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 (acid-alcohol). If the control organisms are too light, leave them in the trichrome longer and shorten the time to two dips in the acid-alcohol solution. Also, remember that the 95% alcohol rinse after the acid-alcohol step should be performed quickly to prevent additional destaining from the acid-alcohol reagent.
3. When you purchase the chromotrope 2R, obtain the highest dye content available. Two sources are Harleco, Gibbstown, NJ, and Sigma Chemical Co., St. Louis, MO. (the dye content is among the highest [85%]). Fast green and aniline blue can be obtained from Allied Chemical and Dye, New York, NY. See also appendix 5.
4. In the final stages of dehydration, the 95% ethanol should be kept as free from water as possible. Coplin jars must have tight-fitting caps to prevent both evaporation of reagents and absorption of moisture.
Procedure Limitations for the Acid-Fast Trichrome Staining Method
1. Although this staining method stains the microsporidia, the range of stain intensity and the small size of the spores may still cause some difficulty in identifying these organisms. Since this procedure results in many other organisms or objects staining in stool specimens, differentiation of the microsporidia from surrounding material is still very difficult. There also tends to be some slight size variation among the spores.
2. If the patient has severe watery diarrhea, there is less artifact material in the stool to confuse with the microsporidial spores; however, if the stool is semiformed or formed, the amount of artifact material is much greater and so the spores are much harder to detect and identify. Also, the number of spores may vary according to the stool consistency (generally, the more liquid the stool, the more spores are present).
3. The workers who developed some of these procedures think that concentration procedures result in an actual loss of microsporidial spores; therefore there is a recommendation to use unconcentrated, formalinized stool. However, there are no data indicating which centrifugation speeds were used in the study.
4. In the UCLA Clinical Microbiology Laboratory, we have generated data (unpublished) to indicate that centrifugation at 500 × g for 10 min dramatically increases the number of microsporidial spores available for staining (from the concentrate sediment). We
