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Special stains - Orcein

  • Hepatitis B
  • Orcein staining Technique
  • Elastic Orcein

Orcein was originally a natural dye obtained from lichens, but is now synthetically produced. It can be used to stain for elastic fibres and Hepatitis B Surface antigens. Demonstration of Hepatitis B surface antigens acts by oxidation of sulphur containing proteins by Potassium permanganate, which form reactive sulphonate residues. These react with the Orcein. In this type of viral Hepatitis the affected Hepatocytes show a typical cytoplasmic ground glass appearance.

Below is how to perform the orcein technique
  1. Take sections to water
  2. Treat with acidified permanganate (9.5 mls 0.5% potassium permanganate + 0.5mls 0.3% sulphuric acid) for 5 minutes.
  3. Bleach in 5% oxalic acid
  4. Wash in water, then in 70% alcohol.
  5. Stain in orcein solution for 45 minutes in water bath at 37ºC.
  6. Differentiate in 1% acid / alcohol until background is pale brown.
  7. Wash in water
  8. Dehydrate, clear and mount

Results

Hepatitis 'B' Surface Antigen

Dark purple

Background

Pale purple


Photo - x10 Orcein Stain

The Elastic fibres method is modified from the Taenzer-Unna Orcein method and is particularly useful for fine, delicate fibres. Orcein appears to be remarkably selective for elastic tissue. The precise mechanism of orcein binding by elastin is not clear, although Van der Waals forces are considered to be involved. Elastic fibres are a dense structure therefore they attract more of the dye and are thus more deeply stained.

Method

1.      Take sections to 70% alcohol.

2.      Stain in orcein at 37°C for 1 hour.

3.      Rinse in 70% alcohol.

4.      Differentiate 1% acid / alcohol until background is clear.

5.      Wash well in distilled water.

6.      Counterstain if required with haematoxylin for 10-20 seconds.

7.      'Blue' in Lithium Carbonate.

8.      Dehydrate, clear and mount

Results

Elastic Fibres

Dark Brown

Other tissues

Brown

Photo - Elastic Orcein x10