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

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

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This particular animation is one of two in[br]this series showing the staining of gram-positive

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bacteria with critical structures of the bacterial[br]surface represented schematically.

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

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they were examined during each step of the[br]staining procedure.

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Prior to staining, the bacteria would be transparent[br]and invisible in the microscope.

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After heat fixing the slide, it is first flooded[br]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[br]and the bacteria would appear blue in the

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microscope if examined at this point in the[br]procedure.

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

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During this step the iodine and crystal violet[br]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[br]dark blue or black after this step.

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

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However, the crystal violet iodine complexes[br]are not washed out of the thick and tortuous

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

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Finally the slide is counter-stained with[br]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[br]however the red color is not apparent because

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

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

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