The final step of the Gram stain procedure involves adding a secondary counterstain, typically safranin. This dye imparts a pink color to the previously decolorized cells, making them distinct while remaining largely unnoticeable in the cells that successfully retained the dark purple crystal violet dye.

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The Gram stain procedure is a multi-step differential staining technique developed by Danish microbiologist Hans Christian Gram in $${}1884$$. It remains one of the most frequently used methods to effectively distinguish between bacteria based on their different types of cell walls by sequentially applying a primary stain, a mordant, a decolorizing agent, and a secondary counterstain.

Gram Stain Procedure

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The first step of the Gram stain procedure involves the application of a primary stain, typically crystal violet, to a heat-fixed smear. This initial application imparts a purple color to all of the cells in the specimen before any further differentiation occurs.

Gram Stain Primary Stain

Following the primary stain in the Gram stain procedure, Gram's iodine is added to serve as a mordant. This substance acts as a trapping agent that forms a complex with the crystal violet dye, causing the dye complex to clump and remain securely contained within the thick layers of peptidoglycan in bacterial cell walls.

Gram Stain Mordant

After applying the mordant in the Gram stain procedure, a decolorizing agent such as ethanol or an acetone-ethanol solution is applied. This agent selectively washes the primary dye out of cells with thinner peptidoglycan layers, rendering them colorless again, while cells with thick peptidoglycan layers generally remain unaffected and retain their original purple color.

Gram Stain Decolorization

Gram Stain Counterstain

When examining a Gram-stained specimen under a microscope, gram-positive cells appear as purple, crystal-violet stained cells. In contrast, gram-negative cells appear as red or pink, safranin-dyed cells.

Gram Stain Visual Results

When interpreting the results of a Gram stain, it is recommended to use fresh bacterial cultures. Older bacterial cells may sustain damage to their cell walls, which can cause them to incorrectly appear gram-negative even if the species is actually gram-positive.

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