Gram Staining Principle- Step by step procedure with explanation, Reagent Preparation

1. How Gram Stain Works?

2. Gram Staining-Principle
• Developed by Christian Gram
• Classification of bacteria into Gram
positive and Gram negative is based on the
difference in the cell wall composition of
Gram positive and Gram negative bacteria.
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3. In Gram positive bacteria
-cell wall is made up of 2
layers
 A thicker outer layer
made of peptidoglycan
and the cell membrane
Low lipid content
In Gram negative
bacteria,
cell wall has three layers
A lipid rich outer wall
A middle layer consists
of thin layer of
peptidoglycan and a cell
membrane.
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4. Step 1:
Prepare thin smear of bacteria on a clean slide
.;;;.;; ;
.
;;;’;;’;’;;’.;;..;;..;;.;.
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5. Step 2:
Cover the smear with few drops of crystal violet for 1
min
.;.;;;.;; ;
. .
;;;’;;’;’;;’.;;..;;.;.;
Step 3:
Wash excess stain with water
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6. Step 4: Add few drops of iodine solution on Here Iodine solution is a
the smear for 1 min mordant.
Mordants are substance
that improves the staining
property of the dye.
Here Iodine favors more
interaction between cell
and the dye (crystal
.;;;.;; ;
. violet). So that, the cells
;;;’;;’;’;;’.;;..;;..;;.;.
are more strongly stained.
Step 5: Rinse in water  Now under the
microscope, all cells are
violet coloured, we cannot
;;;;;.;..;,;,;,l, differentiate Gram
positive and Gram
negative bacteria.
Under microscope

7. On washing with ethanol
Step 6: Wash with 95% ethanol for a minute. or acetone, the smear
either retains the violet
colour or decolorizes.
Those bacteria that
retain violet colour are
Gram positive, and those
that lose violet colour on
95% alcohol alcohol treatment are the
Gram negative.
This difference in
;;;.;; ;
. staining is due to the
;;;’;;’;’;;’.;;..;;..;;..;. difference in the cell wall
components of Gram
positive and Gram
negative bacteria.
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8. In Gram positive bacteria,
After ethanol treatment . the lipid content is very
low and has a thick
peptidoglycan wall. So
ethanol cannot remove
the crystal violet stain
bound to thick
;;;;;.;..;,;,;,l, peptidoglycan wall of
gram positive bacteria. So
it retains violet colour.
Gram positive bacteria Under microscope
In Gram negative bacteria
On ethanol treatment,
lipids are dissolved in
ethanol and peptidoglycan
wall is very thin to hold
;;;;;.;..;,;,;,l,
the stain so gram negative
bacteria lose violet colour.
Gram negative bacteria Under microscope

9. Step 7: Wash excess stain with water This is to visualise Gram
negative bacteria.
Step 8: Stain with 1% safranin for 1
minute. After ethanol treatment
in step-6, only Gram
positive bacteria retain
crystal violet.
We need to stain further
using a counter stain like
safranin to visualise Gram
negative bacteria.
.;
;;;’;;’ ; .;; .;
;’;;’.;;..;..;;..;; ;
;;;;;.;..;,;,;,l,
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10. Step 9: Wash stain in excess water.
The end result after staining with safranin
Gram positive bacteria appear violet or purple (Colour of crystal violet)
Gram negative bacteria appear pink to red (colour of safranin).
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11. Procedure Summary Chart

12. Reagent Preparation
Crystal violet solution: 1g crystal violet dissolved in
100 ml ethanol.
Safranin solution: 1g safranin dissolved in 100 ml
H2O.
Iodine solution: Dissolve 2g potassium iodide (KI) in
100 ml water. To the solution add iodine crystals till
a deep yellow colour develops.
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13. FOR DETAILED NOTES ON
THIS TOPIC VISIT
http://www.biologyexams4u.com/2011/09/microbiology-notes.html
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