Gram stain procedure
1. Fix slides with methanol, flood for 1 min, then air dry
2. Cover the alcohol fixed and dried slide smear with crystal violet solution, allowing the stain to remain for one min.
3. Gently wash off the crystal violet with slow flowing tap water and remove excess water (caution: excessive rinsing could cause organisms to be washed off).
4. Flood the slide with iodine for one min.
5. Gently rinse off the iodine with slow flowing tap water.
6. Decolorize with acetone-alcohol (abot five secs).
7. Rapidly remove the decolorizer with gentle tap water. (Caution: excessive rinsing in this step could cause dye-iodine complex to be washed from gram positove cells).
8. Flood the smear with the counterstain (safranon or basic fuschin) for 30 secs.
9. Rinse the counterstain aith slow flowing tap water.
10. Drain the slide and blot dry with bibulous paper.
Crystal violet binds to cell wall of bacteria after treatment with a weak iodine solution. Some bacteria retain the crystal violet, even after a decolorizer is added. The organisms that retain dye are called Gram-positive (stain purple), and those with a high lipid content lose the purple color and pick up the counter-stain (safranin). These are called Gram-negative.