What buffer is used with the Methyl Green-Pyronin Y reagents?

• A. Acetate Buffer solution (acetic acid, sodium acetate)
• B. Phosphate buffer
• C. Tris buffer
• D. Citrate buffer

Answer: (A)

The staining principle relies on keeping the tissue at a strongly acidic pH so the two dyes differentiate DNA from RNA: methyl green binds DNA to appear green, while pyronin Y binds RNA to appear red. Acetate buffer, made from acetic acid and sodium acetate, provides a stable acidic environment in the pH range of about 4.0–4.5 that best supports this selective binding and preserves color contrast during the stain. Other buffers tend to hold pH closer to neutral or basic ranges or can interact with the dyes in ways that blur the color distinction, leading to poorer differentiation between DNA and RNA.