Page 17 - PowerPoint Presentation

Steps of the Gram Stain

1. Crystal Violet Staining: The sample is first stained with crystal violet, a purple dye that penetrates all
bacterial cells.

2. Iodine Treatment: Iodine is added as a mordant, forming a complex with the crystal violet dye, which
helps to fix the dye inside the cells.

3. Decolorization: The sample is treated with alcohol or acetone. This step is crucial:
•Gram-positive bacteria: Have a thick peptidoglycan layer in their cell walls, which retains the
crystal violet-iodine complex and remain purple.
•Gram-negative bacteria: Have a thinner peptidoglycan layer and an outer membrane. The decolorizer
disrupts the outer membrane and washes out the crystal violet, causing them to become colorless.

4. Counterstaining: The sample is then stained with safranin, a red dye. This stains the now colorless Gram
negative bacteria, making them appear pink, while Gram-positive bacteria remain purple.