(English captions by Andrea Matsumoto, University of Michigan.) This program will illustrate how the gram stain procedure is able to distinguish gram-positive
and gram-negative bacteria by representing the staining events at the ultra-structural level.
This particular animation is one of two in this series showing the staining of gram-positive
bacteria with critical structures of the bacterial surface represented schematically.
The circle at the lower right tracks how the bacteria would appear in the microscope if
they were examined during each step of the staining procedure.
Prior to staining, the bacteria would be transparent and invisible in the microscope.
After heat fixing the slide, it is first flooded with crystal violet for one minute and then
washed.
The stain colors the bacterial cell wall blue and the bacteria would appear blue in the
microscope if examined at this point in the procedure.
Next the slide is flooded with iodine solution for one minute and then washed again.
During this step the iodine and crystal violet combine to form a large complex within the
layers of the cell wall.
Microscopically the bacteria would appear dark blue or black after this step.
The slide is now rinsed with a decolorizing agent, an acetone alcohol solution.
However, the crystal violet iodine complexes are not washed out of the thick and tortuous
layers of the gram-positive cell wall and the organisms remain dark blue in color.
Finally the slide is counter-stained with neutral red or safranin for one minute and
then washed again.
The red stain also confers color to the bacteria however the red color is not apparent because
of the persistent dark blue stain that dominates the microscopic appearance of the bacteria.
So, by virtue of the complex multilayer structure of the gram-positive cell wall, these bacteria
appear dark blue or black in the microscope after this staining.