Gram's stain, laboratory staining technique that distinguishes between two groups of bacteria by the identification of differences in the structure of their cell walls. The Gram stain, named after its developer, Danish bacteriologist Christian Gram, has become an important tool in bacterial taxonomy, distinguishing between so-called gram-positive bacteria, which remain colored after the staining procedure, and gram-negative bacteria, which do not retain dye. In the staining technique, cells on a microscope slide are heat-fixed (killed) and stained with a basic dye, crystal violet, which stains all bacterial cells blue; then they are treated with an iodine-potassium iodide solution that allows the iodine to enter the cells and form a water-insoluble complex with the crystal violet dye. The cells are treated with alcohol or acetone solvent in which the iodine-crystal violet complex is soluble. Following solvent treatment, only gram-positive cells remain stained, possibly because of their thick cell wall, which is not permeable to solvent. After the staining procedure, cells are treated with a counterstain, i.e., a red acidic dye such as safranin or acid fuchsin, in order to make gram-negative (decolorized) cells visible. Counterstained gram-negative cells appear red, and gram-positive cells remain blue. Although the cell walls of gram-negative and gram-positive bacteria are similar in chemical composition, the cell wall of gram-negative bacteria is a thin layer sandwiched between an outer lipid-containing cell envelope and the inner cell membrane, whereas the gram-positive cell wall is much thicker, lacks the cell envelope, and contains additional substances, such as teichoic acids, polymers composed of glycerol or ribitol. The difference in reactivity between gram-positive and gram-negative bacteria is linked with differences in physiological properties of the two groups. Gram-positive bacteria are generally more sensitive to growth inhibition by dyes, halogens, many antibiotics, and to attack by phagocytosis (see endocytosis), and are more resistant to digestion by the enzymes pepsin and trypsin and enzymes in animal sera.
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