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