Gram staining is a differential staining technique that divides bacteria into two groups according to the structure of their cell wall. It was developed by Hans Christian Gram in 1884 and is still one of the first tests used to identify bacteria.The method has four main steps, applied to a heat-fixed smear of bacteria on a slide:1. Flood with crystal violet (the primary stain). All cells turn purple.
2. Add iodine, which forms a large crystal violet-iodine complex inside the cells.
3. Rinse with alcohol (or acetone), the decolouriser.
4. Counterstain with safranin, a red stain.
Gram-positive bacteria have a thick layer of peptidoglycan (murein) in their cell wall. The alcohol dehydrates this layer and traps the purple complex inside, so these cells stay purple/violet. Examples include Staphylococcus aureus and Streptococcus.Gram-negative bacteria have only a thin peptidoglycan layer, surrounded by an outer membrane of lipopolysaccharide. The alcohol dissolves the outer membrane and the purple complex washes out. The colourless cells then take up the safranin and appear red/pink. Examples include Escherichia coli and Salmonella.Knowing the Gram type helps doctors choose treatment. Penicillin acts on peptidoglycan, so it is generally more effective against Gram-positive bacteria. The outer membrane of Gram-negative bacteria acts as an extra barrier that keeps many antibiotics out.
2. Add iodine, which forms a large crystal violet-iodine complex inside the cells.
3. Rinse with alcohol (or acetone), the decolouriser.
4. Counterstain with safranin, a red stain.
Gram-positive bacteria have a thick layer of peptidoglycan (murein) in their cell wall. The alcohol dehydrates this layer and traps the purple complex inside, so these cells stay purple/violet. Examples include Staphylococcus aureus and Streptococcus.Gram-negative bacteria have only a thin peptidoglycan layer, surrounded by an outer membrane of lipopolysaccharide. The alcohol dissolves the outer membrane and the purple complex washes out. The colourless cells then take up the safranin and appear red/pink. Examples include Escherichia coli and Salmonella.Knowing the Gram type helps doctors choose treatment. Penicillin acts on peptidoglycan, so it is generally more effective against Gram-positive bacteria. The outer membrane of Gram-negative bacteria acts as an extra barrier that keeps many antibiotics out.