In the Bakers acid hematein method, differentiation is accomplished by which reagent?

• A. Borax ferricyanide
• B. Sodium sulfide
• C. Ammonium chloride
• D. Acetic acid

Answer: (A)

Differentiation in Bakers acid hematein is done by a reagent that selectively removes the excess hematoxylin, leaving nuclei dark while cytoplasmic and non-nuclear areas are lighter. Borax ferricyanide provides this controlled differentiation: the ferricyanide acts as an oxidizing/bleaching agent in the differentiating bath, so loosely bound or non-nuclear hematoxylin is removed or lightened, while the hematoxylin tightly bound in nuclei remains intensely stained. This yields crisp nuclear detail and good contrast.

Other reagents wouldn’t give that same selective effect: sodium sulfide would reduce or disrupt the stain, ammonium chloride mainly affects ionic conditions without differentiating nuclear from non-nuclear stain, and acetic acid is used in some protocols for acid differentiation but does not accomplish the same controlled differentiation seen with borax ferricyanide in this method.