and allow it to ripen in a lighted room for at least 1 week at room temperature.
Solution 2
Ferrous ammonium sulfate
Ferric ammonium sulfate
Working Solution
Mix equal volumes of solutions 1 and 2. The working solution should be made fresh every week.
70% Ethanol plus Iodine
1. Prepare a stock solution by adding iodine crystals to 70% alcohol until a dark solution is obtained (1 to 2 g/100 ml).
2. To use, dilute the stock solution with 70% alcohol until a dark reddish brown strong-tea color is obtained. As long as the color is acceptable, new working solution does not have to be made. The replacement time will depend on the number of smears stained and the size of the container (1 week to several weeks).
Prepare by combining.
70% Isopropyl or Ethyl Alcohol
100% Ethyl Alcohol (Recommended)
or 95%/5% Commercial Absolute Alcohol (Second Choice)
This is ethyl alcohol that has been denatured with isopropanol and methanol, but is considered commercial absolute alcohol and does not require a license to purchase (unlike absolute ethanol that has not been denatured). However, this product does not dehydrate as well as actual absolute ethanol.
Xylene or Xylene Substitute
Quality Control for Iron Hematoxylin Stain
1. Stool samples used for quality control can be fixed stool specimens known to contain protozoa or preserved negative stools containing PVA to which buffy coat cells (PMNs or macrophages) have been added. A QC smear prepared from a positive sample or a 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.
3. If the xylene or xylene substitute becomes cloudy or there is an accumulation of water in the bottom of the staining dish, discard the old reagents, clean the dishes, dry them 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 should to be changed on an as-needed basis.
6. When the smear is thoroughly fixed and the stain is performed correctly, the cytoplasm of protozoan trophozoites will be blue-gray, sometimes with a tinge of black. Cysts tend to be slightly darker. Nuclei and inclusions (chromatoidal bars, RBCs, bacteria, and Charcot-Leyden crystals) are dark gray-blue, sometimes almost black. The background material usually stains pale gray or blue, providing some color intensity contrast with the protozoa. This contrast is less distinct than that obtained with the trichrome stain, which tends to stain everything with multiple colors (pink, red, purple, green, blue).
7. 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