Hans Christian Gram developed the Gram staining technique in 1884 to classify bacteria. Gram staining involves staining bacteria with crystal violet dye, treating it with iodine to form a complex with the dye, decolorizing with alcohol, and counterstaining with safranin. Bacteria that retain the crystal violet dye after decolorization appear violet and are Gram-positive, while those that lose the dye and appear pink are Gram-negative. The Gram staining characteristics help differentiate between bacteria with thick peptidoglycan cell walls that are Gram-positive from those with thin cell walls and an outer membrane that are Gram-negative.

2. GRAM STAINING
By:
N.Mathan
BP211508
I MSc applied Microbiology
Sacred Heart College(Autonomous)
Tirupattur
To:
Dr.P.Saranraj
Head .Dept.of Microbiology
Sacred Heart College(Autonomous)
Tirupattur

3. Hans christian gram
Hans Christian Gram proposed
Gram staining In 1884
Dhanish Microbiologist.
Why Gram Staining iss
important?
A staining technique used to classify
bacteria; bacteria are stained with
gentian violet(crystal violet) and
then treated with Gram's solution;
after being decolorized with alcohol
and treated with safranine and
washed in water, those that retain the
gentian violet are Gram-positive and
those that do not retain it are Gram-
negative

4. First we wiil see About Gram
negaticce and positicve bacteria.
 Gram positive bacteria :
Gram positive bacteria have a
thick cell wall of peptidoglycan and
other polymers.
Peptidoglycan consists of
interleaving filaments made up of
alternating acetylmuramic acid and
actylglucosamine monomers.
In Gram positive bacteria,
there are teichoic acids.
As well, between the cell wall
and cell membrane, there is a
membrane teichoic acid.
Gram negative Bacteria
 Gram negative bacteria:
have an outer membrane of
phospholipids and bacterial
Lipopolysaccharides outside of
their thin peptidoglycan layer.
The space between the outer
membrane and the peptidoglycan
layer is called the periplasmic
space.
The outer membrane protects
Gram negative bacteria against
penicillin and lysozymes.

5. Gram Positive Bacteria Gram Negative Bacteria

6. Gram Staining
Definition:
A method of staining bacteria
using a violet stain. The gram staining
characteristics (denoted as positive or
negative). A heat fixed bacterial smear
is stained with crystal violet (methyl
violet), treated with 3%
iodine/potassium iodide solution,
washed with alcohol and
counterstained. The method
differentiates bacteria into two main
classes, gram-positive and gram-
negative
Stains

7. Procedure
 Take Clean a Glass slide
 Fix Thin smear on the Glass
slide
Smear
preparation
 Smear to air dry and fixed with
heat.

8.  On a rock flood with crystal
violet for 1 minute
 Wash briefly in Running water
for remove excess crystal violet.
 Flood with grams iodine for 1
minute.
 Wash briefly in running water .
 Decolorize with (95% ethyl
alcohol)
 wash immidietly in the Running
water.
 Add counter stain with
Safranine for 2 mins.
 Observe under oil Immersion
lense
Gram staining

9. Gram positive cocci
Gram negative cocci
Gram positive Bacilli
Gram negative bacilli
Violet
colour
and
spherical
shape
Violet
colour
and
Rod
shape
Pink
color
And
rod
shape
Pink
color
And
rod
shape