Diagnostic Medical Parasitology. Lynne Shore Garcia
added. A QC smear prepared from a positive fecal sample or a fixative sample containing buffy coat cells should be used when new stain is prepared or at least once each week. Cultured protozoa can also be used.
2. A QC slide should be included with a run of stained slides at least monthly; more frequent QC is recommended for those who may be unfamiliar with the method (14).
3. If the xylene becomes cloudy or there is an accumulation of water in the bottom of the staining dish containing xylene, discard the old reagents, clean the dishes, dry thoroughly, and replace with fresh 100% ethanol and xylene or xylene substitute.
4. All staining dishes should be covered to prevent evaporation of reagents (screw-cap Coplin jars or glass lids).
5. Depending on the volume of slides stained, staining solutions will have to be changed on an as-needed basis.
6. When the smear is thoroughly fixed and the staining procedure is performed correctly, the cytoplasm of protozoan trophozoites is blue-green, sometimes with a tinge of purple. Cysts tend to be slightly more purple. Nuclei and inclusions (chromatoidal bars, RBCs, bacteria, and Charcot-Leyden crystals) are red, sometimes tinged with purple. The background material usually stains green, providing a nice color contrast with the protozoa. This contrast is more distinct than that obtained with the hematoxylin stain, which tends to stain everything in shades of gray-blue to black.
7. If appropriate, the microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope).
8. Known positive microscope slides and photographs (reference books) should be available at the workstation.
9. Record all QC results; the laboratory should also have an action plan for “out-of-control” results.
Procedure for Trichrome Stain with Mercury-Based Fixatives (Fig. 3.16)
Note In all staining procedures for fecal and gastrointestinal tract specimens, the term “xylene” is used in the generic sense. Xylene substitutes are recommended for the safety of all personnel performing these procedures.
Figure 3.16 Trichrome staining. Option 1, for use with smears prepared from fixatives containing mercuric chloride. The iodine is used to remove the mercuric chloride, and the subsequent two alcohol rinse steps remove the iodine. Thus, prior to staining, both the mercuric chloride and iodine have been removed from the smear. Options 2 and 3, for use with smears prepared from fixatives containing no mercuric chloride (the user can select option 2 or 3; there is minimal to no difference). doi:10.1128/9781555819002.ch3.f16
1. Prepare the slide for staining as described above.
2. Remove the slide from liquid Schaudinn’s fixative, and place it in 70% ethanol for 5 min.
3. Place the slide in 70% ethanol plus iodine for 1 min for fresh specimens or 5 to 10 min for PVA air-dried smears. The exposure to iodine will remove the mercuric chloride from the smear prior to staining with the actual trichrome dyes (substitution of iodine for mercury).
4. Place the slide in 70% ethanol for 5 min.* This and the next step in 70% ethanol will remove the iodine from the smear.
5. Place it in a second container of 70% ethanol for 3 min.*
6. Place it in trichrome stain for 10 min. The fecal smear no longer contains either mercuric chloride or iodine and is now ready for staining.
7. Place it in 90% ethanol plus acetic acid for 1 to 3 s. Immediately drain the rack (see Procedure Notes), and proceed to the next step. Do not allow slides to remain in this solution. This is the destaining step.
8. Dip the slide several times in 100% ethanol. Use this step as a rinse.
9. Place it in two changes of 100% ethanol for 3 min each.* This is a dehydration step.
10. Place it in xylene or xylene substitute for 5 to 10 min.* This is a dehydration step.
11. Place it in a second container of xylene or xylene substitute for 5 to 10 min.* This step completes the dehydration process (removal of all water on the smear).
12. Mount the slide with a coverslip (no. 1 thickness), using mounting medium (e.g., Permount).
13. Allow the smear to dry overnight or after 1 h at 37°C.
14. Examine the smear microscopically with the 100× objective. Examine at least 200 to 300 oil immersion fields before reporting a negative result (Fig. 3.17).
Figure 3.17 Guess the identity of these intestinal protozoa! See p. 76 for the answers. doi:10.1128/9781555819002.ch3.f17
*Slides may be held for up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.
Procedure for Trichrome Stain with Non-Mercury-Based Fixatives (Iodine-Alcohol Step and Alcohol Rinse Not Required) (Fig. 3.16)
1. Prepare the slide for staining as described above.
2. Place the slide in 70% ethanol for 5 min.*
3. Place it in the trichrome stain for 10 min. Some people prefer to place the dry smear directly into the stain and eliminate step 2 from the protocol.
4. Place it in 90% ethanol plus acetic acid for 1 to 3 s. Immediately drain the rack (see Procedure Notes), and proceed to the next step. Do not allow slides to remain in this solution.
5. Dip the slide several times in 100% ethanol. Use this step as a rinse.
6. Place it in two changes of 100% ethanol for 3 min each.*
7. Place it in xylene or xylene substitute for 5 to 10 min.*
8. Place it in a second container of xylene or xylene substitute for 5 to 10 min.*
9. Mount with a coverslip (no. 1 thickness), using mounting medium (e.g., Permount).
10. Allow the smear to dry overnight or after 1 h at 37°C.
11. Examine the smear microscopically with the 100× objective. Examine at least 200 to 300 oil immersion fields before reporting a negative result.
*Slides may be held for up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.
Results and Patient Reports from the Trichrome Staining Method
Protozoan trophozoites and cysts are readily seen, although helminth eggs and larvae may not be easily identified because of excess stain retention (wet smears from the concentration procedure[s] are recommended for detection of these organisms) (Fig. 3.18 and 3.19).