Bacterial Staining Techniques,Types of Stains,Gram Staining

1. BACTERIAL STAINING
TECHNIQUES
BY :
SREETAMA MOITRA
B.PHARM (3rd SEM)
GURUNANAK INSTITUTE OF PHARMACEUTICAL SCIENCE AND
TECHNOLOGY

2. CONTENTS
• Definition of staining
• Types of stains
• Principles of staining
• Different types of staining
• Advantages of staining
• Disadvantages of staining
• Conclusion
• References

3. Definition of staining
 Bacteria are microscopic organisms.They are also
colourless for the most part.In order to visualize them
to study their structure,shape and other structural
characterics,it becomes necessary to make them more
easily visible.
 Staining is a process where we use different dyes to
color the living or dead microorganisms.

4. Types of Stains
 Stains/dyes - organic compounds which
carries either positive charges or negative
charges or both.
 Types
 Basic stain/dyes – charge on the dye ion is +ve.
 Acidic stain/dyes – charge on the dye ion is -ve
 Neutral stain/dyes – complex salt of a dye acid with
a dye base.

5. Principles of Staining
• The essential steps in the preparation of a fixed
stained smear are –
1)Preparation of the smear
2)Fixation
3)Application of one or more staining solutions

7. Gram Staining
• Gram staining is a method of differentiating
bacterial species into two large groups (Gram
positive and Gram negative)
• This technique was introduced by Christian
Gram in 1884.

8. METHOD CONSISTS OF FOUR
COMPONENTS :
•Primary stain— Crystal violet
• Mordant— Iodine Solution
• Decolourizer— Alcohol
•Counter stain— Safranin

9. Steps of Gram Staining

10. Gram-positive bacteria
• Have a thick peptidoglycan
layer surrounds the cell.
•Retain the color of the primary
stain(crystal violet) after
decolorization with alcohol.
Gram-negative bacteria
•Have a thin peptidoglycan layer
that does
•not retain crystal violet stain.
Instead, it has a thick lipid layer
which dissolved easily upon
decolorization with Acetone-
Alcohol.
•Therefore, cells will be
counterstained with safranin and
turned red.

11. Result of Gram Staining

12. Acid Fast Staining
• Used to identify Mycobacterium tuberculosis
and M. leprae.
• These bacteria have cell walls with high lipid
content; in particular, mycolic acid.
• They can be stained by harsh procedures which
uses heat and phenol to drive basic fuchsin into
the cells.
• Once basic fuchsin has penetrated, M.
tuberculosis and M. leprae are not easily
decolorized by acid alcohol.

13. Advantages of Staining
Procedure
 Better Visualization
 Identification and Classification
 Identification of Cellular Structures
 It is simple and cost-effective

14. Disadvantages of Staining
Procedure
 Inability to determine motility, distortion of cell
size/shape
 Through simple staining, we cannot classify a
particular type of organism.
 It does not give much information rather than the
morphological characteristics of bacteria.

15. Conclusion
Therefore, we can conclude that a simple staining
method is the easiest way to colour the microscopic
object as it uses a single basic stain. The results of
simple staining are based on the type of basic stain
that has been used.
On the other hand differential staining helps us to
know the differences between cells of different
species and also within the same species.

16. REFERENCEs
• Pelzar, Chan Kreig, Microbiology, Tata
McGraw Hilledn.,5th edition,Chapter-4 :The
microscopic examination of
microorganisms,Pg-65-68
• Prescott Harley Klein's Microbiology 7th
Edition, Chapter-2 : The Study of Microbial
Structure: Microscopy and Specimen
Preparation, Pg-25-28