- Hans Christian Gram developed the Gram staining technique in 1884 while examining lung tissue from pneumonia patients under the microscope. He discovered that certain bacterial cells retained dye differently than others. - Gram staining is a common differential staining technique that classifies bacteria as either Gram positive or Gram negative based on differences in their cell wall structure. Gram positive bacteria have a thick peptidoglycan layer that retains the primary stain, while Gram negative bacteria have a thin layer and outer membrane that washes away the primary stain. - The Gram staining procedure involves staining a smear with crystal violet, adding a mordant, decolorizing with alcohol, and counterstaining with safranin. Gram positive bacteria appear purple/blue

1. Dr.Yogesh Bhagrava Sir
By Adil Ali
Msc Microbiology 1st Semester
Submitted To
Gram Staining

2. Content
 History
 Introduction
 Principle
 Requirements
 Reagent
 Functions of Reagent
 The Gram stain procedure
 Result
 Precautions
 Reference

3. Gram staining developed by
Hans Christian Gram,a
Danish Doctor working in
Berlin
While examining lungs tissue from a patients who
had died of pneumonia, he discovered that certain
stains were preferentially taken up and retained by
bacterial cells.
Gram devised his technique to enable bacteria to be
seen more readily in stained section of lung tissue.
He published his method in 1884, and included in his
short report the observation that the typhus bacillus did
not retain the stain.
Hans Christian Gram
1853-1938
History

4. Introduction
Gram Staining is the common, important and most used
differential staining techniques in microbiology.
This test differentiate the bacteria into Gram Positive
and Gram negative Bacteria, which helps in the
classification and differentiations of microorganisms.
Gram staining involves four process staining with a
water-soluble dye called Crystal Violet, Gram Iodine,
Decolorization and counterstaing usually with
safranin.

5. Principle
When the bacteria is stained with primary stain Crystal Violet and
fixed by the mordant, some of the bacteria are able to retain the
primary stain and some are decolorized by alcohol.
The cell walls of gram positive bacteria have a thick layer of
protein-sugar complexes called peptidoglycan and lipid content is low.
Decolorizing the cell causes this thick cell wall to dehydrate and
shrink, which closes the pores in the cell wall and prevents the stain
from exiting the cell.
So the ethanol cannot remove the Crystal Violet-Iodine complex that
is bound to the thick layer of peptidoglycan of gram positive bacteria
and appears blue or purple in colour.

6. In case of gram negative bacteria, cell wall also takes up the CV-
Iodine complex but due to the thin layer of peptidoglycan and thick
outer layer which is formed of lipids, CV-Iodine complex gets washed
off.
When they are exposed to alcohol, decolorizer dissolves the lipids in
the cell walls, which allows the crystal violet-iodine complex to leach
out of the cells. Then when again stained with safranin, they take the
stain and appears red in color.

7. Requirements
 A clean grease free slide
 Bacterial cell suspension
 Tissue Paper
 Nichrome wire loop
 Spirit lamp/Bunsen burner
 Oil immersion Microscope

8. Reagents
 Primary Stain (Crystal Voilet)
 Mordant (Gram’s Iodine)
 Decolorizing (Alcohol 95%)
 Counterstain (Fuschin or Safranin)

9. Functions of Reagents
 Crystal Violet - It is a primary stain and a basic dye it stains all
Microorganisms.
 Gram’s Iodine – Gram’s Iodine acts as a mordant and it forms a complex
with crystal violet that is CV-I complex. This complex increase affinity
between cell and stain.
 Alcohol – It is a decolorizing agent as well as a lipid solvent. It tries to
decolorize the cell by removing the CV-I complex from the cell.
 Fuschin or Safranin – It acts as a counterstain. It stains the cells that are
decolorized by alcohol. Only gram negative bacteria get decolorize and
this counterstain gives pink to these cells.

10. The Gram Stain Procedure
 Take a clean grease free slide.
 Let the smear should be air dried.
 Heat fixation is mostly used to a fix the bacteria to the slide so that
they don’t rise out during the staining procedure.
 Prepare the smear with the help of nichrome wire on the clean glass slide.

11.  Primary Staining: The smear is covered with crystal violet,
for one minute and wash with water.

12.  Mordant: It is then covered with Gram’s iodine, kept for 1
minute, and washed with water.
Crystal Violet – Iodine Complex (CV-I complex)

13. Decolourization: The smear is covered with alcohol and is washed
with water immediately with in 5second.

14. Counter staining: The smear is then covered with safranin, kept
for 30 seconds and wash with water.
• Let it dry the smear
• Mount the smear
• Observe under oil immersion microscope

17. Result
GRAM POSITIVE: Blue/Purple Color
GRAM NEGATIVE: Red/Pink Color

18. Examples
G+Ve G-Ve
Streptococcus pneumoniae Salmonella Typhi
Mycobacterium tuberculosis E.Coli
Difference between Gram positive and Negative bacteria
Gram positive bacteria (G+) have cell walls that contain thick
layers of peptidoglycan (90% of cell wall). These stain purple Gram
negative bacteria (G-) have walls with thin layers of peptidolyacn
(10% of cell wall), and high lipid content.

20. Precautions
 Gram stain materials will stain skin and other materials.
 Wear chemical splash goggles and chemical-resistant gloves.
 Wash hands thoroughly with soap and water before leaving the
laboratory.
 Follow all laboratory safety guidelines. Please review current Material
Safety Data Sheets for additional safety, handling and disposal
information.
 Check the reagent for the expire date before the using.
 Care should be taken when sterilizing the inoculating loop and
when flame-fixing slides.

21. Reference
 Infection,Immunity and Inflammation – University of Mosul
 Bio Practicals – Nagaraj Balasubramanian
 Stains & Staining – Dr. Rakesh Sharda
 Microbiology – Pelczar
 Jotscroll.com
https://www.jotscroll.com/gram-stain-procedure-gram-staining-
steps-and-results
 Microbiology info.com
https://microbiologyinfo.com/gram-staining-principle-procedure-
interpretation-examples-and-animation/
 Slideshow
 FLINN SCIENTIFIC BIO FAX

22. Thank you