Staining of a Gram-Negative Bacterium

This short animation demonstrates a gram stain of gram-negative bacterium. This resource was developed by Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009. Copyright 2009-2010, Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/.

(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.