The microscope should be calibrated (within the last 12 months) (recommended but not always required, depending on the use and care of the microscope), 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 Iron Hematoxylin Stain with Mercury-Based Fixatives
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.
1. Prepare the slide for staining as previously described (for SAF smears or smears prepared from other non-mercury single-vial preservatives, proceed to step 4).
2. Place the slide in 70% ethanol for 5 min.
3. Place the slide in the iodine-70% ethanol (70% alcohol to which is added enough D’Antoni’s iodine to obtain a strong-tea color) solution for 2 to 5 min. The iodine is designed to remove the mercury from the smear.
4. Place it in 70% ethanol for 5 min (this rinse step removes the iodine). Begin the procedure for SAF-fixed slides at this point.*
5. Wash the slide in running tap water (constant stream of water into the container) for 10 min.
6. Place the slide in iron hematoxylin working solution for 4 to 5 min.
7. Wash the slide in running tap water (constant stream of water into the container) for 10 min.
8. Place the slide in 70% ethanol for 5 min.*
9. Place the slide in 95% ethanol for 5 min.*
10. Place the slide in two changes of 100% ethanol for 5 min each.*
11. Place the slide in two changes of xylene for 5 min each.*
12. Add Permount to the stained area of the slide, and cover with a coverslip.
Note An alternative method to using mounting medium is given on p. 53.
13. 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.
Procedure for Iron Hematoxylin Stain with Non-Mercury-Based Fixatives
1. Prepare the slide for staining as described above.
2. Place it in 70% ethanol for 5 min.*
3. Wash the slide in running tap water (constant stream of water into the container) for 10 min.
4. Place the slide in iron hematoxylin working solution for 4 to 5 min.
5. Wash the slide in running tap water (constant stream of water into the container) for 10 min.
6. Place the slide in 70% ethanol for 5 min.*
7. Place the slide in 95% ethanol for 5 min.*
8. Place the slide in two changes of 100% ethanol for 5 min each.*
9. Place the slide in two changes of xylene for 5 min each.*
10. Add Permount to the stained area of the slide and cover it with a coverslip.
Note An alternative method to using mounting medium is given on p. 53.
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 Iron Hematoxylin Staining Method
Protozoan trophozoites and cysts are readily visible, 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). Yeasts (single and budding cells and pseudohyphae) and human cells (macrophages, PMNs, and RBCs) can be identified. The following quantitation chart can be used for examination of permanent stained smears with the oil immersion lens (100× objective; total magnification, ×1,000).
| Quantitation of parasites, cells, yeasts, and artifacts | |
| Quantity | No. per 10 oil immersion fields (×1,000) |
| Few Moderate Many | ≤2 3–9 ≥10 |
1. Report the organism and stage (do not use abbreviations).
Examples: Entamoeba coli trophozoites
Dientamoeba fragilis trophozoites
2. Quantitate the number of Blastocystis spp. organisms seen (rare, few, moderate, many). Do not quantitate other protozoa.
Example: Many Blastocystis spp.
3. Note and quantitate the presence of human cells.
Example: Moderate WBCs, few macrophages, few RBCs, rare Charcot-Leyden crystals
4. Report and quantitate yeast cells.
Example: Many budding yeast cells and few pseudohyphae
NOTE Because yeast can continue to grow if the stool is not immediately preserved, some laboratories do not report yeast, since the report can be misleading. They elect to call the physician and discuss the findings. Another option is to add a report comment indicating that reports of yeast (budding and/or pseudohyphae) might be misleading due to a lag time between stool passage and specimen fixation.
5. Save positive slides for future reference. Label prior to storage (name, patient number, organisms present). Most laboratories hold their negative smears for several weeks and then discard them; slide boxes that are rotated can be used to batch, store, and discard negative smears.
Procedure
