3.27). The 100× oil immersion objective gives better-quality images. Immersion oils used for light microscopy may be autofluorescent, and special low-fluorescence immersion oil should be used.
3. If the fluorescence is not clear or definitive, a suspicious slide can be restained with a modified acid-fast stain and reexamined using light microscopy and the 100× oil immersion objective.
4. If protected from sunlight, auramine O slides can be kept on the bench at room temperature for up to 2 to 3 weeks, with only minor loss of fluorescence (photo bleaching).
Modified Trichrome Stain for the Microsporidia (Weber—Green) (41)
The diagnosis of intestinal microsporidiosis (Enterocytozoon bieneusi, Encephalitozoon intestinalis) has depended on the use of invasive procedures and subsequent examination of biopsy specimens, often by electron microscopy. However, the need for a practical method for the routine clinical laboratory has stimulated some work in the development of additional methods. Slides prepared from fresh, formalin-fixed, or stools preserved in the Universal Fixative can be stained by a chromotrope-based technique and can be examined under light microscopy. This staining method is based on the fact that stain penetration of the microsporidial spore is very difficult; therefore, the dye content in the chromotrope 2R is greater than that routinely used to prepare Wheatley’s modification of Gomori’s trichrome method, and the staining time is much longer (90 min) (Fig. 3.28 and 3.29) (39, 41). At least several of these stains are available commercially from a number of suppliers.
Figure 3.28 (Left) Microsporidian spores in a nasopharyngeal specimen stained with a Ryan modified trichrome stain; (right) microsporidian spores in stool stained with a Weber modified trichrome stain. The spores range from about 1.5 to 2.0 µm in diameter. Note the horizontal lines, indicating the presence of a polar tubule. doi:10.1128/9781555819002.ch3.f28
Figure 3.29 (Left) Microsporidian spores in a urine sediment, stained with calcofluor white stain; (right) Encephalitozoon intestinalis spores stained with an organism-specific fluorescent antibody reagent. Note that some of the spores are intracellular. A urine specimen tends to be “cleaner” than stool; therefore the spores may be easier to see and identify in urine or any specimen that contains less artifact material than stool. doi:10.1128/9781555819002.ch3.f29
The specimen can be fresh stool or stool that has been preserved in 5 or 10% formalin, SAF, or the newer single-vial-system fixatives (Universal Fixative—no PVA). Actually, any specimen other than tissue thought to contain microsporidia could be stained by this method.
Trichrome Stain (Modified for Microsporidia) (Weber—Green) (41)
*(10 times the normal trichrome stain formula)
1. Prepare the stain by adding 3.0 ml of acetic acid to the dry ingredients. Allow the mixture to stand (ripen) for 30 min at room temperature.
2. Add 100 ml of distilled water. Properly prepared stain is dark purple.
3. Store in a glass or plastic bottle at room temperature. The shelf life is at least 24 months.
Acid-Alcohol
Prepare by combining the two solutions.
Quality Control for the Modified Trichrome Staining Method (Weber—Green)
1. Unfortunately, the only way to perform acceptable QC procedures for the modified trichrome staining method is to use actual microsporidial spores as the control organisms. Obtaining these positive controls may be somewhat difficult. It is particularly important to use the actual organisms, because the spores are difficult to stain and they are very small (1 to 2.5 µm).
2. A QC slide should be included with each run of stained slides, particularly if the staining setup is used infrequently.
3. All staining dishes should be covered to prevent evaporation of reagents (screw-cap Coplin jars or glass lids).
4. Depending on the volume of slides stained, staining solutions must be changed on an as-needed basis.
5. When the smear is thoroughly fixed and the stain is performed correctly, the spores are ovoid and refractile, with the spore wall being bright pinkish red. Occasionally, the polar tube can be seen either as a stripe or as a diagonal line across the spore. The majority of the bacteria and other debris tend to stain green. However, some bacteria and debris still stain red.
6. The specimen is also checked for adherence to the slide (macroscopically).
7. The microscope should be calibrated (within the last 12 months), 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). Although recalibration every 12 months may not be necessary, this will vary from laboratory to laboratory, depending on equipment care and use.
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 Modified Trichrome Staining Method (Weber—Green)
1. Using a 10-µl aliquot of unconcentrated (concentrated recommended), preserved liquid stool (5 or 10% formalin or SAF or one of the non-PVA single-vial preservatives), prepare the smear by spreading the material over an area 45 by 25 mm.
2. Allow the smear to air dry.
3. Place the smear in absolute methanol for 5 min.
4. Allow the smear to air dry.
5. Place in trichrome stain for 90 min.
6. Rinse in acid-alcohol for no more than 10 s.
7. Dip slides several times in 95% alcohol. Use this step as a rinse.
8. Place in 95% alcohol for 5 min.
9. Place in 100% alcohol for 10 min.
10. Place in xylene substitute for 10 min.
11. Mount with a coverslip (no. 1 thickness), using mounting medium.
12. Examine smears under oil immersion (×1,000), and read at least 300 fields; the examination time will probably be at least 10 min per slide.
