What is the secondary stain used in Gram staining?

In the Gram staining procedure, the secondary stain used is saffranin. This step is crucial for differentiating between Gram-positive and Gram-negative bacteria. After the initial staining with crystal violet, which stains all cells, the addition of iodine acts as a mordant, helping to fix the crystal violet stain inside the cells. The subsequent decolorization step with alcohol or acetone washes out the crystal violet from Gram-negative bacteria but not from Gram-positive bacteria, which retain the purple color.

Following decolorization, saffranin is applied as the secondary stain. It provides contrast by staining the now colorless Gram-negative bacteria pink or red, allowing observers to distinguish between the two types of bacteria based on their color. This differential staining is foundational for bacterial classification and further microbiological analysis.