The Procedure: To prepare for the Gram Stain, cells from the culture are spread in a thin layer on a glass slide. After air-drying, the slide is quickly passed through a flame in order to fix the cells to the slide.
In the first step of the Gram Stain, the smear of cells is flooded with the dye crystal violet for 1 minute. This stains all of the cells purple.
In the second step of the procedure, the slide is flooded with iodine solution for 1 minute. The cells remain purple in color.
The third step is the decolorizing step, where the slide is briefly flooded with alcohol for approximately 20 seconds. This removes the purple dye from gram-negative cells, while any gram-positive cells remain purple. Caution! Decolorizing too long, will give you false results.
The fourth and final step of the Gram Stain is to counter-stain the smear with the dye safranin for 1-2 minutes. The gram-negative cells, will take up the safranin and be red in color, while the gram-positive cells will still remain purple.
The Theory: The Gram Stain works, because bacteria can be divided into two main groups based on the morphology of their cell wall. Bacterial cells are either gram-positive (purple) or gram-negative (red).
At the beginning of the procedure, all cells are stained purple as they take up the crystal violet and the iodine. These two dyes form an insoluble complex inside of the cells. The purpose of the alcohol in the decolorizing step is to extract this complex from the cell. However, this is only possible in gram-negative cells. Why?
Gram-positive cells have a many layers of peptidoglycan forming a very thick cell wall. When the alcohol is added to the smear, these layers dehydrate, which closes any pores present in the wall, trapping the crystal violet-iodine complex inside.
The gram-negative cells have a much more complex structure, including a fatty outer-membrane surrounding a much thinner peptidoglycan cell wall. The alcohol is able to easily penetrate the outer membrane and extract the dye complex, removing the purple color from the cell.
Finally, when the red safranin dye is added to the smear, only the gram-negative cells are capable of taking it up and appearing red.
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