href="#u95f7992a-c24b-5bad-aae4-1b9fc08921bc">appendix 5.
4. In the final stages of dehydration, the 100% ethanol and the xylenes (or xylene substitutes) 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. If the xylene or xylene substitute becomes cloudy after addition of slides from 100% alcohol, return the slides to fresh 100% alcohol and also replace the xylene or xylene substitute with fresh stock.
Procedure Limitations for Modified Trichrome Staining Methods (Weber or Ryan)
1. Although this staining method stains the microsporidia, the range of stain intensity and the small size of the spores 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 the spores are much harder to detect and identify. Also, the number of spores varies according to the stool consistency (generally, the more liquid the stool, the more spores will be present). However, remember that there is also a dilution factor if the stool is very liquid.
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, etc., 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 In the UCLA Clinical Microbiology Laboratory, we have generated data (unpublished) to indicate that centrifugation at 500 × g for 10 min dramatically increases the number of microsporidial spores available for staining (from the concentrate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). This is the same method we use for centrifugation of all stool specimens, regardless of the suspected organism.
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 in which metal or plastic screens are used for filtration.
Modified Trichrome Stain for the Microsporidia (Kokoskin—Hot Method) (38)
Changes in temperature from room temperature to 50°C and in the staining time from 90 to 10 min have been recommended as improvements for the modified trichrome staining methods. The procedure is as follows.
1. Using a 10-µl aliquot of concentrated, preserved stool (5 or 10% formalin, SAF, 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 min.
4. Allow the smear to air dry.
5. Place in trichrome stain for 10 min at 50°C.
6. Rinse in acid-alcohol for no more than 10 s.
7. Dip the slide 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 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 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.
Acid-Fast Trichrome Stain for Cryptosporidium and the Microsporidia
The detection of Cryptosporidium spp. and the microsporidia from stool specimens has depended on two separate stains. However, 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
