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Method 1622/1623 Microscopy
Prototype

Introduction

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This is the EPA Method 1622/1623 Microscopy Module on Giardia and Cryptosporidium. The U.S. Environmental Protection Agency (EPA) is required to evaluate the risk to public health posed by drinking water contaminants, including waterborne parasites, such as Giardia and Cryptosporidium. Method 1622/1623 uses filtration, immunomagnetic separation (IMS) and microscopy to detect Giardia and Cryptosporidium. This module was developed to assist both new and experienced analysts in the proper identification and enumeration of Giardia and Cryptosporidium. This module also targets the characteristics of interfering organisms.


narration: Welcome to the EPA Method 1622\/1623 Microscopy module on Giardia<\/i> and Cryptosporidium<\/i>. Method 1622\/1623 is the current method for detecting these specific waterborne parasites. Method 1622 detects Cryptosporidium<\/i> only, whereas, Method 1623 was developed to address utilities concerns regarding the presence of either Giardia<\/i> and Cryptosporidium<\/i>. Both methods use microscopy as the means for identification and enumeration. Therefore, the ability to properly identify Giardia<\/i> and Cryptosporidium<\/i> and distinguish both organisms from interfering organisms is critical to the proper implementation of Method 1622\/1623. Animation


Lesson Information

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Approximate Length: 1 hour

Identification and enumeration by microscopy is the endpoint of Method 1622/1623. Narration, animation, photographs and video are used to explain key diagnostic features of both Giardia and Cryptosporidium. After examining the positive specimens, variations, cross-reacting organisms and autofluorescing organisms will be highlighted. Finally, how to enumerate an environmental field slide, where both cross-reacting and real organisms could be observed, is covered.

Knowledge Reviews and summaries will help highlight key points. You will have two attempts (one for True/False questions) to correctly answer each Knowledge Review. A self-directed Quiz follows the module.


narration: Microscopy is a critical skill for analysts performing Method 1622\/1623. This module will help guide the analyst through both the science and art of Giardia<\/i> and Cryptosporidium<\/i> identification. You will have the chance to simulate focusing through an organism to see the series of planes visible at the microscope. Diagnostic features of both positive and unknown specimens are covered followed by proper enumeration techniques. This module is not intended to substitute for time and experience at the microscope but supplement it. Enjoy the microscopy journey. 3 pictures: woman looking through microscope, Giardia and Cryptosporidium


Learning Objectives

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By completing this lesson you should be able to:

  • Locate proper plane of focus
  • Locate Giardia and Cryptosporidium by fluorescence (FITC)
  • Identify and describe Giardia and Cryptosporidium by DAPI
  • Identify and describe internal morphology of Giardia and Cryptosporidium using DIC
  • Measure Giardia cysts and Cryptosporidium oocysts
  • Characterize cross-reacting and autofluorescing organisms
  • Exhibit proper scanning technique
  • Enumerate an environmental field slide


narration: On completion of this module and subsequent practice, participants will be able to identify and describe Giardia<\/i> and Cryptosporidium<\/i> as defined in Method 1622\/1623. The objectives are listed and highlight the difference between Giardia<\/i> and Cryptosporidium<\/i> and cross-reacting organisms.


Determining the Correct Plane of Focus

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The Giardia and Cryptosporidium are located on the bottom of the well. Find this plane of focus before examining a slide according to the following standard operating procedure.

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  1. Place the microscope on 20X objective
  2. Place a Method 1623 positive control slide on the stage
  3. Move the stage and slide so that the edge of the well is below the 20X objective
  4. Examine the edge of the well and focus using the coarse adjustment
  5. Move the stage to position the objective over the middle of the well
  6. Without adjusting the coarse adjustment, locate an organism
  7. Focus this organism using the fine adjustment
  8. Examine other portions of the well – most organisms should be in focus
  9. Constant focusing up and down is necessary to locate organisms which may have lifted off the bottom of the well slide
Select the link for
Tricks and Tips.
narration: Before any slides can be examined, the proper plane of focus must be determined. Ocular adjustments and Kohler illumination must also be performed to reach the maximum contrast and resolution. During Method 1623 processing, the Giardia<\/i> and Cryptosporidium<\/i> cysts and oocysts are dried onto a well slide and should be found on the bottom of the well. Locate this plane of focus before adjusting the microscope and before examining a slide. Most organisms will be located in this plane but constantly adjusting the fine adjustment while examining a slide is necessary to find any organisms which may be in a slightly different plane.

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Tricks and Tips

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  • Always focus cysts/oocysts on 20X at the end of a microscope session. You will be in the correct plane of focus when you return to the scope.


  • If the stage is completely lowered, then engage the 40X objective and use the coarse adjustment to bring the stage up close to the 40X objective, WATCHING from the side. DO NOT perform this without watching. It could crash the slide into the 40X objective, which will break the lens, unless you have retractable objectives. This gets you close to the correct plane of focus; switch to 20X objective and continue to focus using the fine adjustment while viewing through the oculars.




Identification of Giardia under FITC

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Proper identification is imperative for accurate and precise enumeration. Under FITC Giardia will exhibit the following characteristics:

  • Brilliant apple-green color
  • Brightly highlighted wall
  • Fluorescence - crisp and sharp
  • Fluorescence - consistent thickness around the organism
  • Fluorescence over the complete cyst is lacking
  • No splattering of fluorescence when focusing

After identifying Giardia the shape must be documented on the examination results form.
narration: The wall of Giardia<\/i> cysts are stained with FITC labeled antibodies. A properly stained organism is the first indication that the organism may be Giardia<\/i>. The fluorescence is crisp and sharp; highlighting the edge of the cyst. As the analyst focuses through the organism, the edge stays consistent around the cyst. Giardia under FITC


Giardia Examination Results Form

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Giardia Benchsheet


Knowledge Review

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Under FITC Giardia will exhibit all of the following characteristics except:

A. Splattering of fluorescence when focusing
B. Crisp and sharp fluorescence
C. Brilliant apple-green color
D. Bright green fluorescing rim contrasting with less intense center



This completes the Prototype.