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.