Staining of a Gram-Positive Bacterium

This short animation demonstrates a gram stain of gram-positive 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 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.