Gram Stain

A Gram stain is a diagnostic stain used to differentiate between two types of bacterial cells: Gram-negative and Gram-positive cells.

The general structure of a Gram stain consists of four steps. The first step involves a primary stain (usually crystal violet). The primary stain is the stain that becomes insoluble in some structure of interest during the second step, the mordant (usually iodine). Upon becoming insoluble, it is unable to be washed off, in the third step, by a decolorizing agent (usually 95% ethanol). After decolorizing, the last step involves the counterstain (usually safranin), which provides color for cells that have the primary stain washed away from the structure of interest.

This general structure for a Gram stain can be applied to the common Gram stain used to identify bacteria. Crystal violet, the primary stain, will stain all bacterial cells (whether they are Gram-negative or Gram-positive). Applying iodine as a mordant insolubilizes the crystal violet in the peptidoglycan matrix of the cell wall. However, because Gram-negative cell walls have a thinner layer of peptidoglycan than their Gram-positive counterparts, the crystal violet is washed away easily by 95% ethanol or some other decolorizing agent. Staining the cells safranin for a short time will give cells a slight pink color that does not override the violet of stained Gram-positive cells, while highlighting the stainless Gram-negative cells. Therefore, Gram-positive cells should stain purple/violet because of the crystal violet they entrap in their cell walls, and Gram-negative cells should stain pink from the safranin.

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