(English captions by Andrea Matsumoto, University of Michigan.) This program will explain how the gram stain procedure is able to distinguish between gram-positive
and gram-negative bacteria by representing the staining events at the ultra-structural level.
This is one animation, from a series of two, that specifically shows the staining of gram-negative
bacteria with the 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.
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 larger complex within the
layers of the cell wall.
Microscopically the bacteria appear dark blue or black after this step.
The slide is now rinsed with a decolorizing agent, an acetone alcohol solution.
Because of the relative simplicity of the gram-negative cell wall, the crystal violet-iodine
complexes can be washed away with this treatment and the organisms once again appear transparent
at this stage because the dark stain has been removed.
Finally the slide is counter-stained with neutral red or safranin for one minute and
then washed for the final time.
The red stain confers red color to the bacteria and this color dominates the microscopic appearance.
So by virtue of the simpler structure of the gram-negative cell wall these bacteria appear
red in the microscope after this staining procedure.