Destain (remove the stain) by dropping the 95% alcohol/acetone mixture down the slanted slide one drop at a time. At first, a lot of violet color will rinse away. Continue adding drops until only a faint violet color is seen in the alcohol rinse. You should be able to see some color in the smear on the slide. If not, you have destained too much. The alcohol/acetone removes the crystal violet stain from the gram-negative bacteria. The gram-positive bacteria will not be destained. 8. Cover the smear with safranin for 30 to 60 seconds. This will stain the destained gram-negative bacteria a pink/red color. The gram-positive bacteria will be unaffected by the safranin. 9. Briefly rinse the smear with water as above. Blot it lightly with blotting paper and let it dry at room temperature. 10. Examine each slide using the highest magnification on your microscope. Questions:

1. Which of the steps/reagents above distinguishes between Gram-positive and Gram-negative cells?
2. What cell structure is responsible for the difference in staining detected by the Gram stain procedure?
3. What is the purpose of doing a differential stain?
4. What is the most important step in the Gram staining procedure? Why?
5. What is the purpose of the 95% ethanol step in the Gram staining procedure?

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