Species of microsporidia have been associated with severe infections in AIDS patients. Encephalocytozoon hellum (keratoconjunctivitas), E. cuniculi (hepatitis 7 peritonitis), and Enterocytozoon bieneusi (diarrhea) are among the most commonly reported. In 1992, Weber et al. published a modified trichrome staining procedure for microsporidial spores in duodenal aspirates. A slightly modified procedure was recommended by the CDC. In 1993, Ryan et al. described a trichrome-blue modification of the Weber et al. procedure. The trichrome-blue staining procedure can be used to detect microsporidial species in urine, stool, and nasopharyngeal specimens. Spores stain pink-red with a polar or central clear zone, whereas yeast cells and pseudohyphae stain a grayish blue color.
|601A||Ryan modification of Trichrome-Blue|
|3719A||95% Reagent Alcohol|
|930E||Xylene Substitute (d-limonene)|
Thin smears should be made from formalin preserved samples, collected in the usual manner. Smears should be air dried and fixed in absolute alcohol for 5 minutes prior to staining.
- Place slide in Trichrome-Blue stain for 90 minutes at room temperature, or 30 minutes at 37C.
- Place slide in Trichrome Decolorizer for 1 to 3 seconds.
- Briefly rinse in 95% alcohol.
- Place in two changes of 100% alcohol for 3 minutes each.
- Place in two changes of xylene* for 10 minutes each.
- Mount with a coverslip using Permount.
*an acceptable xylene-substitute can be used
Note: Staining times may vary to suit the individual.
Preparations should be read at 1000x magnification. At least 100 fields should be read before reporting a negative result. Microsporidia are oval, and measure 0.7-1.1 x 1.1-1.7 µm. The spore wall stains a bright pinkish-red. The spore is transparent or will exhibit a pinkish belt-like stripe across the center.
Sources of Error
- Do not stain smears which have only been air dried. Smears must also be “fixed”.
- Smears should not be too thick.
- After staining it is essential that the back surface of the slide is wiped clean.