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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's iodine - fixes the stain in gram positive bacteria Ethanol or acetone - washes the stain from gram negative bacteria Safranin - counterstain, will restain gram negative bacteria while not interfering with the previous stain in gram positive bacteria The mechanism is as follows, gram positive bacterial membrane pores shrink in response to the ethanol or acetone and prevent the stain from being washed.