The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative bacteria. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking which resists the decolorization. The differences in cell wall composition of Gram-positive and Gram-negative bacteria account for the Gram staining differences. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink the gram-positive bacteria remain blue.įind information and process for the Preparation of Gram Staining Regent Principle of Gram Stain Image 2: Cell wall of Gram Positive and Gram Negative Bacteria.The gram-negative bacteria appear colorless and gram-positive bacteria remain blue. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, gram-positive bacteria.Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine (CV-I) complex all cells continue to appear blue.Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple.
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(methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material). Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol.An easy way to remember the steps of the Gram stain