The Gram Stain is the most
widely employed staining method
in microbiology.
It is a differential stain because
it divides bacteria into 2 classes:
gram positive and gram negative.
In the first step of the gram stain
procedure,
cells from a fresh culture
are transferred to a clean slide
and allowed to dry.
If the cells are on an augur plate,
they should first be transferred
to a liquid medium for dilution.
The cells should form a thin,
barely visible film.
This can be achieved
by smearing cells
obtained from the surface
of an augur medium
or from a liquid culture.
Fresh cultures must be used
because as cells age,
they lose their ability
to retain the stain.
The cells are then fixed to the slide
by passing slightly
above the flame of a Bunsen burner.
After passing above the flame,
the slide should feel warm
when touched to the back of the hand;
but should not be too hot.
The fixed cells are then stained
with the basic dye, crystal violet,
for 30 to 40 seconds.
The slide is then rinsed with water
to remove excess stain.
At this point, all cells appear purple
under the microscope.
Next, a solution of Grams iodine
is added and retained on the slide
for about 1 minute.
The iodine combines
with the crystal violet
to form a dye-iodine complex,
thereby decreasing its solubility
within the cell.
At this point,
the cells still appear purple.
The cells are then de-colorized
by washing with ethanol or acetone.
This is the differential step.
Gram positive bacteria retain
the crystal violet,
whereas gram negative bacteria do not.
The ethanol or acetone
should be added dropwise
with the slide tilted at an angle
until the drop coming off
the edge of the slide
just starts to become colorless.
Even gram positive cells can lose
the crystal violet iodine complex
during prolonged excessive
de-coloration.
Excess ethanol is then
washed off with water.
When viewed under the microscope,
gram positive cells appear purple
and gram negative cells are colorless.
Finally, the rinsed cells are covered
with the counter stain safranin
for 20 to 30 seconds.
This stains the gram negative bacteria
pink.
After rinsing with water,
the slide is dried with filter paper.
When viewed microscopically,
the gram positive bacteria are purple
and the gram negative bacteria are pink.
Generally, the gram stain correlates
with the cell wall structure
among the bacteria.
The ethanol is thought to shrink
the thick peptidoglycan
in gram positive cells,
thus retaining the dye.
The thick dehydrated
peptidoglycan layer
of gram positive bacteria
appears to be a permeability barrier
preventing the loss
of the crystal violet iodine complex.
In contrast, the peptidoglycan
in gram negative bacteria is very thin,
and has large pores.
Ethanol may extract lipids
and increase the porosity
thus removing the crystal violet
iodine complex.