Differential Staining Technique

Differential staining distinguishes microorganisms based on cell wall properties, aiding bacterial identification and classification.

Gram-staining classifies bacteria as gram-positive or gram-negative by their ability to retain crystal violet after alcohol decolorization.

A thin smear of microbial culture is spread on a slide, air-dried, and heat-fixed.

The smear is stained with crystal violet, washed with water, and treated with iodine, forming a crystal violet-iodine complex.

Alcohol is used to decolorize this smear until the runoff is clear, followed by a rinse with water.

Gram-positive bacteria retain the purple stain due to their thick peptidoglycan layer.

In contrast, gram-negative bacteria lose the purple stain as alcohol disrupts the outer membrane and washes off the crystal violet-iodine complex.

Safranin is used to counterstain gram-negative bacteria pink, while gram-positive bacteria remain purple.

Acid-fast staining detects Mycobacterium species, whose waxy, mycolic acid-rich cell walls resist decolorization.

The acid-fast bacteria retain carbol fuchsin, a red dye, while non-acid-fast cells are usually counterstained with methylene blue.