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(English captions by Andrea Matsumoto, University of Michigan.) This program will explain how the gram stain procedure is able to distinguish between gram-positive

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and gram-negative bacteria by representing
the staining events at the ultra-structural level.

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This is one animation, from a series of two,
that specifically shows the staining of gram-negative

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bacteria with the critical structures of the
bacterial surface represented schematically.

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The circle at the lower right tracks how the
bacteria would appear in the microscope if

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they were examined during each step of the
staining procedure.

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Prior to staining the bacteria would be transparent
and invisible.

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After heat fixing the slide, it is first flooded
with crystal violet for one minute and then

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washed.

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The stain colors the bacterial cell wall blue
and the bacteria would appear blue in the

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microscope if examined at this point in the
procedure.

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Next the slide is flooded with iodine solution
for one minute and then washed again.

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During this step the iodine and crystal violet
combine to form a larger complex within the

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layers of the cell wall.

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Microscopically the bacteria appear dark blue
or black after this step.

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The slide is now rinsed with a decolorizing
agent, an acetone alcohol solution.

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Because of the relative simplicity of the
gram-negative cell wall, the crystal violet-iodine

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complexes can be washed away with this treatment
and the organisms once again appear transparent

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at this stage because the dark stain has been
removed.

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Finally the slide is counter-stained with
neutral red or safranin for one minute and

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then washed for the final time.

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The red stain confers red color to the bacteria
and this color dominates the microscopic appearance.

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So by virtue of the simpler structure of the
gram-negative cell wall these bacteria appear

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red in the microscope after this staining
procedure.