Gram staining is a differential staining technique that classifies bacteria as Gram positive or Gram negative based on whether they retain or lose the primary stain, crystal violet, after treatment with a decolorizing agent like alcohol. The difference is based on cell wall composition - Gram negative bacteria have a thinner peptidoglycan layer and higher lipid content, making their cell wall more porous. The Gram staining procedure involves staining with crystal violet, iodine as a mordant, alcohol as a decolorizer, and safranin as a counterstain. Gram positive bacteria retain the crystal violet stain while Gram negative bacteria take up the safranin counterstain, appearing purple or pink under a microscope respectively. Es

1. Topic- GRAM STAINING
Presented by –
Name- Madhurima Nandy
Stream- B.PHARM
Year- 5th semester, 3rd year.
Institute- Guru Nanak institute of
pharmaceutical science and technology

3. CONTENT:
SL.NO. TOPIC SLIDE NUMBER
1 Introduction. 4
2 Classification of staining
technique.
5
3 Gram staining introduction. 6
4 Principle. 7
5 Mechanism. 8-9
6 Procedure. 10
7 Diagramatic representation
of procedure.
11
8 Result and conclusion. 12
9 Reference. 13

4. INTRODUCTION :
Staining is the process to identify or to
visualise the microorganism with the use
of some organic chemicals known as dyes.
Staining is mainly done for identification
of different morphological and distingui-
shable characteristics of cells.

5. CLASSIFICATION OF STAINING
TECHNIQUES:
STAINS
DIFFERENTIAL
STAIN
GRAM STAIN
ACID-FAST
STAIN
SIMPLE
STAIN
NEGATIVE
STAIN
ENDOSPORE
STAIN

6. GRAM STAINING INTRODUCTION:
Gram staining is a differential staining tech-
nique by which bacteria are classified as “Gram
positive or Gram negative” depending upon
whether they retain or lose the primary stain –
crystal violet, when subjected to treatment with
a decolorizing agent such as alcohol. Christian
Gram in 1884 discovered this technique.

7. •The difference between Gram positive &
Gram negative bacteria is based on the cell
wall composition. The cell wall of Gram
negative bacteria possess higher percentage
of lipids (10 – 20%) to that in the cell wall of
Gram positive bacteria (1 – 2 %).
•The peptidoglycan layer in cell wall of Gram
positive is thick than that of Gram negative
bacteria.
PRINCIPLE:

8. MECHANISM:
• The smear is subjected to Crystal violet – Primary
stain, Grams iodine – Mordant, Alcohol –Decolorizing
agent and Safranin – Counter stain.
• The primary stain (CV) forms a complex with the
mordant Gram’s iodine (CV-I complex) in the cell.
When the bacterial cells are treated with decolorizing
agent – alcohol, lipids from the cell wall of Gram
negative bacteria are extracted, this results in
increased porosity of the cell wall.

9. MECHANISM CONTINUITION:
• Therefore CV-I complex escapes the cell and thus
Gram negative organisms are decolorized. To render the
colourless Gram negative bacteria visible, counter stain
– safranin is applied where the cells take up the colour
of safranin, hence appear red in colour.
• In contrast, the cell wall of Gram positive bacteria
because of their low lipid content becomes dehydrated
during treatment with alcohol.
• The cells shrinked in size results in decreased pore size
and the CV-I complex cannot be extracted but it is
retained in the cell. Therefore , Gram positive cell
appear purple violet in colour.

10. Prepare a smear by the given culture on a clean sterile glass slide and
allow it to dry.
Flood the smear with crystal violet for 30 seconds and rinse with water.
Flood the smear with gram iodine (trapping agent) and rinse it with
water
Apply 95% ethanol as decolourising agent , gram positive bacteria
remain purple.
The counter stain safranin adds a pink colour to decolourised gram
negative bacteria without affecting the colour of gram positive bacteria.
Rinse with water, drain, blot and air dry. Examine under microscope.
PROCEDURE:

11. DIAGRAMATIC REPESENTATION
OF THE PROCEDURE:

12. RESULT AND CONCLUSION:
So, we can conclude that Gram staining is
important in differentiating Gram positive and
Gram negative bacteria in which the Gram
positive bacteria stained purple colour while
Gram negative organisms stained pink.
Escherichia coli is Gram negative while bacillus
sp., Staphylococcus aureus and actinomycetes
are Gram positive bacteria.

13. REFERENCES:
For the above presentation we have referred
the book- “Microbiology “, fifth edition
Written by- Michael J. Pelczar , JR.
-E.C.S. Chan.
-Noel R. Krieg.
Published by- Mc Graw Hill Education.
Page number: 65 - 68