Gram staining principle and procedure

Investigative procedure in microbiology

Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria.

It may also be used to diagnose a fungal infection.^[1] The name comes from the Danish bacteriologistHans Christian Gram, who developed the technique in 1884.^[2]

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls.

Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet.

5 steps of gram staining

Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on addition of ethanol. They are stained pink or red by the counterstain,^[3] commonly safranin or fuchsine. Lugol's iodine solution is always added after addition of crystal violet to form a stable complex with crystal violet that strengthen th