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 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.