In a Gram staining procedure, what is the correct order of the staining steps?

The correct sequence of the Gram staining procedure is essential for distinguishing between Gram-positive and Gram-negative bacteria based on their cell wall characteristics. The first step is the application of the primary stain, which is usually crystal violet. This stain initially colors all the bacterial cells.

Next, a mordant, commonly iodine, is added. This mordant forms a complex with the primary stain, making it more difficult to wash out of the cells. The mordant helps to ensure that the crystal violet adheres to the cell walls.

Following the mordant, a decolorizer, which is typically alcohol or acetone, is applied. This step is critical as it differentiates the two types of bacteria. In Gram-positive bacteria, the thick peptidoglycan layer retains the crystal violet-iodine complex, whereas in Gram-negative bacteria, the thinner peptidoglycan layer allows the decolorizer to wash away the stain.

Finally, a counterstain, usually safranin, is applied. This stain colors the decolorized Gram-negative bacteria, allowing them to be visualized against the Gram-positive bacteria that remain stained with crystal violet.

This order of staining steps—primary stain, mordant, decolorizer, followed by counterstain