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Gram Staining
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Created by Hans Christian Gram
What is Gram Staining?
Gram staining is a common experiment that is used to differentiate
between two large groups of bacteria. The bacteria are differentiated
by their cell wall components.
The procedure distinguishes between two groups : Gram Positive
and Gram Negative by staining them red or violet.
Gram Positive bacteria have a thicker layer of peptidoglycan in their
cell wall, so they stain violet because the substance retains the crystal
violet stain.
Gram negative bacteria have a thinner layer of peptidoglycan in
their cell wall , so they stain red because they cannot retain the
crystal violet stain.
The Process!
1. Staining!
Crystal Violet Dye
The cells are stained with
crystal violet dye.
Next, Gram’s solution
(iodine and potassium
iodide) is added to form a
larger molecule than the
original crystal violet dye
and iodine. This becomes
insoluble in water.
Gram’s
Solution
2. Decolourising!
Decolouriser
A decolouriser such as ethanol alcohol
or acetone is added to the sample. This
shrinks and tightens the peptidoglycan
layer.
The crystal violet dye and Gram’s
solution cannot penetrate the tightened
peptidoglycan layer so instead it is
trapped in the cell of the Gram Positive
bacteria.
On the other hand, the outer layer of the
Gram negative bacteria has degraded
and the thin peptidoglycan layer isn’t
able to retain the dye or solution so the
colour is lost.
Gram Positive:
Colour retained
Gram Negative:
colour lost
3. Counterstaining!
Safranin
A counterstain, often Safranin ( a
weakly water soluble substance) is
added to the sample, staining it
red.
Since Safranin is lighter than
crystal violet, it doesn’t disrupt the
colouration of Gram positive
bacteria.
The decolourised Gram negative
cells are however stained red.