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Introduction
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Welcome to the Environmental Protection Agency (EPA) on-line training module detailing the immunomagnetic separation or IMS procedure used in Method 1623. The IMS procedure is a separation and purification procedure designed to remove Cryptosporidium oocysts and Giardia cysts from extraneous debris in a water sample. This procedure is critical to ensure accurate and precise recoveries of the organisms.
Narration:
Welcome to the EPA on-line training module detailing the immunomagnetic separation or IMS procedure used in Method 1623. The IMS procedure is a separation and purification procedure designed to remove Cryptosporidium oocysts and Giardia cysts from extraneous debris in a water sample. This procedure is critical to ensure accurate and precise recoveries of the organisms.
Lesson Information
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The IMS procedure used in Method 1622/1623 requires consistent and meticulous lab technique. 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.
Credit for each photograph is indicated with a letter which refers to the photographer listed on the acknowledgment page at the end of the lesson.
Computer requirements:
800 x 600 screen resolution, minimum
Sound turned on for narration
Macromedia Flash 7, required for interactive graphics and video segments
Computer audio on to hear narration
Module features:
The capital T icon in the menu bar displays narration
Printer icon prints the complete module
Knowledge Reviews at the end of each section help highlight and reinforce key learning points
Narration:
The IMS procedure used in Method 1622/1623 requires consistent and meticulous lab technique. 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, the student will be able to:
- Describe the process for complete transfer of the target organisms in the sample, and any sub-samples, between 3 types of tubes and a slide
- Describe the process for optimizing the performance of mixing equipment including rotator, magnet housings, vortexer and pipettes
- Describe the process for preparing solutions and suspensions for precision and efficiency
- Identify troubleshooting techniques based on the IMS section of Method 1622/1623
- Identify techniques designed to improve the recovery from difficult matrices
Additional objectives are detailed at the beginning of each section in the module.
Narration:
Upon completion of the module, the student will be able to properly perform and troubleshoot the IMS procedure maximizing recovery and precision between samples, analysts, and laboratories.
Definition and Purpose
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Immunomagnetic separation (IMS) is a selective purification procedure which uses magnetically responsive particles coated with antibodies that selectively attach to Cryptosporidium and Giardia. The bead-organism complexes are separated from the debris in the sample using a magnet. The 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 samples, IMS is a very dynamic and critical processing step in Method 1622/1623.
Narration:
Immunomagnetic separation, or IMS, is a selective purification procedure which uses magnetically responsive particles coated with antibodies that selectively attach to Cryptosporidium and Giardia.
Technology (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. The IMS kit most commonly used by laboratories performing Method 1622/1623 exhibits the following properties:
- Magnetism only when exposed to a magnetic field (no residual magnetism)
- Minimal non-specific binding
- Polymer shell, eliminating toxic exposure to iron
- Uniform size (approx. 5 µM), spherical shape and surface area
Narration:
The beads used are polymer particles which contain iron and exhibit the properties listed.
The beads are coated with anti-Cryptosporidium and anti-Giardia monoclonal antibodies which are genus specific but not species specific.
Technology (2 of 3)
ims0050 | page 6 of 8
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The beads are added to a water sample concentrate along with specific buffer solutions.
The samples are mixed allowing the bead-organism complexes to form.
After mixing, the samples are exposed to a magnet which removes the bead-organism complexes from the sample.
Narration:
IMS beads are mixed with a water sample concentrate and bead-organism complexes are removed using a magnet.
Technology (3 of 3)
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The beads are dissociated from the organisms to prevent their interference with microscopy. Method 1622/1623 currently uses an acid dissociation protocol.
Hydrochloric acid is added to the microcentrifuge tube 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 bonds from reforming.
The beads are removed using the magnet, leaving behind a suspension containing the organisms which is then placed on a well slide.
Narration:
The beads are dissociated from the organisms to prevent their interference with microscopy. Method 1622/1623 currently uses an acid dissociation protocol.
Hydrochloric acid is added to the microcentrifuge tube 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 bonds from reforming.
The beads are removed using the magnet, leaving behind a suspension containing the organisms which is then placed on a well slide.
ims0060 | page 8 of 8
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This concludes the prototype for the Method 1622/1623
Immunomagnetic Separation Module.