The Gram stain is a fundamental method of bacterial classification that serves as the first step in identifying an unknown bacterium. Developed in 1884 by Hans Christian Gram, this technique provides a standardized way to categorize nearly all species. It is a form of differential staining, meaning it uses multiple chemical reagents to exploit the physical and chemical differences in bacterial cell walls.
The Four Essential Reagents
The Gram stain relies on the sequential application of four specific chemical reagents, each with a distinct function in the staining process. The procedure begins with the primary stain, which is a solution of crystal violet, a purple dye that permeates the cell wall of every bacterium on the slide. Following this, a solution of Gram’s iodine is applied, acting as a mordant. The iodine reacts with the crystal violet to form a large, insoluble Crystal Violet-Iodine (CV-I) complex inside the cell.
The third reagent is the decolorizer, typically a solution of \(95%\) ethanol, acetone, or a mixture of both. This solvent is responsible for the differential aspect of the stain, as it selectively removes the primary stain from only one group of bacteria. Finally, the counterstain, safranin, is applied to the slide. Safranin is a reddish-pink dye that provides a contrasting color to stain any cells that lost the initial purple complex during the decolorization step.
Step-by-Step Staining Procedure
The procedure begins with the preparation of a thin smear of bacteria on a glass slide, which is then fixed, often using heat, to adhere the cells to the glass so they are not washed away. The first chemical application involves flooding the fixed smear with crystal violet for approximately one minute to ensure all bacterial cells are stained purple. After rinsing the excess primary stain with water, the slide is covered with Gram’s iodine for a minute to allow the mordant to act.
Following another gentle water rinse, the decolorization step is performed, which is the most time-sensitive phase of the procedure. The alcohol or acetone solution is applied for only a few seconds, typically between five and fifteen, and immediately rinsed off with water to halt the solvent’s action. The technician must stop the decolorizing agent as soon as it ceases to wash purple color from the slide. The final step is the application of the counterstain, safranin, which is left on the slide for about thirty seconds to one minute before a final wash.
Distinguishing Gram-Positive and Gram-Negative Bacteria
The fundamental principle underlying the Gram stain’s differential capability is the structural difference in the bacterial cell wall, primarily the thickness of the peptidoglycan layer. Gram-positive bacteria possess a thick, mesh-like layer of peptidoglycan that constitutes approximately \(50%\) to \(90%\) of their cell envelope. When the decolorizer is added, it dehydrates this thick peptidoglycan, causing the pores to shrink and contract. This physical change effectively traps the large, insoluble Crystal Violet-Iodine complex within the cell wall of Gram-positive bacteria, preventing the purple color from washing out.
In contrast, Gram-negative bacteria have a significantly thinner peptidoglycan layer, often making up only about \(10%\) of the cell envelope, and also possess an outer lipopolysaccharide membrane. The decolorizing solvent dissolves this outer lipid membrane and easily passes through the thin peptidoglycan layer, allowing the CV-I complex to escape and leaving the cell colorless. Once the colorless Gram-negative cells are exposed to the safranin counterstain, they take up the pink/red dye, while the Gram-positive cells remain purple.