Introduction

BioGnost’s Sperm-Diff kit enables rapid, simple, and high-quality staining of sperm cells based on the May Grünwald-Giemsa (Romanowsky) staining principle for optimal analysis of sperm morphology. Sperm staining is based on a modified Romanowsky principle of differential staining using an acidic (eosin) and a basic (thiazine) staining reagent following smear fixation. Sperm-Diff 1 reagent is a fixative that fixes the protein and membrane structures of sperm cells, making them suitable for binding eosin and thiazine dyes. In the next step, Sperm-Diff 2 reagent is applied, which contains an acidic dye, Eosin Y, that binds to basic cellular components (cytoplasmic proteins), causing the cytoplasm and certain sperm structures to take on an eosinophilic pink‑red color. Sperm-Diff 3 reagent contains basic dyes from the thiazine dye group (methylene blue, azures) that bind to acidic cellular regions (nucleic acids and other anionic structures), causing nuclei and some intracellular details to take on a basophilic blue or purple color. The interaction of eosin, thiazine dyes, and DNA produces characteristic Romanowsky shades of purple that allow clear differentiation of the sperm head, midpiece, and tail, as well as other cells present in the ejaculate (e.g., leukocytes, immature germ cells).