“Harris Hematoxylin,” What Harris Really Wrote and the Mechanism of Hemalum Stains

Abstract In current textbooks, Harris “hematoxylin” stain includes differentiation in acid alcohol, followed by bluing of sections. However, Harris (1900) did not mention differentiation, but strongly recommended progressive staining with an acidified or diluted solution of his formula or with Mayer's acid hemalum. A review of the chemistry of hematoxylin and hemalum stains showed binding of unoxidized hematoxylin by tissues. Oxidation of hematoxylin yields anionic hematein, which does not contain a quinonoid ring. Two hematein ions form a chelate with an Al+++ cation. Hemalum is bound to tissues by coordinate and hydrogen bonds. Dye binding by nuclei occurs mainly via non-ionic bonding. Protons of acids added to the hemalum solution or used in differentiation compete with the metal for binding sites. When appropriate amounts of acid are added to the hemalum solution, nuclei are stained selectively. Acidified hemalum solutions require only two steps, staining and washing, and yield perfectly reproducible ...