The second dye used in gram staining is ___. methylene bluecrystal violetiodinesafranin

The correct answer is safranin.

In the Gram staining procedure, the second dye, safranin, acts as a counterstain after the crystal violet has been applied. The process begins with the application of crystal violet, which temporarily stains all bacterial cells. Then, iodine is used as a mordant, enhancing the retention of the crystal violet stain in Gram-positive bacteria by forming a complex. Following the iodine step, a decolorization step typically using ethanol or acetone is performed, which removes the crystal violet from Gram-negative bacteria but leaves it in Gram-positive bacteria. Finally, safranin is applied, which stains the now colorless Gram-negative bacteria, allowing for dual differentiation. This process is critical for identifying bacteria and determining their characteristics based on their cell wall structure.