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 color) solution for 2 to 5 min.
4. Place it in 50% ethanol for 5 min. Begin the procedure for SAF- or non-mercury-fixed slides at this point.*
5. Wash the slide in running tap water (constant stream of water into the container) for 3 min.
6. Place the slide in 4% ferric ammonium sulfate mordant for 5 min.
7. Wash the slide in running tap water (constant stream of water into the container) for 1 min.
8. Place the slide in 0.5% aqueous hematoxylin for 2 min.
9. Wash the slide in tap water for 1 min.
10. Place the slide in 2% phosphotungstic acid for 2 to 5 min.
11. Wash the slide in running tap water for 10 min.
12. Place the slide in 70% ethanol (plus a few drops of saturated aqueous lithium carbonate) for 3 min.
13. Place the slide in 95% ethanol for 5 min.*
14. Place the slide in two changes of 100% ethanol for 5 min each.*
15. Place the slide in two changes of xylene for 5 min each.*
16. Add Permount to the stained area of the slide, and cover it with a coverslip.
Note An alternative method to using mounting medium is as follows.
A. Remove the slide from the last container of xylene, place it on a paper towel (flat position), and allow it to air dry. Remember that some of the xylene substitutes may take a bit longer to dry.
B.
