Introduction to Lab Ex. Differential Stains
Gram Staining
Introduction to Lab Ex. Differential Stains – Gram Staining
Basic classification of bacteria is based on the cell wall structure.
There are 2 main groups: Gram positive and Gram negative.
Gram staining is a differential staining technique that provides an
easy differentiation of bacteria into one of two groups.
The staining technique, developed in the late 1700’s by
Christian Gram classifies the rigid cell walled bacteria into one of
two groups based on whether they are able to resist the decolorizing
action of an alcoholic solution.
Those that resist decolorization by 95% ethanol are arbitrarily
termed Gram positive and those that do not are Gram negative
(the terms positive and negative have nothing to do with charges
of the cell but based on differences in the cell wall structure of
these two groups of bacteria).
The characteristic compound found in all true bacterial cell walls
is peptidoglycan. The amount of PPG is among one of the
differences between the GP and GN cell walls.
Gram-positive cell walls
Thick peptidoglycan
90% peptidoglycan
Teichoic acids
1 layer
Not many
• In acid-fast cells,
contains mycolic acid
Gram-negative cell
Thin peptidoglycan
5-10% peptidoglycan
No teichoic acids
3 layers
Outer membrane has
lipids, polysaccharides
• No acid- fast cells
(mycolic acid)
Figure 4.13b, c
The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).
The Gram stain
Thin smear/heat fix
Gram stain:
a. Flood slide with crystal violet and let
for 1 minute.
b. Drain off crystal violet and rinse off
with distilled water; flood slide
with Gram's iodine for 1 minute.
c. Rinse off Gram's iodine with distilled
d. Hold the slide on an angle (preferably
a clothes pin) and drop 95% ethyl
onto it until the
alcohol leaving the slide no
longer has a purple tint; be sure to drop
alcohol onto the upper portion of the
so that the smears are subjected
to uniform
decolorization. Be careful not to
"decolorize" dye from the clothes pin!!
e. Rinse with distilled water and flood the
slide with safranin and let stain for 2-3
f. Rinse with distilled water and blot dry
The crucial step in the staining process is the decolorizing step.
The most accepted theory about the rationale for the Gram staining
process is the one proposed by Salton.
This theory relies on the fact that the PPG is found in layers and the
stain molecules are trapped within the many layers of the GP CW
when they form the complex with the mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of stain captured in
those CWs is much lesser.
When the cells are treated with the decolorizer – the ethanol – this
causes denaturation of the proteins in the outer membrane of the
GN CWs resulting in gaping holes in these CWs that lead to the
removal of the crystal violet-iodine complexes easily, leaving these
cells unstained.
The counterstain -safranin- thus is used to make these cells visible.
There are 4 conditions to be followed for a valid Gram staining
Young cultures - must be young within 18-24hrs old
(older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
Thin smear
thicker or uneven smears will result in uneven staining
and decolorization
Fresh reagents - of proper strength
Control cultures - for a known GP bacterium and GN culture
(S.aureus & E.coli)
Demos: Gram stained slides of
Neisseria, Streptococcus, Pseudomonas, Actinomyces species.

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