![]() Image 3: Procedure of Gram Staining note the color change after each step Do not wipe or blot the slide, as this can remove cells. Once two minutes have passed, tilt the slide and drain off the excess methanol and let the slide air dry. Alternatively, dip the slide into a Coplin jar filled with methanol. Place or hold the slide over a paper towel and flood the slide with absolute methanol for two minutes. Alternatively, the smear can be fixed using methanol. Heat application helps the cell adhesion (fixation) to the glass slide and prevents its loss during rinsing.Īllow the slide to cool to the touch before applying the stain. Air dry the sample, and once the sample gets air dried, heat fix the smear by passing it through a bunsen burner three times.Spread the sample to an even-thin film over a circle of 15 mm diameter.If performing a Gram stain from a bacterial colony, first put a drop or a few loopful of water and emulsify the bacterial colony in the water drop. Transfer a loop of the sample (for example, sputum, CSF, or pus) to the microscope slide.In contrast, the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added. Thus ethanol treatment traps the large CV-I complexes within the cell.Īfter decolorization, the gram-positive cell remains purple. The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol.With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin peptidoglycan layer exposed.The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the lipids of both gram-positive and gram-negative bacteria membranes. 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 Gram-positive and Gram-negative cell walls. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking, which resists decolorization. The differences in Gram-positive and Gram-negative bacteria cell wall composition 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 is a dark blue to purple dye.Heating the slide causes cell distortion, could increase cell debris, and may cause erroneous Gram stain results.). Methanol fixation preserves the morphology of host cells and bacteria. (Methanol fixation is recommended rather than heat fixation. Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol.Come In and Stain! is an easy way to remember the steps of the Gram stain
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