- Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria.
- In a Gram stain test, gram-positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or fuchsine) added after the crystal violet gives all gram-negative bacteria a red or pink coloring.
For gram positive bacteria, the thick peptidoglycan layer in the cell wall that encases their cell membrane retains the stain, making definitive identification possible.
Gram-negative bacteria cannot retain the violet stain after the decolorization step; alcohol used in the decolorization process degrades the outer membrane of gram-negative cells making the cell wall more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take up the counterstain (safranin orfuchsine) and appear red or pink.