Proper technique ensures that the entire area on the slide is read only once with no overlap or deletions. This provides the most accurate and precise enumeration.
Scan the entire well slide systematically in a top-to-bottom or side-to-side pattern using a minimum of 200X magnification and FITC fluorescence. Organisms which resemble Giardia or Cryptosporidium by FITC fluorescence are examined using DAPI and DIC.
Each row or column should be equal to or slightly less than the height or width of one field of view.
Tricks and Tips
narration: Proper scanning technique is necessary for accurate and precise enumeration. Use the positive control slide or OPR to practice. An analyst scans the entire well in a top-to-bottom or side-to-side fashion with each column or row the same width or height. Scanning should be completed using epifluorescence and FITC at a minimum magnification of 200X. Once an object that needs further investigation is observed, the analyst switches to DAPI and at least 400X magnification. Finally, the analyst switches to 1000X DIC to complete the examination and characterization.
The end result of Method 1622/1623 is the total number of cysts/oocysts counted on the slide which is used to calculate the number per L.
After conducting the appropriate examination of the positive control slide, the field slide is scanned following the correct procedure. The cysts and oocysts counted must meet the criteria for Giardia and Cryptosporidium stated in the Method.
- Correct size and shape
- Brilliant apple-green fluorescence
- No atypical features when viewed with DAPI or on DIC
Each organism is described, measured and recorded on the examination results form. If the organism does not meet the definition, the reason is stated.
Play the video to begin a trial count of 2 rows.
narration: After practicing describing positive control Giardia<\/i> and Cryptosporidium<\/i>, counting an OPR slide, and meeting the Analyst Verification criteria, an analyst is prepared to examine a field slide. A careful record of organisms examined and the final determinations regarding those organisms is kept by the analyst. During scanning or when scanning is complete, a second analyst examines the organisms determined to be Giardia<\/i> and\/or Cryptosporidium<\/i> and\/or cross-reacting organisms. Once multiple analysts agree that the organisms meet the criteria of the Method, the examination results form is completed and the slide is archived.
When an analyst is scanning correctly, which of the following is true?