Which step in the Gram stain is the critical step in differentiating gram-positive cells from gram-negative cells? A) safranin B) alcohol-acetone C) iodine D) crystal violet

The correct answer is: B) alcohol-acetone

Gram staining includes next steps:

• Fixation - usually by heating or by using methanol

• Primary stain (crystal violet) - it stains all cells blue/purple

• Mordant - added to form a crystal violet iodine

• Decolorization - distinguishes gram-positive from gram-negative cells. Acetone or ethanol are usually used. Acetone/ethanol extracts blue dye from Gram - bacteria (the lipid-rich, thin wall) more than from the, Gram + bacteria (lipid poor, thick walled). The Gram - bacteria appear colorless and Gram + bacteria remain blue.

• Safranin-the red dye stains the decolorized Gram - bacteria.