The document outlines the gram staining technique, which differentiates bacteria into gram-positive and gram-negative based on their cell wall composition. Developed by Hans Christian Gram in 1884, the method utilizes specific reagents such as crystal violet, iodine, and safranin to achieve color differentiation. Applications include diagnosing diseases and selecting culture media appropriate for the bacterial type.

1. GRAM STAINING
TECHNIQUE
By Mehak Saeed
MSc. Biotechnology
University Of Karachi

2. INTRODUCTION
Gram staining
The Gram’s staining is an important technique in microbiology, used to differentiate
between Gram-positive organisms and Gram-negative organisms.

3. HISTORY
➢ In 1884, Danish scientist “Hans Christian Gram” developed the technique while
working with “Carl Friedlander” in the morgue of the city hospital in Berlin.

4. REAGENTS USED IN GRAM STAINING
➢ Primary stain:
CrystalViolet
➢ Mordant:
Iodine
➢ Decolorizer:
Alcohol and Acetone(95%)
➢ Counter stain:
Safranin

5. PRINCIPLE
➢ When the bacteria is stained with crystal violet and fixed by mordant, some bacteria
retain the primary stain while some are decolorized by alcohol.
➢ Gram-positive bacteria have thick layer of peptidoglycan and low lipid content. So,
ethanol cannot remove the crystal violet-iodine complex bound to cell wall and bacteria
appears blue or purple in color.
➢ Gram-negative bacteria have thin layer of peptidoglycan and high lipid content.The
decolorizer ethanol, dissolves the lipids in the cell wall and allows crystal violet-iodine
complex to leached out of the cell. So, bacteria appear red in color when stained with
safranin.

6. APPLICATIONS OF GRAM STAINING
➢ To differentiate between gram-positive and gram-negative bacteria.
➢ To determine the chemical composition of cell wall of bacteria.
➢ To select suitable culture media based on gram stain findings.
➢ To rapid presumptive diagnosis of diseases of such as bacterial meningitis.

7. PROCEDURE

8. RESULT INTERPRETATION
 Gram-positive:
Blue/purple color
 Gram-negative:
Red color

9. EXAMPLES
➢ Gram-positive Bacteria:
Actinomyces , Bacillus , Clostridium , Enterococcus ,
Corynebacterium, etc.
➢ Gram-negative Bacteria:
E.coli , Salmonella , Shigella , Pseudomonas , Helicobacter ,
Moraxella , etc.

10. Thank You