a method is now available that stains both organisms, an important improvement since dual infections have been demonstrated in AIDS patients (44). This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination (30, 45, 46) (Fig. 3.30). Also, it appears that modified trichrome stains and staining with fluorochromes are equally useful in the diagnosis of microsporidiosis; however, a combination of the two methods may be more sensitive in cases where the number of spores is very small (41).
Figure 3.30 Cystoisospora (Isospora) belli oocyst and microsporidian spores in an acid-fast trichrome stain. Note the size differential. doi:10.1128/9781555819002.ch3.f30
The specimen can be fresh stool or stool that has been preserved in 5 or 10% formalin, SAF, or some of the newer single-vial-system fixatives. Actually, any specimen other than tissue thought to contain microsporidia could be stained by this method.
Trichrome Stain (Modified for Microsporidia) (44)
*(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, and adjust the pH to 2.5 with 2.0 N HCl. Properly prepared stain is dark purple. The staining solution should be protected from light.
3. Store in a glass or plastic bottle at room temperature. The shelf life is at least 24 months.
Carbol Fuchsin Solution
Phenol solution
Saturated alcoholic fuchsin solution
Add the mixture of phenol and water to 25.0 ml of the saturated alcoholic fuchsin solution.
Acid-Alcohol
Prepare by combining the two solutions.
Quality Control for the Acid-Fast Trichrome Staining Method
1. Unfortunately, the only way to perform acceptable QC procedures for this method is to use actual microsporidian 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 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 blue; however, some bacteria and debris 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 if it has received heavy use), 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 varies 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 the Acid-Fast Trichrome Staining Method
1. Using a 10-µl aliquot of concentrated (10 min at 500 × g), preserved liquid stool (5 or 10% formalin, SAF, one of the single-vial preservatives, zinc based or Universal Fixative), prepare the smear by spreading the material over an area 45 by 25 mm (47, 48).
2. Allow the smear to air dry.
3. Place the smear in absolute methanol for 5 or 10 min.
4. Allow the smear to air dry.
5. Place in carbol fuchsin solution for 10 min (no heat required).
6. Rinse briefly with tap water.
7. Decolorize with 0.5% acid-alcohol.
8. Briefly rinse with tap water.
9. Place in trichrome stain for 30 min at 37°C.
10. Rinse in acid-alcohol for no more than 10 s.
11. Dip the slide several times in 95% alcohol. Use this step as a rinse (no more than 10 s).
12. Place in 95% alcohol for 30 s.
13. Allow the slide to air dry.
14. Examine the smear under oil immersion (×1,000) and read at least 300 fields; the examination time will probably be at least 10 min per slide.
Results and Patient Reports from the Acid-Fast Trichrome Staining Method
The microsporidial spore wall should stain pink, with the interior of the spore being clear or perhaps showing a horizontal or diagonal stripe that represents the polar tube; a vacuole may also be visible in some spores. The Cryptosporidium oocysts stain bright pink or violet. The results of this staining procedure should be reported only if the positive control smears are acceptable. The production of immunoassay reagents should provide a more specific and sensitive approach to the identification of the microsporidia in fecal specimens.
1. Report the organism.
Examples: Microsporidial spores present. The following information can also be added to the report to assist the physician in treating and following the patient:
The organisms are probably Enterocytozoon bieneusi or Encephalitozoon intestinalis (if from fecal specimens or urine).
The organisms are probably Encephalitozoon intestinalis (identification to species highly likely) (generally the organism involved in disseminated cases from the gastrointestinal tract to kidneys; organisms are recovered in urine).
Procedure Notes for the Acid-Fast Trichrome Staining Method
1. It is mandatory that positive control smears be stained and examined each time patient specimens are stained and examined.
2. Because of the difficulty in achieving stain penetration through the spore wall, prepare thin smears and do not reduce the staining time in trichrome. Also, make sure the slides are not left too long in the decolorizing agent