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 Notes for the Iron Hematoxylin Staining Method
1. The single most important step in the preparation of a well-stained fecal smear is good fixation. If this has not been done, the protozoa may be distorted or shrunk, may not be stained, or may exhibit an overall gray or blue-gray color with poor internal morphology.
2. Slides should always be drained between solutions. Touch the end of the slide to a paper towel for 2 s to remove excess fluid before proceeding to the next step. This will maintain the staining solutions for a longer period. The slides can also be drained against the edge of each container before being moved to the next container.
3. Incomplete removal of mercuric chloride (Schaudinn’s fixative and PVA fixative prepared with a mercuric chloride base) may cause the smear to contain highly refractive crystals or granules, which may prevent finding or identifying any organisms present. Since the 70% ethanol–iodine solution removes the mercury complex, it should be changed at least weekly to maintain the strong-tea color. A few minutes are usually sufficient to keep the slides in the iodine-alcohol; too long a time in this solution may also adversely affect the staining of the organisms.
4. When using non-mercury-based fixatives, the iodine-alcohol step (used for the removal of mercury) and the subsequent alcohol rinse (used for the removal of iodine) can be eliminated from the procedure. The smears for staining can be prerinsed with 70% alcohol and then placed in the water step prior to the hematoxylin stain as the first step in the staining protocol.
5. For staining large numbers of slides, the working hematoxylin solution may be diluted and affect the quality of the stain. If dilution occurs, discard the working solution and prepare a fresh working solution.
6. The shelf life of the stock hematoxylin solutions may be extended by keeping the solutions in the refrigerator at 4°C. Because of crystal formation in the working solutions, it may be necessary to filter them before preparing a new working solution.
7. In the final stages of dehydration (steps 9 to 11), the 100% ethanol and the xylenes should be kept as free from water as possible. Coplin jars must have tight-fitting caps to prevent both evaporation of reagents and absorption of moisture. If the xylene becomes cloudy after addition of slides from the 100% ethanol, return the slides to fresh 100% ethanol and replace the xylene with fresh stock.
8. If the smears peel or flake off, the specimen might have been inadequately dried on the slide (in the case of fixed specimens containing PVA), the smear may have been too thick, or the slide may have been greasy (fingerprints). However, slides generally do not have to be cleaned with alcohol prior to use.
9. On examination, if the stain appears unsatisfactory and it is not possible to obtain another slide to stain, the slide may be restained. Place the slide in xylene to remove the coverslip, and reverse the dehydration steps, adding 50% ethanol as the last step. Destain the slide in 10% acetic acid for several hours, and then wash it thoroughly first in water and then in 50 and 70% ethanol. Place the slide in the iron hematoxylin stain for 8 min, and complete the staining procedure (2, 3).
Procedure Limitations for the Iron Hematoxylin Staining Method
1. The permanent stained smear is not recommended for staining helminth eggs or larvae; these structures are often too dark (excess stain retention) or distorted. However, they are occasionally recognized and identified. The wet smear preparation from the concentrate is the recommended approach for identification of helminth eggs and larvae.
2. The smear should be examined with the oil immersion lens (100×) for the identification of protozoa, human cells, Charcot-Leyden crystals, yeast cells, and artifact material. Quantitation of these cells and other structures is normally done from the examination of the permanent stained smear, not the wet smear preparations (direct wet smear, concentration wet smear).
3. This high-magnification (oil immersion; total magnification, ×1,000) examination is recommended for protozoa, particularly for confirming species identification.
4. With low magnification (10× objective), one might see eggs or larvae; however, this is not recommended as a routine approach.
5. In addition to helminth eggs and larvae, C. belli oocysts are best seen in wet preparations (concentration wet smears prepared from formalin-preserved, not preserved stool containing PVA).
6. Cryptosporidium and Cyclospora oocysts are generally not recognized on an iron hematoxylin-stained smear (modified acid-fast stains or the fecal immunoassay for Cryptosporidium spp. is recommended).
Iron Hematoxylin Stain (Tompkins-Miller Method) (20)
A longer iron hematoxylin method was described by Tompkins and Miller (20). Since differentiation of overstained slides is critical in most iron hematoxylin staining procedures, Tompkins and Miller have described a method that employs phosphotungstic acid to destain the protozoa and that gives excellent results, even in unskilled hands.
1. Prepare the slide for staining as described above (for SAF or non-mercury-based smears, 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