![]() It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Do not smear.īacteria have a cell wall made up of peptidoglycan. Blot gently and allow the slide to dry.However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red. The counterstain stains both gram-negative and gram-positive cells. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red.Rinse with water to stop decolorization.The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The important aspect is to ensure that all the color has come out that will do so easily. This can be done in a steady stream, or a series of washes. Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol.This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test. Add iodine (Gram’s iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute.The heat-fixed cells should look purple at this stage. Allow to stain for approximately 1 minute. Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. ![]() Do not let the glass become hot to the touch. Heat fix the slide by passing it (cell side up) through a flame to warm the glass.
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