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Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet.
The Gram staining is one of the most crucial staining techniques in microbiology. ... The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye.
Exposing gram negative cells to the decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. This allows the cells to subsequently be stained with safranin.
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