What role does safranin play in Gram staining?

Safranin serves as a counterstain in the Gram staining procedure, specifically for gram-negative bacteria. In a typical Gram stain, the first step involves applying a primary stain, such as crystal violet, which will adhere to the thick peptidoglycan layer found in gram-positive bacteria, making them appear purple. Following this, iodine is used to form a complex with crystal violet, which helps in retaining the dye in gram-positive cells during the subsequent steps.

However, gram-negative bacteria have a much thinner peptidoglycan layer and an additional outer membrane that does not retain the crystal violet during the decolorization step with alcohol or acetone. As a result, these cells lose the purple color and become colorless. The application of safranin at this stage provides a contrasting color (red or pink) to the decolorized gram-negative bacteria, allowing them to be visualized under the microscope.

This process is crucial because it helps differentiate between the two groups of bacteria based on their cell wall composition, with gram-negative bacteria taking on the safranin color and appearing red or pink, while gram-positive bacteria remain purple. Thus, the role of safranin as a counterstain in Gram staining highlights the distinction between the