Heat-fix the bacteria to a glass slide. Transfer the bacteria from a collected specimen sample using a sterile swab or inoculating loop. Spread the bacteria on a pre-cleaned glass slide. Heat the slide over a Bunsen burner flame for a few seconds to affix the bacteria to the slide. Place the slide on a rack over a sink.
Pour five to 10 drops of crystal violet solution to the glass slide and wait for about one minute. This will stain the bacteria violet.
Rinse the slide off using distilled water. Pour 5 to 10 drops of Gram's iodine solution on the slide and wait one minute. The Gram's iodine solution will affix the crystal violet stain to the bacteria if the bacteria has a thick cellular membrane.
Rinse the Gram's iodine solution off using distilled water. Pour five to 10 drops of acetone and wait about 10 seconds. The acetone will remove crystal violet stain from bacteria with thin cellular walls but will not remove the crystal violet solution from bacteria with thick cellular walls.
Pour five to 10 drops of safranin counterstain solution on the glass slide and wait one minute. The safranin will stain the cellular wall of the bacteria red if the crystal violet stain was removed using the acetone.
Rinse the safranin solution using distilled water. A thin-cell-walled bacteria will retain the safranin solution. A thick-cell-walled bacteria will still retain the crystal violet stain, so the safranin solution will be rinsed off.
Place the slide under the microscope under oil immersion and observe the bacteria. Gram-positive bacteria will appear violet while Gram-negative bacteria will appear red.