Gram staining is a microbiological technique to differentiate between gram-positive and gram-negative bacteria.

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