rel="nofollow" href="#ulink_d643c204-6c42-5aaa-97bf-b2918aea6f4b">Modified Trichrome Stain for the Microsporidia (Ryan—Blue) (39)
A number of variations to the modified trichrome stain (Weber—green) have been tried in an attempt to improve the contrast between the color of the spores and the background staining. Optimal staining was achieved by modifying the composition of the trichrome solution. This stain is also available commercially from a number of suppliers.
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. Actually, any specimen other than tissue thought to contain microsporidia could be stained by this method.
Trichrome Stain (Modified for Microsporidia) (Ryan—Blue)
*(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 1.0 M 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.
Acid-Alcohol
Prepare by combining the two solutions.
Quality Control for the Modified Trichrome Staining Method (Ryan—Blue)
1. Unfortunately, the only way to perform acceptable QC procedures for this 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 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 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 the Modified Trichrome Staining Method (Ryan—Blue)
1. Using a 10-µl aliquot of concentrated (10 min at 500 × g), preserved liquid stool (5 or 10% formalin, SAF, one of the non-PVA single-vial preservatives, zinc based, or Universal Fixative), 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 or 10 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 the slides several times in 95% alcohol. Use this step as a rinse (no more than 10 s).
8. Place in 95% alcohol for 5 min.
9. Place in 95% alcohol for 5 min.
10. Place in 100% alcohol for 10 min.
11. Place in xylene substitute for 10 min.
12. Mount with a coverslip (no. 1 thickness), using mounting medium.
13. 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.
Results and Patient Reports from Modified Trichrome Staining Methods (Weber or Ryan)
The microsporidial spore wall should stain pinkish to red, with the interior of the spore being clear or perhaps showing a horizontal or diagonal stripe which represents the polar tube. The background appears green or blue, depending on the method. Bacteria, some yeast cells, and some debris stain pink to red; the shapes and sizes of the various components may be helpful in differentiating the spores from other structures. 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 be added to the report to assist the physician in treating and following the patient:
The organisms are most probably Enterocytozoon bieneusi or Encephalitozoon intestinalis (if from fecal specimen or urine).
The organisms are most probably Encephalitozoon intestinalis (identification to species highly likely) (generally the organism involved in disseminated cases from the gastrointestinal tract to other body sites).
Procedure Notes for Modified Trichrome Staining Methods (Weber or Ryan)
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 getting the stain to penetrate 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 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 (dye content among the highest [85%]). Fast green and aniline blue can be obtained from Allied Chemical and Dye, New York, NY. See also
