Gomori's One-StepTrichrome Stain
Gomori,Sheehan and Hrapchak in: Histotechnology
A Self-Instructional Text; ASCP Press. American Society of Clinical
Pathologists Chicago 1990
- To identify an increase in collagenous connective tissue fibers, or to differentiate between collagen and smooth muscle fibers
- 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
- Any well-fixed tissue may be used. Bouin's solution is used
as a mordant to intensify the color reactions.
- 56 to 58 °C oven
- Coplin jars
- Erlenmeyer flasks
- graduated cylinders
- 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.
- 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
- 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
- Deparaffinize sections and hydrate to distilled water.
- Rinse well in distilled water.
- Mordant sections in Bouin's solution for I hour at
- Remove slides from the oven, allow to cool, and wash
in running water until the yellow color disappears.
- Rinse in distilled water.
- Stain sections in Weigert's hematoxylin for 10 minutes.
- Wash in running water for 10 minutes.
- Stain sections for 15 to 20 minutes in Gomori's trichrome
- Differentiate for 2 minutes in 0.5% acetic acid.
- Dehydrate, clear, and mount with synthetic resin.
- Nuclei..... black
Cytoplasm, keratin, muscle fibers ..... red
Collagen and mucus ..... green or blue
- 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.