Transcript Slide 1

Introduction to Lab: Differential Stains
Gram Staining
Introduction to Lab 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.
Differential Stains – Gram
• The staining technique, developed in the
late 1700’s by Christian Gram classifies the
rigid cell walled bacteria into one of two
• based on whether they are able to resist
the decolorizing action of an alcoholic
Differential Stains – Gram
• Those that resist decolorization by 95%
ethanol are arbitrarily termed Gram
positive and those that do not are Gram
• (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
Gram-negative cell
Thick peptidoglycan
90% peptidoglycan
Teichoic acids
1 layer
Not many
• In acid-fast cells,
contains mycolic
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).
stain fix
Gram stain:
a. Flood slide with crystal violet and let stain
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 water.
d. Hold the slide on an angle (preferably with
clothes pin) and drop 95% ethyl alcohol
onto it until the alcohol leaving the slide no
longer has a purple tint; be sure to drop the
alcohol onto the upper portion of the slide
so that the smears are subjected to uniform
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 with
bibulous paper.
The crucial step in the staining process is the decolorizing step.
The most accepted 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
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.