Gomori's One-StepTrichrome Stain

Gomori,Sheehan and Hrapchak in: Histotechnology A Self-Instructional Text; ASCP Press. American Society of Clinical Pathologists Chicago 1990
Purpose
To identify an increase in collagenous connective tissue fibers, or to differentiate between collagen and smooth muscle fibers
Principle
In the one-step trichrome procedure, a plasma stain (chromotrope 2R) and a connective tissue fiber stain (fast green FCF, light green, or aniline blue) are combined in a solution of phosphotungstic acid to which glacial acetic acid has been added. Phosphotungstic acid favors the red staining of muscle and cytoplasm. The tungstate ion is specifically taken up by collagen, and the connective tissue fiber stain is subsequently bound to this complex, coloring the collagen green or blue, depending on the counterstain used.
Fixative
Any well-fixed tissue may be used. Bouin's solution is used as a mordant to intensify the color reactions.
Equipment
Technique
Cut paraffin sections at 4 to 5 µm.
Quality Control
Practically every tissue has an internal control, so no other control sections are needed; however, if a control is desired, uterus, small intestine, appendix, or fallopian tube will provide good material.
Reagents
  • Bouin's Solution
  • Picric acid, saturated aqueous solution .... 75.0 mL
  • Formaldehyde, 37% to 40% ….. 25.0 mL
  • Glacial acetic acid ….. 5.0 mL
  • Weigert's Iron Hematoxylin
  • Solution A
  • Hematoxylin..... 10.0 g
  • Alcohol, 95% ..... 1,000.0 mL
  • Solution B
  • Distilled water ..... 475.0 mL
  • Hydrochloric acid, concentrated ..... 5.0 mL
  • Ferric chloride, 29% solution ..... 20.0 mL
  • Working Solution
  • Mix equal parts of solutions A and B.
  • Gomori's Trichrome Stain
  • Chromotrope 2R (Baker No. 5-F703) . . . . . 0.6 g
  • Fast green FCF, light green or aniline blue ..... 0.3 g
  • Phosphotungstic acid ..... 0.8 g
  • Glacial acetic acid . . . . . 1.0 mL
  • Distilled water ..... 100.0 mL
  • Store this solution in the refrigerator.
  • 0.5% Acetic Acid Solution
  • Glacial acetic acid ..... 0.5 mL
  • Distilled water ..... 99.5 mL
Procedure
  2. Deparaffinize sections and hydrate to distilled water.
  3. Rinse well in distilled water.
  4. Mordant sections in Bouin's solution for I hour at 56 °C.
  5. Remove slides from the oven, allow to cool, and wash in running water until the yellow color disappears.
  6. Rinse in distilled water.
  7. Stain sections in Weigert's hematoxylin for 10 minutes.
  8. Wash in running water for 10 minutes.
  9. Stain sections for 15 to 20 minutes in Gomori's trichrome stain.
  10. Differentiate for 2 minutes in 0.5% acetic acid.
  11. Dehydrate, clear, and mount with synthetic resin.
Results
Nuclei..... black
Cytoplasm, keratin, muscle fibers ..... red
Collagen and mucus ..... green or blue
Remarks
Sweat et al state that coloration of fine connective tissue fibers is affected by the dye solution pH, with maximum binding occurring around pH 1.3. The pH of Gomori's trichrome is about 2.5, which decreases affinity for anions by approximately 50%, so these investigators suggest that by replacing the acetic acid with hydrochloric acid, a pH of approximately 1.3 can be obtained. The intensity of coloration of the fine connective tissue fibers can be varied by altering the pH.