery may result from the improper preparation of the pellet or the incorrect addition of beads and buffers.






Summary

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Congratulations! You have completed the EPA training module on IMS. Key points addressed in the module include:

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.


equipment with full MPC-6



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:

ABrian Strohecker, Invitrogen Corporation
BGeorge 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.