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.