Virus Stain (Orcein Method)

Virus Stain (Orcein Method)

Virus Stain (Orcein Method)


Virus Stain (Orcein Method) is a weak acid that can be dissolved in alcohol. When combined with an acidic solution, it transforms into a dark orange-red color. In the presence of HBsAg (Hepatitis B surface antigen), the surface antigen of the hepatitis B virus, orcein in an acidulated alcoholic solution can effectively stain the antigen. This unique staining procedure involves the oxidation of disulfide bonds in the cystine of HBsAg, resulting in the display of distinct colors. The following is a step-by-step guide to the orcein staining procedure, highlighting the main components and precautions for accurate results.

Main Components of Virus Stain (Orcein Method):

  • Potassium Permanganate Solution: Contains potassium permanganate as the primary ingredient.
  • Sulfuric Acid Solution: Comprises sulfuric acid as the main ingredient.
  • Oxalic Acid Solution: Contains oxalic acid as the primary ingredient.
  • Acidified Orcein Ethanol Solution: Comprising orcein as the main ingredient.

Sample Requirement

Before performing the staining procedure, ensure that the tissue section is fully fixed.

Test Procedure

Dewaxing and Washing:

  • Start by dewaxing the tissue slices and washing them with distilled water.
  • Ensure the pieces are clean and free from any contaminants.

Staining with Potassium Permanganate Solution and Sulfuric Acid Solution:

  • Mix the potassium permanganate solution and sulfuric acid solution in a 1:1 ratio.
  • Stain the tissue slices with the mixture for approximately 5 minutes.
  • Rinse the tissue section slightly with distilled water.

Bleaching with Oxalic Acid Solution:

  • Bleach the stained tissue slices in the oxalic acid solution for 2 to 3 minutes, until the color disappears.

Rinse and Ethanol Wash:

  • Rinse the tissue slices with running water for 2 to 3 minutes.
  • Submerge the pieces into 75% ethanol and wash them gently.

Acidified Orcein Ethanol Staining:

  • Immerse the tissue slices in the acidified orcein ethanol solution for 3 to 5 hours.
  • Allow the staining process to complete.

Ethanol Wash:

  • Dip the tissue slices in 75% alcohol solution twice, each time for 10 to 30 seconds, until there is no dye precipitate left.

Dehydration, Transparentization, and Sealing:

  • Perform conventional dehydration and transparentization.
  • Seal the tissue slices with neutral resin.

Result Interpretation

Positive HBsAg staining appears a reddish-brown to dark-brown color, displaying a fine granular or homogeneous form. The positive material may form a circular or oval lump or fill the entire cytoplasm. In some instances, it may appear as a ring-shaped or crescent structure around the nucleus. Occasionally, the staining may be visible at the edge of the liver cell cytoplasm, exhibiting a lacey pattern. The elastic fibers also stain dark brown, whereas the rest of the background appears relatively pale gray-brown. Liver nuclei, however, remain uncolored.


  • Allow Reagents to Reach Room Temperature: Before using the reagents, ensure they are fully restored to room temperature after removal from the refrigerator.
  • Reuse of Acidified Orcein Ethanol Solution: The acidified orcein ethanol solution can be reused several times until the background of the slices becomes too deep to use. Close the lid securely when staining.
  • Professional Use and Interpretation: This staining procedure should be carried out by professionals who can accurately interpret the results. Read the instruction manual carefully before use and ensure personal hygiene protection.
  • Proper Disposal: Dispose of waste according to the hospital or environmental protection department’s guidelines after using the reagents.
  • Check Outer Packaging for Information: The production date, production batch number, and expiration date are provided on the outer packaging.


  • Chinese Medical Association. Specifications for Clinical Technical Operations – Pathology Volumes. People’s Military Medical Publishing House.
  • Ling Qibo. Histochemistry and Special Staining in Practical Pathology [M]. Guangdong Higher Education Press.


The orcein staining procedure is an effective method for detecting HBsAg in tissue samples. By following the specified steps and precautions, accurate and reliable results can be achieved, aiding in the diagnosis, and understanding of hepatitis B virus infections.

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