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(English captions by Andrea Matsumoto, University of Michigan.) This program will illustrate how the gram
stain procedure is able to distinguish 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 particular animation is one of two in
this series showing the staining of gram-positive

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bacteria with 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 in the microscope.

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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 large complex within the

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layers of the cell wall.

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Microscopically the bacteria would 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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However, the crystal violet iodine complexes
are not washed out of the thick and tortuous

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layers of the gram-positive cell wall and
the organisms remain dark blue in color.

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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 again.

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The red stain also confers color to the bacteria
however the red color is not apparent because

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of the persistent dark blue stain that dominates
the microscopic appearance of the bacteria.

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So, by virtue of the complex multilayer structure
of the gram-positive cell wall, these bacteria

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appear dark blue or black in the microscope
after this staining.