Gram-positive / gram-negative staining is based on blue and red dye to stain bacterial cells. How does this work?

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Explanation:

One of the processes gram staining is a common method used to differentiate two large groups of bacteria based on their different cell wall components. The Gram staining procedure distinguishes between the Gram positive and Gram negative groups by coloring these cells red or violet. The positive gram microorganisms contain progressive bacteria stain violet due to the presence of a thick layer of peptidoglycan on their cell walls, which retains the violet crystal with which these cells are stained. Alternatively, Gram-negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the violet crystal during the fading process.

The process of Gram staining includes three procedures: staining with a water-soluble dye called crystal violet, discoloration and contratinction, usually with safanine. In line for the differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker layer of peptidoglycan) retain the violet crystal stain during the fading process, while Gram negative bacteria lose the violet crystal stain and instead stain with safranine in the final staining process. The process involves three steps:

The cells are stained with crystal violet dye. Next, a solution of Gram iodine (iodine and potassium iodide) is added to form a complex between the violet crystal and the iodine. This complex is a larger molecule than the original violet crystalline stain and iodine and is insoluble in water.

A bleach such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and squeezing it. The large violet-iodine crystal complex cannot penetrate this tight peptidoglycan layer, and is therefore trapped in the cell in Gram positive bacteria. To compare, the external membrane of Gram-negative bacteria degrades and the thinner peptidoglycan layer of Gram-negative cells cannot retain the violet-iodine crystal complex and the color is lost.

A counter stain, such as weakly water-soluble safranine, is added to the sample, staining it red. As safranine is lighter than violet crystal, it does not interrupt the purple coloration in Gram positive cells. However, discolored Gram negative cells stain red.

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