Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

The "one step trichrome stains", of which there are many variants, all rely on molecular size competition for tissue reactive sites in conditions of controlled pH.  For the correct colour balance to be achieved, it may be necessary to slightly alter the concentration of the two dyes, this is preferred to attempting to differentiate the end result.

Technical Points

Step 6 - The acidity of this stain is sufficient to differentiate the nuclear stain.  Staining is progressive should be checked after 15 mins and controlled microscopically.

Method

1.      Bring sections to distilled water

2.      Stain nuclei with Celestin Blue 5 mins

3.      Rinse in distilled water

4.      Stain in haematoxylin 5 mins

5.      Wash well in running tap water 5 mins

6.      Stain with Gomori's stain 15 mins

7.      Rinse with distilled water

8.      Dehydrate, clear and mount.

Results

                        muscle fibres......................red

                        collagen............................green

                        nuclei...............................blue black

Reagent Formulae

1. Celestin Blue

5% ammonium ferric sulphate (iron alum)……100 ml

Celestin Blue (CI 51050)………………………………0.5 g

Add the celestin blue to the ammonium ferric sulphate and boil for 3 minutes.

Filter when cool.

Store refrigerated. 

2. Gomori's trichrome stain:
     distilled water --------------------- 200.0 ml
     chromotrope 2R (CI 16570) ------ 1.2 g
     light green SF (CI 42095) --------- 0.6 g
     dodecatungstophosphoric acid --- 1.6 g
     glacial acetic acid    2.0 mls
Dissolve each reagent separately in 50 mls aliquots of the distilled water, then mix all four solutions together.  Allow to stand overnight, filter into a reagent bottle.  The stain keeps well.

ReferencesGomori G, (1950), Am. J. Clin. Path., 20, 665