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).
*Slides may be held for up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.
Note An alternative method to using mounting medium is as follows (2).
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 than xylene itself does.
B. Approximately 5 to 10 min before you want to examine the slide, place a drop of immersion oil on the dry fecal film. Allow the oil to sink into the film for a minimum of 10 to 15 min. If the smear appears to be very refractile on examination, you have not waited long enough for the oil to sink into the film or you need to add a bit more oil to the film.
C. Once you are ready to examine the slide, place a no. 1 (22- by 22-mm) coverslip onto the oiled smear, add another drop of immersion oil onto the top of the coverslip (as you would normally do for any slide with a coverslip), and examine with the oil immersion lens (100× objective).
D. Do not eliminate adding the coverslip; the dry fecal material on the slide often becomes very brittle after dehydration. Without the addition of the protective coverslip, you might scratch the surface of the oil immersion lens. Coverslips are much cheaper than oil immersion objectives!
Iron Hematoxylin Stain
The iron hematoxylin stain is one of a number of stains that allow one to make a permanent stained slide for detecting and quantitating parasitic organisms. Iron hematoxylin was the stain used for most of the original morphologic descriptions of intestinal protozoa found in humans (3) (Fig. 3.20). On oil immersion power (×1,000), one can examine the diagnostic features used to identify the protozoan parasites. Although there are many modifications of iron hematoxylin techniques, only two methods are outlined below: the Spencer-Monroe (19) and Tompkins-Miller (20) procedures. Both methods can be used with either fresh specimens or those preserved with SAF, preservatives containing PVA, single-vial systems, or the Universal Fixatives.
Figure 3.20 Intestinal protozoa stained with iron hematoxylin stain. (Top row) Dientamoeba fragilis trophozoite (left), Giardia lamblia (G. duodenalis, G. intestinalis) cysts (right); (middle row) Iodamoeba bütschlii cyst (note the large glycogen vacuole) (left), Entamoeba histolytica trophozoite (note ingested RBCs) (right); (bottom row) Entamoeba coli cyst (note more than five nuclei) (left), Entamoeba histolytica/E. dispar cyst (right). doi:10.1128/9781555819002.ch3.f20
The specimen usually consists of fresh stool smeared on a microscope slide, which is immediately fixed in Schaudinn’s fixative; stool in fixative containing PVA smeared on a slide and allowed to air dry; SAF-preserved stool smeared on an albumin-coated slide and allowed to air dry; single-vial-preserved stool smeared on an albumin-coated slide and allowed to air dry; or stool smeared on a slide and allowed to air dry (Universal Fixative, no adhesive is required).
Iron Hematoxylin Stain (Spencer-Monroe Method) (19)
Solution 1
Place solution in a stoppered clear flask or bottle, 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.
