In the Masson Trichrome, Biebrich scarlet is removed from collagen by which reagent?

• A. Acetic acid
• B. Phosphotungstic acid
• C. Hydrochloric acid
• D. Sodium thiosulfate

Answer: (B)

In Masson Trichrome, the differentiating step uses a reagent that selectively removes the red dye (Biebrich scarlet) from collagen while leaving other tissues stained red for the final color contrast. Phosphotungstic acid serves this purpose. It binds and differentiates such that Biebrich scarlet is stripped from collagen, allowing the collagen to take up the final blue stain (often aniline blue) in the last step. This creates the characteristic color separation: collagen appears blue, while muscle and cytoplasm remain red, and nuclei stain dark. Other reagents like acetic acid, hydrochloric acid, or sodium thiosulfate aren’t the differentiating agent that removes Biebrich scarlet from collagen in this protocol.