Why Do You Decolorize Crystal Violet?

A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.

What is the purpose of the Decolorizer?

The decolorizer dissolves the lipids, increasing cell-wall permeability and allowing the crystal violet-iodine complex to flow out of the cell. The color of the counterstain must contrast with that of the primary stain. A counterstain such as safranin stains gram-negative cells red.

What does decolorization after staining with crystal violet do?

This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly. Rinse with water to stop decolorization.