Safranine is the fourth and final chemical used in a Gram stain. It is a positively charged dye that is red in color.
In the previous steps in the Gram stain, cells have been stained with crystal violet and treated with Gram’s iodine to increase binding of the purple stain to the cells. This is followed by use of Gram’s decolorizer, which washes the crystal violet/iodine complex out of Gram negative cells but fails to do so with Gram positive cells. This means after Gram’s decolorizer is used, Gram positive bacteria are purple and Gram negative bacteria are clear.
To see Gram negatives under the microscope, a second stain must be used. These counterstains are a different color from the primary stain (here crystal violet). Safranine is the counterstain in the Gram stain procedure. Its red/pink color allows Gram negative bacteria to be seen, and their lighter red/pink makes them easily distinguishable from the purple of Gram positive bacteria.