What Does Acetone Alcohol Do to a Gram Stain?

What Does Acetone Alcohol Do to a Gram Stain
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The Gram stain is a differential staining procedure that shows which bacteria are Gram-positive or Gram-negative based on their stain color. Acetone alcohol is one reagent used in this process to provide the color differentiation. Gram-positive bacteria have a thick peptidoglycan layer and stain purple, while Gram-negative microorganisms have a little to no peptidoglycan layer and stain pink.

Primary Stain-Crystal Violet

Once a slide of bacterial sample is prepared, crystal violet is used to stain the sample first. At this point both Gram-positive and Gram-negative bacteria will appear purple. Usually the crystal violet is applied on the slide for 30 seconds before washing off any excess stain with water. The crystal violet can adhere a little to the peptidoglycan layers so not all the primary stain will be washed off with the water.

Mordant-Iodine

Iodine is then added to the sample for one minute. It acts as a mordant, which serves to fix dyes in a staining process. Iodine performs this function by binding to the crystal violet and creating an insoluble complex which adheres much better to the thick peptidoglycan layer found in Gram-positive bacterial cells than just the crystal violet alone. There is not a washing step with water after adding the iodine.

Decolorizer-Alcohol

Either acetone or ethyl alcohol can be used as the decolorizing agent. The alcohol dissolves lipids found in the outer cell membrane of Gram-negative bacteria, allowing the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer. The alcohol is added for 10 to 20 seconds; it is poured on the slide until all the iodine is washed away and the run-off is colorless. At this point in the Gram stain process, Gram-negative bacteria are colorless while Gram-positive bacteria still retain the crystal violet. Once finished the slide needs to be rinsed with water to stop the decolorizing effect.

Counterstain-Safranin

Safranin is then added to increase the visibility and contrast to the colorless Gram-negative bacteria. The stain makes these bacteria appear pink under the microscope. Since the stain is added to the whole sample, it also stains Gram-positive bacteria as well, but the darker of the crystal violet hides the lighter safranin pink color. Once the slide sample has been flooded with safranin for about a minute, water is used to wash off any excess stain that did not adhere to the bacterial cells.

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