Giemsa (azure B) stain in a Coplin jar. Remove stain from stock stain in bottle with a clean, dry pipette.
2. Add 40 ml of phosphate buffer (pH 7.0 to 7.2) containing 0.01% Triton X-100.
3. Place fixed specimen smears and control smears in Giemsa stain for 30 min.
4. Remove slides, dip in phosphate buffer, stand slides on end, and allow to drain and air dry.
5. Examine with oil or mount slides.
Giemsa Staining for Microsporidia
1. Internal morphology may be difficult to interpret (spores with polar tubules).
2. Giemsa stain may be more relevant for specimens other than stool (urine, sputum, eye).
3. Perform modified trichrome (Ryan blue or Weber green) stains on stool.
Potential Problems with Stain Interpretation
In the procedures in which the spores are stained, spores and fungi may appear very much alike. If rare organisms are seen, it may be almost impossible to differentiate microsporidia from various fungi, particularly if no budding organisms are seen. Although the microsporidia are now classified with the fungi, they will continue to be covered in both sections of this book.
Although a second type of stain can be used (Giemsa, which stains the spores), the organisms may be very difficult to differentiate from cellular debris. Consequently, few laboratories rely on the Giemsa stain alone, especially when staining specimens for Pneumocystis or the microsporidia (Fig. 6.5).
Figure 6.5 Pneumocystis jirovecii trophozoites within the cyst wall (stained with Giemsa stain). Note that the cyst wall is not visible when Giemsa stain is used. Other trophozoites can also be seen in the background. doi:10.1128/9781555819002.ch6.f5
Other methods such as fluorescent-antibody and immunoperoxidase techniques have been used for P. jirovecii (13–17). The development of tissue culture procedures for P. jirovecii may lead to additional diagnostic procedures for this infection. Immunoassay reagents are also available (14–18) and are being widely used in many diagnostic laboratories.
When noninduced sputum and the direct fluorescent-antibody technique were used, the sensitivity was 55%; this is within the range reported in the literature for the diagnosis of Pneumocystis pneumonia from induced sputum (19). Although detection of P. jirovecii has increased with the use of PCR, particularly in sputum specimens, some workers recommend that sputum samples of HIV-infected patients be tested by both PCR and immunofluorescence. The results of PCR in this patient group could be misleading without careful clinical evaluation (20). However, the level of sensitivity seen using PCR and other molecular tests indicates that these procedures should be considered for patients in whom Pneumocystis pneumonia is suggestive clinically and the specimen is negative by the immunofluorescence test (21, 22).
Quality Control Measures
Quality control measures for all stains should include the following, and control slides must be incorporated into all stain procedures.
1. Examine a control slide for each stain prior to examination of specimen stains. The stain intensity of controls will be a guide to the stain appearance of organisms in specimens.
2. Examine stained specimen smears by systematically moving from field to field until the majority of the smear has been covered (total area for silver stain).
3. In Giemsa stains, Pneumocystis trophozoite clumps of various sizes may be detected. In large clumps, it may be difficult to differentiate individual organisms. Look at organisms at the edges of clumps, and look for small, more dispersed clumps.
4. In silver- or other cyst wall-stained smears, look for the various cyst forms, including those that show dark centers, cup-shaped cysts, and cysts with foldlike lines (they look like “punched-in” ping-pong balls). If dark-staining organisms appear more oval, look carefully for budding forms, which indicate that the organisms are yeasts. It is important to review thinner areas of the smear for microsporidial spores.
A. P. jirovecii cysts: 70% should have delicately stained walls, usually brown or gray. They appear somewhat transparent, with structures described as “parentheses” staining black; these curved structures are usually thick (much thicker than the cyst wall) rather than thin like a line drawing.
B. Other fungi and actinomycetes: gray to black. Microsporidial spores stain dark gray to black; some of the spores may contain the polar tubule (horizontal or diagonal stripe), but they are more difficult to see in the silver stain than in the modified trichrome stains.
C. Glycogen, mucin, and red blood cells: rose taupe to gray.
D. Background: pale green (from the light green counterstain).
5. Detection of P. jirovecii in specimen smears from one type of stain always suggests a careful examination of the other stain, hopefully to confirm the identification.
6. Retain all positive stained specimen slides and control slides for reference.
Reporting Smear Results
1. Report P. jirovecii as follows (high dry power; total magnification, ×400).
A. “No Pneumocystis jirovecii seen.”
B. “Pneumocystis jirovecii seen” (no quantitation should be included).
2. Report other fungi that may be present as follows (low power; total magnification, ×100).
A. “No mycotic elements seen.”
B. “Budding yeasts” or “Budding yeasts and pseudohyphae resembling Candida species” are quantitated as few, moderate, or many.
C. Hyphae are reported with no quantitation:
a. “Septate hyphae seen.”
b. “Nonseptate hyphae seen.”
3. Report actinomycetes (oil immersion; total magnification, ×1,000) as follows.
A. “No filamentous branching bacteria seen.”
B. “Filamentous branching bacteria seen” (no quantitation).
4. Report microsporidia (oil immersion; total magnification, ×1,000) as follows.
A. “No microsporidial spores seen.”
B. “Microsporidial spores seen; identification to genus not possible.” However, if the specimen is stool or other gastrointestinal tract specimens, the organisms
