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Gram stain

The Gram stain is a differential staining technique developed by Hans Christian Gram in 1884. It classifies bacteria into Gram-positive and Gram-negative groups based on differences in their cell wall structure, especially the thickness of the peptidoglycan layer. This technique is fundamental in microbiology for bacterial identification and clinical diagnosis.

Principle of Gram Stain

Gram-positive bacteria have a thick peptidoglycan layer that retains the primary stain (crystal violet–iodine complex) even after decolorization with alcohol/acetone.

Gram-negative bacteria have a thin peptidoglycan layer and a higher lipid content. The alcohol dissolves the lipids, making the cell wall porous and allowing the primary stain to be washed out. They are then counterstained by safranin (or fuchsine) and appear pink/red under the microscope.

Procedure of Gram Stain

1. Smear Preparation – Prepare a thin smear of bacterial culture on a glass slide, air dry, and heat-fix.

2. Primary Stain – Flood slide with crystal violet for about 1 minute, then rinse with water.

3. Mordant – Add Gram’s iodine for 1 minute (forms a crystal violet–iodine complex), rinse with water.

4. Decolorization – Wash with 95% ethanol or acetone-alcohol for 10–20 seconds, then rinse immediately.

Gram-positive: retain purple color

Gram-negative: lose color (become colorless)

5. Counterstain – Flood with safranin for 30–60 seconds, rinse with water.

6. Microscopy – Observe under oil immersion (100× objective).

Gram Stain of Negative Bacilli Bacteria

Appearance: Gram-negative bacilli (rod-shaped bacteria) appear pink/red rods after staining because they do not retain the crystal violet stain and instead take up the counterstain (safranin).

Examples: Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Proteus species.

Significance: Identifying Gram-negative bacilli is critical in clinical microbiology because many are pathogens responsible for gastrointestinal, respiratory, and urinary tract infections.

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