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Answer Posted / santhosh kumar guptha
Classic Gram staining techniques involves following steps:
Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material).
Application of the primary stain (crystal violet).Crystal violet stains all cells blue/purple
Application of Mordant: The iodine solution (mordant) is added to form a crystal violet iodine (CV-I) complex; all cells continue to appear blue.
Decolorization Step: The decolorization step distinguishes gram-positive from gram-negative cells.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. The gram negative bacteria appear colorless and gram positive bacteria remain blue
Application of counter stain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.
Principle of Gram Stain
Cell wall of Gram Positive and Gram Negative Bacteria
The differences in cell wall composition of Gram positive and Gram negative bacteria accounts for the Gram staining differences. Gram positive cell wall contain thick layer ofpeptidoglycan with numerous teichoic acid cross linking which resists the decolorization.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. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.When added, iodine interacts with CV+ to form large crystal violet iodine (CV-I) complexes within the cytoplasm and outer layers of the cell. 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.The outer membrane of the gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.The highly cross-linked and multi-layeredpeptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.
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