This document discusses Gram staining of bacteria. Gram staining is an important differential staining technique that classifies bacteria into two groups - Gram positive and Gram negative - based on their cell wall structure. Gram positive bacteria appear violet after Gram staining due to their thick peptidoglycan layer retaining the crystal violet dye, while Gram negative bacteria appear red as their thinner peptidoglycan layer is decolorized, revealing the safranin counterstain. The document outlines the reagents, steps, and expected colors of the Gram staining procedure and compares the cell wall structures of Gram positive and Gram negative bacteria.

2. SARDAR VALLABHAI PATEL UNI
OF AGRICULTURE AND
TECHNOLOGY
PRESENTATION ON GRAM STAINING
OF BACTERIA
SUBMITTED BY:
ANUPAM KUMAR
SUBMITTED TO:-
PROF. KAMAL KHILADI
DEPTT- NEMATOLOGY

3. 3

4. 4
Gram StainGram Stain::
It is the most importantIt is the most important
differential stain used indifferential stain used in
bacteriology becausebacteriology because
it classified bacteria intoit classified bacteria into
two major groupstwo major groups::
a)a)Gram positiveGram positive::
Appears violet after
Gram’s stain
b)b) Gram negativeGram negative::
Appears red after
Gram’s stain
GRAM
STAIN

5. OBJECTIVES
 Describe reagents used in Gram stain &
purpose of these reagents.
 Color expected of Gram Pos & Gram Neg
after performing the procedure.
 Explain reason of differential stain by Gram
Pos & Gram Neg
 Describe cell wall structure of Gram Pos &
Negative bacteria.

6. REAGENTS USED IN GRAM STAIN
1. Gram Crystal Violet 0.5%
2. Gram Iodine
a. Potassium Iodide 2%
b. Resublimed Iodine 1%
3. Gram Decolorizer
a. Methanol 80%
b. Acetone 20%
4. Gram Safranine 1%
Gram negative
Gram positive

7. REAGENTS USED IN GRAM STAIN
1. CRYSTAL VIOLET
• Primary stain.
• Violet colored, stains all micro-organisms.
2. GRAM IODINE ( substance that combine with stain & there by fixes it in
material).
• Mordant
• Forms Crystal violet iodine complexes.
3. DECOLORIZER
• Acetone + Methanol
• Removes Crystal violet iodine complex from thin peptidoglycan layers
• Dissolves outer layer of Gram negative organism.

8. Time Frame
1) 1 minute
2) 1 minute
3) 15 seconds
4) 1 minute
Rinse with water between each step
STEPS IN GRAM STAINING

9. The Gram Stain Procedure
Step 1 - Prepare a Smear
Watch what happens to the “Bacteria” at each step
“Bacteria”
 Suspend some of the material to be stained in a
drop of water on a microscope slide,
 spread the drop to about the size of a nickel.
 Allow to air dry.
 Heat fix by gently warming

10. The Gram Stain Procedure
STEP 2- CRYSTAL VIOLETthe Primary
Stain
 Flood the Smear with Crystal Violet
 Allow to stand for 1 min.
 Rinse with water to remove excess stain.

11. The Gram Stain Procedure
Step 3 - Apply the Mordant
 Flood the Smear with Iodine solution.
 Allow to stand 2 mins.

12. The Gram Stain Procedure
Step 4 - Rinse
Rinse with water to remove excess Iodine

13. The Gram Stain Procedure
Step 5 - Decolorize
 Drip Decolorizer (80% Methanol +20% Acetone)
across the slide about 5 sec
 The effluent should appear pale or clear

14. The Gram Stain Procedure
Step 6 - Rinse
Rinse with water to remove excess alcohol

15. The Gram Stain Procedure
Step 7 - Counterstain
 Flood the slide with Safranin solution
 Let stand for 2 minutes

16. The Gram Stain
Step 8 - Rinse, Dry and Observe
Gram-Positive Gram-Negative
 Rinse with water to remove excess stain.
 Blot dry
 Observe under Oil Immersion

17. CELL WALL OF GRAM POS & NEG

18. CELL WALL IN GRAM +VE AND
GRAM –VE BACTERIA
Cell Wall Structures Gram Positive
organisms
Gram Negative
organisms
Inner cytoplasmic membrane Present Present
Peptidoglycan layer Thick Thin
Teichoic Acid Present Absent
Outer membrane layer Absent Present
Lipids & lipoprotein content Low High
Peri-plasmic space Absent Present