×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 PVA-preserved, material).
6. Cryptosporidium and Cyclospora oocysts are generally not recognized on a trichrome-stained smear (modified acid-fast stains or the immunoassay reagent kits are recommended). Microsporidial spores do not stain sufficiently for recognition by the regular trichrome method; modified trichrome stains are required.
Trichrome Stain (Modified for Use with SAF-Preserved Fecal Specimens) (Dr. Norbert Ryan, VIDRL Modification)
It is generally recognized that stained fecal films are the single most productive means of stool examination for intestinal protozoa. The permanent stained smear facilitates detection and identification of cysts and trophozoites and affords a permanent record of the protozoa encountered. Small protozoa missed by direct smear and concentration techniques are often seen on the stained smear. Many reference texts mention that the combination of trichrome and SAF is unsuitable, because the green background stain of trichrome dominates, perhaps interacting with the albumin used to bind material to the slide. In this method the trichrome stain has been modified to reduce background staining. The modification is based on the theory that phosphotungstic acid functions both as a mordant and as a “colorless” stain of medium size. By increasing the content of this agent the intensity of background staining is reduced, without compromising the staining of structures with small pore size such as protozoa and tissue cells.
Note All other sections related to the trichrome stain presented above are relevant for the VIDRL modification of the trichrome stain for SAF-preserved fecal specimens presented here.
Trichrome Stain (VIDRL Modification for SAF-fixed smears)
1. Prepare the stain by adding 1.0 ml of acetic acid to the dry components. Allow the mixture to stand (ripen) for 15 to 30 min at room temperature.
2. Add 100 ml of distilled water. Properly prepared stain is purple.
3. Store in a glass or plastic bottle at room temperature. The shelf life is 24 months.
Procedure for Trichrome Stain (VIDRL Modification)
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. Place the slide in 70% ethanol for 5 min.*
2. Place it in a second container of 70% ethanol for 3 min.*
3. Place it in trichrome stain (VIDRL modification) for 10 min. The fecal smear no longer contains either mercuric chloride or iodine and is now ready for staining.
4. Place it in 90% ethanol plus 0.5% acetic acid for 1 to 3 s. Immediately drain the slide (see Procedure Notes), and proceed to the next step. Do not allow slides to remain in this solution. This is the destaining step.
5. Place the slide in 95% ethanol for 1 to 3 s. Use this step as a rinse.
6. Place it in two changes of 100% ethanol for 3 min each.* This is a dehydration step.
7. Place it in xylene or xylene substitute for 10 min.* This is a dehydration step.
8. Mount the slide with a coverslip (no. 1 thickness), using mounting medium (e.g., Permount).
9. Allow the smear to dry overnight or after 1 h at 37°C.
10. 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