Gram staining procedure

Learn the fundamental differences between Gram-positive bacteria (two-layer structure with thick peptidoglycan) and Gram-negative bacteria (three-layer structure with thin peptidoglycan and outer membrane). This structural variance is the basis for differential staining.

Flood the bacterial smear with crystal violet stain and allow it to remain for 1 minute. This primary stain colors all bacteria purple.

Add iodine solution to the stained bacteria. The iodine forms a crystal violet-iodine complex that is too large to escape the cell membrane, fixing the purple color.

Wash the slide with alcohol for approximately 20 seconds. Alcohol destabilizes outer membranes in Gram-negative bacteria and enlarges peptidoglycan pores, allowing the crystal violet-iodine complex to escape. Gram-positive bacteria retain the complex due to their thick peptidoglycan wall.

Flood the slide with safranine stain for 1 to 2 minutes. Gram-negative bacteria (now colorless) take up the pink safranine color, while Gram-positive bacteria (still purple) are protected by their thick peptidoglycan layer.

Wash the slide with water to remove excess safranine. Observe the final staining: Gram-positive bacteria appear purple and Gram-negative bacteria appear pink, allowing for bacterial classification and identification.