Canadian Society for Medical Laboratory Science (CSMLS) Practice Exam

What is the counterstain used in Gram's stain?

1. Eosin

2. Safranin

3. Iodine

4. Giemsa

The correct answer is: Safranin

The counterstain used in Gram's stain is safranin. In the Gram staining process, after the initial application of the crystal violet stain, iodine is added, which forms a complex with the crystal violet, enhancing its retention in the gram-positive cells. This is followed by a decolorization step, usually with alcohol, which removes the crystal violet from gram-negative bacteria, rendering them colorless. At this stage, safranin is applied as the counterstain, which stains the decolorized gram-negative bacteria, allowing them to be visualized under the microscope. It provides contrast, enabling the distinction between gram-positive organisms, which retain the purple color from crystal violet, and gram-negative organisms, which take up the pink hue from safranin. This differentiation is fundamental in microbiology for identifying and categorizing bacterial species based on their cell wall characteristics.