The Gram stain is a differential staining procedure that shows which bacteria are Gram-positive or Gram-negative based on their stain color. Acetone alcohol is one reagent used in this process to provide the color differentiation. Gram-positive bacteria have a thick peptidoglycan layer and stain purple, while Gram-negative microorganisms have a little to no peptidoglycan layer and stain pink.

Once a slide of bacterial sample is prepared, crystal violet is used to stain the sample first. At this point both Gram-positive and Gram-negative bacteria will appear purple. Usually the crystal violet is applied on the slide for 30 seconds before washing off any excess stain with water. The crystal violet can adhere a little to the peptidoglycan layers so not all the primary stain will be washed off with the water.

Iodine is then added to the sample for one minute. It acts as a mordant, which serves to fix dyes in a staining process. Iodine performs this function by binding to the crystal violet and creating an insoluble complex which adheres much better to the thick peptidoglycan layer found in Gram-positive bacterial cells than just the crystal violet alone. There is not a washing step with water after adding the iodine.

Either acetone or ethyl alcohol can be used as the decolorizing agent. The alcohol dissolves lipids found in the outer cell membrane of Gram-negative bacteria, allowing the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer. The alcohol is added for 10 to 20 seconds; it is poured on the slide until all the iodine is washed away and the run-off is colorless. At this point in the Gram stain process, Gram-negative bacteria are colorless while Gram-positive bacteria still retain the crystal violet. Once finished the slide needs to be rinsed with water to stop the decolorizing effect.

Safranin is then added to increase the visibility and contrast to the colorless Gram-negative bacteria. The stain makes these bacteria appear pink under the microscope. Since the stain is added to the whole sample, it also stains Gram-positive bacteria as well, but the darker of the crystal violet hides the lighter safranin pink color. Once the slide sample has been flooded with safranin for about a minute, water is used to wash off any excess stain that did not adhere to the bacterial cells.