Diagnostic Medical Parasitology. Lynne Shore Garcia

      Quality Control for Direct Smear

      1. Check the working iodine solution each time it is used or periodically (once a week). The iodine and Nair’s methylene blue solutions should be free of any signs of bacterial or fungal contamination.

      2. The iodine should be the color of strong tea (discard if it is too light).

      3. Protozoan cysts stained with iodine should contain yellow-gold cytoplasm, brown glycogen material, and paler refractile nuclei. The chromatoidal bodies may not be as clearly visible as they are in a saline mount. Human WBCs (buffy coat cells) mixed with negative stool can be used as a quality control (QC) specimen. These human cells, when mixed with negative stool, mimic protozoan parasites. The human cells stain with the same color as that seen in the protozoa.

      4. Protozoan trophozoite cytoplasm should stain pale blue and the nuclei should stain a darker blue with the methylene blue stain. Human WBCs mixed with negative stool should stain the same colors as seen with the protozoa.

      5. The microscope should be calibrated (within the last 12 months), and the original optics used for the calibration should be in place on the microscope when objects are measured. Some microbiologists feel that calibration is not required on a yearly basis; however, if the microscope receives heavy use, is in a position where it can be bumped, or does not receive routine maintenance, yearly calibration is recommended. The calibration factors for all objectives should be posted on the microscope or close by for easy access.

      6. All QC results should be appropriately recorded; the laboratory should also have an action plan for “out-of-control” results.

      Procedure for Direct Wet Smear

      1. Place 1 drop of 0.85% NaCl on the left side of the slide and 1 drop of iodine (working solution) on the right side of the slide. If preferred, two slides can be used instead of one. One drop of Nair’s methylene blue can also be placed on a separate slide, although this technique is much less common.

      2. Take a small amount of fecal specimen (the amount picked up on the end of an applicator stick when introduced into the specimen), and thoroughly emulsify the stool in the saline and iodine preparations (use separate sticks for each).

      3. Place a 22-mm coverslip (no. 1) on each suspension.

      4. Systematically scan both suspensions with the 10× objective. The entire coverslip area should be examined under low power (total magnification, ×100).

      5. If something suspicious is seen, the 40× objective can be used for more detailed study. At least one-third to one-half of the coverslip should be examined under high dry power (total magnification, ×400), even if nothing suspicious has been seen.

      6. Another approach is to prepare and examine the saline mount and then add iodine at the side of the coverslip. The iodine will diffuse into the stool-saline mixture, providing some stain for a second examination. Remember, the iodine will kill any organisms present; thus, no motility will be seen after the iodine is added to the preparation. The use of iodine is not mandated, but is based on personal preference.

      Results and Patient Reports from Direct Wet Smear

      Protozoan trophozoites and/or cysts and helminth eggs and larvae may be seen and identified. In a heavy infection with Cryptosporidium spp., Cyclospora cayetanensis, or Cystoisospora (Isospora) belli, oocysts may be seen in a direct smear; however, some type of modified acid-fast stain or fecal immunoassay is normally used to detect Cryptosporidium spp., particularly when few oocysts are present. Cyclospora oocysts are often confirmed using autofluorescence or the modified acid-fast stain. Spores of the microsporidia are too small, and the shape resembles other debris within the stool; therefore, they are not readily visible in a direct smear.

      1. Motile trophozoites and protozoan cysts may or may not be identified to the species level (depending on the clarity of the morphology) and should be confirmed using the permanent stained smear.

      Examples: Giardia lamblia (G. duodenalis, G. intestinalis) trophozoites

      Entamoeba coli cysts

      2. Helminth eggs and/or larvae may be identified.

      Examples: Ascaris lumbricoides eggs

      Strongyloides stercoralis larvae

      3. Cystoisospora (Isospora) belli oocysts may be identified; however, Cyclospora and Cryptosporidium oocysts are generally too small to be recognized or identified without subsequent immunoassays or modified acid-fast staining.

      Example: Cystoisospora (Isospora) belli oocysts

      4. Artifacts and/or other structures may also be seen and reported as follows.

      Note These crystals and cells are quantitated; however, the quantity is usually assessed when the permanent stained smear is examined under oil immersion.

      Examples: Moderate Charcot-Leyden crystals

      Few RBCs

      Moderate PMNs

      Procedure Notes for Direct Wet Smear

      1. In preserved specimens, the formalin, sodium acetate-acetic acid-formalin (SAF), or Universal Fixative replaces the saline and can be used as a direct smear; however, no organism motility will be visible (organisms are killed by the fixatives). Consequently, the direct wet smear is usually not performed when the specimen (already preserved) arrives in the laboratory. The technical time is better spent performing the concentration and permanent stained smear. This approach is recommended for specimens submitted to the laboratory in preservative (2).

      2. As mentioned above, some workers prefer to make the saline and iodine mounts on separate slides and on 2- by 3-in. slides. Often, there is less chance of getting fluids on the microscope stage if separate slides (less total fluid on the slide and under the coverslip) or larger slides are used. Selection of slide size and the use of iodine depend on the personal preference of laboratory personnel.

      3. The microscope light should be reduced for low-power observations, since most organisms are overlooked with bright light due to limited contrast of the internal morphology. This is particularly true when the preparation is being examined without the use of iodine. Illumination should be regulated so that some of the cellular elements in the feces show refraction (lower the condenser). Most protozoan cysts and some coccidian oocysts are refractile under these light conditions.

      Procedure Limitations for Direct Wet Smear

      1. As mentioned above, because motility is lost when specimens are placed in preservatives, many laboratories are no longer performing the direct wet smear (the primary purpose is to see motility) but are proceeding directly to the concentration and permanent stained smear procedures as a better, more cost-effective use of personnel time, as well as a more clinically relevant approach.