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Gram Staining: A. Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for thinly prepared slides, it is usually acceptable to pour the stain on and off immediately. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. Note that the objective of this step is to wash off the stain, not the fixed culture. B. Add the iodine solution on the smear, enough to cover the fixed culture. Let stand for 10 to 60 seconds.
Gram-positive bacteria have a thicker peptidoglycan layer in their cell wall outside the cell membrane, which retains the crystal violet stain during the alcohol wash, so long as it is timed correctly. The counter stain may also be absorbed by gram-positive bacteria but the darker crystal violet stain predominates.