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The Gram Stain is the most [br]widely employed staining method

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in microbiology.

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It is a differential stain because [br]it divides bacteria into 2 classes:

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gram positive and gram negative.

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In the first step of the gram stain [br]procedure,

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cells from a fresh culture [br]are transferred to a clean slide

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and allowed to dry.

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If the cells are on an augur plate, [br]they should first be transferred

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to a liquid medium for dilution.

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The cells should form a thin,[br]barely visible film.

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This can be achieved[br]by smearing cells

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obtained from the surface [br]of an augur medium

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or from a liquid culture.

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Fresh cultures must be used [br]because as cells age,

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they lose their ability [br]to retain the stain.

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The cells are then fixed to the slide [br]by passing slightly

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above the flame of a Bunsen burner.

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After passing above the flame, [br]the slide should feel warm

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when touched to the back of the hand; [br]but should not be too hot.

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The fixed cells are then stained [br]with the basic dye, crystal violet,

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for 30 to 40 seconds.

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The slide is then rinsed with water [br]to remove excess stain.

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At this point, all cells appear purple [br]under the microscope.

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Next, a solution of Grams iodine [br]is added and retained on the slide

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for about 1 minute.

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The iodine combines [br]with the crystal violet

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to form a dye-iodine complex, [br]thereby decreasing its solubility

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within the cell.

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At this point, [br]the cells still appear purple.

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The cells are then de-colorized [br]by washing with ethanol or acetone.

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This is the differential step.

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Gram positive bacteria retain [br]the crystal violet,

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whereas gram negative bacteria do not.

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The ethanol or acetone [br]should be added dropwise

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with the slide tilted at an angle [br]until the drop coming off

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the edge of the slide [br]just starts to become colorless.

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Even gram positive cells can lose [br]the crystal violet iodine complex

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during prolonged excessive[br]de-coloration.

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Excess ethanol is then[br]washed off with water.

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When viewed under the microscope, [br]gram positive cells appear purple

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and gram negative cells are colorless.

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Finally, the rinsed cells are covered [br]with the counter stain safranin

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for 20 to 30 seconds.

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This stains the gram negative bacteria [br]pink.

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After rinsing with water, [br]the slide is dried with filter paper.

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When viewed microscopically, [br]the gram positive bacteria are purple

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and the gram negative bacteria are pink.

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Generally, the gram stain correlates [br]with the cell wall structure

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among the bacteria.

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The ethanol is thought to shrink [br]the thick peptidoglycan

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in gram positive cells, [br]thus retaining the dye.

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The thick dehydrated [br]peptidoglycan layer

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of gram positive bacteria [br]appears to be a permeability barrier

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preventing the loss [br]of the crystal violet iodine complex.

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In contrast, the peptidoglycan [br]in gram negative bacteria is very thin,

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and has large pores.

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Ethanol may extract lipids[br]and increase the porosity

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thus removing the crystal violet [br]iodine complex.