Trichrome Blue Staining

Introduction

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.

Reagents

PRODUCT #
DESCRIPTION
601A Ryan modification of Trichrome-Blue
3719A 95% Reagent Alcohol
374B Reagent Alcohol
3720A Trichrome Decolorizer
134B Xylene 
930E Xylene Substitute (d-limonene)

Specimen Collection

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.

Procedure

  1. Place slide in Trichrome-Blue stain for 90 minutes at room temperature, or 30 minutes at 37C.
  2. Place slide in Trichrome Decolorizer for 1 to 3 seconds.
  3. Briefly rinse in 95% alcohol.
  4. Place in two changes of 100% alcohol for 3 minutes each.
  5. Place in two changes of xylene* for 10 minutes each.
  6. Mount with a coverslip using Permount.

*an acceptable xylene-substitute can be used

Note: Staining times may vary to suit the individual.

Notes

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

  1. Do not stain smears which have only been air dried. Smears must also be "fixed".
  2. Smears should not be too thick.
  3. After staining it is essential that the back surface of the slide is wiped clean.