Gram staining
After 24 hours of growth from nutrient broth, the growth suspension is taken using an inoculation needle and it is placed on a glass slide. A smear is made and it is heat fixed. Then it is treated with solutions like crystal violet and kept for 1 min. Then it is rinsed with distilled water and 2 drops of iodine is added and kept for 60 sec. Then it is decolorized with few drops of alcohol because large amount of alcohol makes even a gram positive cell to gram negative. After adding alcohol, it is again washed with water and saffranin is added and it is kept for 1 min.
It is again washed with distilled water, air dried and observed under microscope. Bacteria stained by the gram method fall under two groups. Gram positive bacteria which retain violet colour and the Gram negative bacteria stained by sarafnin and hence appear pink in colour.