Wheatley's Gomori Trichrome

Introduction

Gomori's trichrome is used to produce permanently stained slides from PVA fixed stool samples. SAF and MIF preserved specimens may also be used but there are alternate staining procedures that give better results with these fixatives.

Reagents

PRODUCT # DESCRIPTION
374B Reagent alcohol
628A  D'Antoni's iodine
3716A 70% reagent alcohol
602A Gomori's trichrome
3105A 0.45% acetic acid in 90% reagent alcohol
134B Xylene
930E D-Limonene (Xylene substitute)

Specimen Collection

Prepare slides in the usual manner, i.e. Mix the PVA/stool sample and place some of the mixture on a paper towel to remove some of the PVA. Apply some of the fixed material from the paper towel and spread edge to edge on a slide. Thoroughly dry the slide overnight at room temperature or several hours at 37çC. The slide must be thoroughly dried to prevent the film from coming off during staining.

Procedure

  1. Remove the mercury from mercury-fixed samples by immersing the slides in 70% reagent alcohol/iodine solution for five to ten minutes. Specimens fixed with non-mercury fixatives (e.g. Zinc-based fixatives) may omit this step.
  2. Immerse in two changes of 70% reagent alcohol for 5 minutes and 2 to 5 minutes respectively.
  3. Stain in trichrome stain for ten minutes.
  4. Dip twice in 90% reagent alcohol with 0.5% acetic acid. This is a decolorizing step and should be omitted if the slide is already under-stained.
  5. Pass through two changes of reagent alcohol for 2 to 5 minutes.
  6. Place in two changes of xylene* for 5 to 10 minutes each.
  7. Mount the coverslip and examine under oil immersion.

*an acceptable xylene-substitute (e.g. d-limonene) can be used

Sources of Error

  1. Inadequately fixed samples will not stain correctly and will have distorted morphology.
  2. Incomplete removal of the mercury will leave artifacts on the slide and make it difficult to read.
  3. Incomplete removal of iodine after soaking in 70% reagent alcohol/iodine will give the slide a greenish cast and make it difficult to read.
  4. Avoid over-decolorizing in Step #4.
  5. Permanently stained smears are not recommended for staining helminth eggs or larvae. Wet smears after concentration are preferred.
  6. Quality control slides should be included with each run.

Bibliography

  1. Garcia, L.S., and D.A. Bruckner, 1993. Diagnostic Medical Parasitology, 2nd ed. American Society for Microbiology, Washington D.C.