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The Gram Stain is the most 
widely employed staining method

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in microbiology.

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It is a differential stain because 
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 
procedure,

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cells from a fresh culture 
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, 
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,
barely visible film.

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This can be achieved
by smearing cells

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obtained from the surface 
of an augur medium

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or from a liquid culture.

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Fresh cultures must be used 
because as cells age,

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they lose their ability 
to retain the stain.

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The cells are then fixed to the slide 
by passing slightly

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above the flame of a Bunsen burner.

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After passing above the flame, 
the slide should feel warm

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

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The fixed cells are then stained 
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 
to remove excess stain.

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

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

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for about 1 minute.

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The iodine combines 
with the crystal violet

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

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within the cell.

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At this point, 
the cells still appear purple.

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

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This is the differential step.

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Gram positive bacteria retain 
the crystal violet,

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whereas gram negative bacteria do not.

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The ethanol or acetone 
should be added dropwise

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with the slide tilted at an angle 
until the drop coming off

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the edge of the slide 
just starts to become colorless.

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Even gram positive cells can lose 
the crystal violet iodine complex

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during prolonged excessive
de-coloration.

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Excess ethanol is then
washed off with water.

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When viewed under the microscope, 
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 
with the counter stain safranin

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for 20 to 30 seconds.

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This stains the gram negative bacteria 
pink.

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

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When viewed microscopically, 
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 
with the cell wall structure

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among the bacteria.

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The ethanol is thought to shrink 
the thick peptidoglycan

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in gram positive cells, 
thus retaining the dye.

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The thick dehydrated 
peptidoglycan layer

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of gram positive bacteria 
appears to be a permeability barrier

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preventing the loss 
of the crystal violet iodine complex.

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

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and has large pores.

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Ethanol may extract lipids
and increase the porosity

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thus removing the crystal violet 
iodine complex.