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Pouchet’s Staining Protocol for Bile Pigments
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Pouchet’s Staining Protocol for Bile Pigments

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


In the presence of trichloracetic acid, bile pigments are oxidised by ferric chloride to form a greenish mixture of biliverdin and cholecyanin.

The cotton dye Sirius red F3B, although similar to Congo red (Puchtler et al), has a high affinity for collagen, and may be substituted for acid fuchsin in Van Gieson variants.

The picrate ions of the counterstain tend to preserve the green oxidation state of the bile pigments, and thus act as an excellent complementary reagent in this method.

This technique will demonstrate both coarse and fine deposits. Bile is most commonly seen in liver parenchyma cells as small, yellow-brown globules in a haematoxylin and eosin stained section.

Technical Points

1.  A known positive control section must be used to ensure correct demonstration has been achieved.

2.  (step 5) - The delicate red and yellow counterstaining is easily lost in watery and alcoholic solutions.


1.      Bring sections to distilled water

2.      Oxidise in Fouchet's reagent 5 mins

3.      Rinse well in distilled water

4.      Stain with Sirius red stain 30 mins

5.      Blot dry

6.      Dehydrate rapidly, clear and mount


  • bile
  • background....................yellow

Reagent Formulae

1.  Fouchet's reagent
            25% aq trichloracetic acid     100.0 ml
            60% aq ferric chloride           20.0 ml
            distilled water                      80.0 ml
            Mix the solutions.
CARE - corrosive hazard.

Trichloracetic acid is highly corrosive.

2.  Sirius red stain
            1% aq Sirius red F3B (CI 35780)     10.0 ml
            sat aq picric acid (CI 10305)         90.0 ml
            Mix the solutions.  The stain keeps well. 

CARE - explosive.
Picric acid is highly toxic, with a very bitter taste.  Picric acid is also a percussion explosive when dry.  Reagent bottles containing solutions of picric acid must be maintained scrupulously clean, taking especial care of any residue around the neck and stopper of the container bottle.


Fouchet A,(1917),C.r.Séanc.Soc.Biol.,80,826

Puchtler H, Sweat F, Levine M,(1962),J.Histochem.Cytochem.,10,355

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