Staining and it's types. Procedure of various types of staining. Briefly discussed with detailed illustrations.

1.  Prepared by : Saikat Bagchi (18601916055) & Sahil Singh (18601916056)
 Prepared under the support and guidance of :
Mrs. Sumana Roy, Mrs. Priyanka Ray, Mrs. Prerona Saha, Mr. Dipanjan Mondal

2. S. NO. TOPIC NAME SLIDE NO.
1. Introduction to Microbiological Staining #3
2. Advantages Of Staining #4
3. Introduction to Dyes #5
4. Dyes and its types #6-7
5. Simple Staining #8-9
6. Differential Staining #10-13
7. Summary #14
8. Bibliography #15
9. Acknowledgement #16

3.  Staining:- Used in microscopy, it is a procedure in which a dye or reagent or a
combination of them is used to color the constituents of cells
and tissues to highlight the structures in biological tissues for
morphological and anatomical evaluation of microbial organisms.

4. 1) Why must staining techniques typically be employed in microbiology?
Ans) The cytoplasm of bacteria, fungi, protozoa, and other microorganisms is
transparent, and it would be very difficult to observe these organisms
without the benefit of staining.
2) What are the advantages of this procedure?
Ans) The advantages of this procedure are as follows:
a) The cells are made more clearly visible after they are coloured.
b) Differences between cells of different species and within the same
species can be demonstrated by use of appropriate staining solutions
(differential or selective staining).

5. Introduction to dyes : -
Definition: Chemicals containing chromophore groups (groups with conjugated double
bonds that give the dye its color) , i.e. a natural or synthetic substance used to add a color or
to change the color of something.
Classification:
NOTE: The chromophore is a region in the molecule where the energy difference between two separate molecular
orbitals falls within the range of the visible spectrum.
DYES
IONIC DYES NON IONIC DYES
BASIC
DYES
ACIDIC
DYES
COVALENT DYES
Results of a Gram stain. The Gram-positive cells (purple) are
Staphylococcus aureus; the Gram-negative cells (reddish-pink) are Escherichia coli.

6. IONIC DYES
Dyes that bind cells by ionic interactions are probably the most commonly used dyes. These
ionizable dyes may be divided into two general classes based on their nature of their
charged group.
1. Acidic Dyes: -
I. They possess groups such as carboxyl (-COOH), and phenolic hydroxyls (-OH)
II. In their ionized form, they have a negative charge and bind to positively charged cell structures.
III. Examples: Eosin, Rose Bengal, Acid Fuschin.
2. Basic Dyes: -
I. They are dyes with positively charged groups (usually some form of pentavalent nitrogen).
I. They bind to negatively charged molecules such as nucleic acids, proteins, and the surface of the bacterial
cells.
II. Examples: Methylene Blue, Basic Fuschin, Crystal Violet, Safranin, Malachite Green.
1

7. NON IONIC
DYES
1. Dyes that bind through their covalent bonds or because of their solubility characteristics
are known as non ionic dyes.
2. The principal advantage of using this dye is that the staining effectiveness of non
ionizable dyes remains unaltered and unaffected by pH change.
3. The nature and degree of the dye of the charge on the cell components remain
unchanged.
4. Examples: Schiff's Reagent(for detection of aldehydes), Sudan III (Sudan Black)( fat
soluble dye used to stain fat particles).
2

8. Simple Staining
 Definition : The coloration of bacteria by applying a single solution of stain to a fixed smear
is termed as simple staining.
1. The fixed smear is flooded with a dye solution for a specified period of time.
2. After which this solution is washed off with water and the slide blotted dry.
3. The cells usually stain uniformly.
4. However, with some organisms, particularly when methylene blue is used, some granules in the
interior of the cell may appear more deeply stained than the rest of the cell, indicating a different
type of chemical substance.
5. Basic dyes like crystal violet, methylene blue, and carbolfuchsin are
frequently used in simple staining to determine the size, shape,
and arrangement of prokaryotic cells.
1

10. DIFFERENTIAL STAINING
 Gram Staining: -
Staining procedures that make the differences between bacterial cells or parts of a bacterial cell are termed
differential staining.
A. This technique was introduced by Hans Christian Gram in 1884.
B. In this process, the fixed bacterial smear is subjected to the following
staining reagents in the order listed crystal violet, iodine, solution,
alcohol (decolorizing agent), and safranin or some other suitable
counterstain.
C. Bacteria stained by the Gram method fall into two groups: Gram
positive bacteria, which retain the crystal violet and hence appear deep
in color.
D. The Gram negative bacteria, which lose the crystal violet, are
counterstained by safranin, and hence appear red in color.
Hans Christian Gram
(1853-1938)

11. Procedure

12. 2. Application of
Carbol Fuschin
1. Heat fixation
3.Rinse the
slide with water

13. 4. Application of
alcohol(decolorizer)
5. Rinse the slide
with water
6. Apply
counterstain

14. SUMMARY OF STAINING BASED ON THEIR PRINCIPLE OF APPLICATION

15.  BIBLIOGRAPHY
1. Preparation and staining of specimens, Pages(25-28); PRESCOTT,
HARLEY, & KLEIN’S MICROBIOLOGY, SEVENTH EDITION, Published by
McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc.
2. Microbial size and Microscopy, Pages(26-32); SCHAUM’S OUTLINE OF
MICROBIOLOGY, SECOND EDITION, I. Edward Alcamo, Ph.D., Jennifer
M. Warner, Ph.D.
3. Bacteria, Pages(27-30); Pharmaceutical Microbiology, Edited By W.B.
Hugo, A.D. Russell, Sixth Edition.
4. Bacterial Morphology and Staining, Pages(31-71); Laboratory Exercises in
Microbiology, Harley Prescott, Fifth Edition.