Differentiation in an H&E stain is achieved by using which of the following?

• A. A weak acid
• B. A strong acid
• C. An oxidizing agent
• D. A buffering salt

Answer: (A)

Differentiation in an H&E stain is achieved by using a weak acid in alcohol. After hematoxylin has stained the tissue, this dilute acid solution removes only the loosely bound hematoxylin, leaving the nuclei deeply stained while reducing background. This creates crisp, contrasting nuclear detail. Using a strong acid would over-decolorize and blur or remove too much stain, an oxidizing agent would alter the dye chemistry rather than selectively differentiate, and a buffering salt would mainly stabilize pH without removing excess stain.