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 In the UCLA Clinical Microbiology Laboratory, we have generated data (unpublished) to indicate that centrifugation at 500 × g for 10 min dramatically increases the number of microsporidial spores available for staining (from the concentrate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 use the same protocol for centrifugation of all stool specimens, regardless of the suspected organism. The acid-fast trichrome procedure presented here also recommended the use of centrifuged fecal specimens.
5. Avoid the use of wet-gauze filtration (an old, standardized method of filtering stool prior to centrifugation) with too many layers of gauze that may trap organisms and not allow them to flow into the fluid to be concentrated. It is recommended that no more than two layers of gauze be used. Another option is to use the commercially available concentration systems that use metal or plastic screens for filtration.
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