Staining methods. various staining methods according to there identification and visualisation

1. Staining Techniques
Prepared by
Mr. Sakhare V. G.
Assistant Professor
SBSPMs B. Pharmacy College Ambajogai

2.  Identification of bacteria using staining techniques ( simple, Grams & Acid fast
staining )
 Staining means the identification of bacteria.
 There are different types of bacteria which can not see with naked eyes so we
used microscope to see them and use dye solution ( as an indicator for
identification of bacteria)
 Staining is technique used in microscopy to enhance contrast in the
microscopic image.
 Stains and dyes are frequently used in biological tissues for viewing, often with
the aid of different microscopes.
 Stains may be used to define and examine bulk tissues (highlighting, for
example, muscle fibers or connective tissue), cell populations (classifying
different blood cells, for instance), or organelles within individual cells.
Bacteria have nearly the same refractive index as water, therefore, when they
are observed under a microscope they are opaque or nearly invisible to the
naked eye.
 Different types of staining methods are used to make the cells and their
internal structures more visible under the light microscope.

3. What is stain ?
 A stain is a substance that adheres to cell giving the cell color.
 The presence of color gives the cell significant contrast so are much more
visible. Different stains have different affinities for different microorganism, so
different parts of organism.
 They are used to differentiate different types of organisms or to view specific
parts of organisms.
Staining Reaction
 To study the shape, Size, Arrangement and properties & Differentiate specific
groups of microorganism biological stains are used. The stain is an organic
compound containing benzene ring with Chromophore or Auxochrome.
1. Chromophore: Its part of molecule which is exposed to light/Visible light will
absorb and reflect certain color.
2. Auxochrome: Auxochrome is a group of atoms which is functional & has
capability to alter the capacity of the Chromophore to reflect the color.
 Different staining techniques are used for Visualization, Differentiation
and separation of bacteria in terms of morphological characteristics and
cellular structure.

4. Why we need to stain Bacteria?
 The bacteria are transparent and colorless, so they would be invisible to naked
eyes if observed under a microscope thus bacteria should be stained with
certain dyes in order to visualize bacterial cell or their internal structure using
the light microscope.
Dyes
 Dyes or stain organic compound in the form of salt, Composed of Positive and
negative ion one of these ions are responsible for colored called chromogen.
1. Acidic Dyes ( Anionic Dyes) : In which the chromogen is negative ion
(Anion). The acidic dye bind to positively charged cell structure like Some
protein used to provide background staining like capsule staining.
Acidic Dye has in the form Na+ Dye.
Ex. Eosin, Nigrosin, Acid Fuschin, India Ink, Picric acid Etc.
2. Basic Dyes ( Cationic Dyes) : In which the chromogen is positive ion
(Cation). Basic dye has in form of Dye+ Cr-.
Ex. Crystal Violet, Methylene blue, Safranin, Basic fuschin, Etc.
3. Neutral Dyes: It is complex mixture of Acidic dyes with Basic dyes. The
Neutral dye stain nucleic acid and cytoplasm.
Ex. Eosinate of Methylene blue and Geimsa stain.

5. Classification of staining methods

6. Simple staining
 In these staining we observe the morphological characteristics such as Shape
and size of bacteria.
 In these staining the single stain dye such as crystal violet Methylene blue,
Carbol fuchsin, Safranin, Malachite green is used.
 In simple staining the bacterial smear is stained with single stain the basic
stain with positively charged chromogen are used.
 The bacterial nucleic acid and certain cell wall components carry a negative
charge that strongly binds to the cationic chromogen.
 The purpose of simple staining is to elucidate the morphology of bacterial cell.
The basic dyes are commonly used for monochrome staining these dyes are
available in salts of acids.
Ex. Methylene blue chloride.
 When Methylene blue rehydrates, it ionizes to form methylene blue and
chloride ion. The positively charged ions having the coloring property.
MB. Cl MB+ + Cl-
Methylene Blue Chloride Methylene Blue
Chloride

8. Firstly take glass slide, Cover slip, Loop & Culture Media and Microscope
All Equipments are washed using Ethanol and drying under flame sterilization
Inoculum loop streak on culture media and bacteria attached to Loop
Streak on glass slide
Now add some drops of Indicator or dyes on surface of slide Eg. Crystal Violet
Allow to dry slide & then wash the slide under tap water for removing excess stain
Put the Coverslip on stain & Placed on the Microscope
Now see the bacteria under microscope and observed the size& shape of bacteria
The Blue colored spherical shape or Rod shaped bacteria is seen

9. Negative Staining
 In these staining we also observe the natural size and shape of bacteria.
 In these staining Eosin, Nigrosin is used as stain and smear is prepared.
 In this method the bacteria are stained with a single stain of Acidic stain and
smear is prepared.
 The acidic stain (negative chromogen) does not penetrate the cell because of the
negative charge on the surface of bacteria hence unstained cells are easily
observed against the colored background.
 This technique is also useful for demonstration of Bacterial cells.
Eg. Eosin, Nigrosin, Congo red, Rose Bengal stain are acidic stain used in
Negative stain.
Advantages
 It's a direct method and positive staining for study of morphology of cells. This
is because heat fixation is not required for the cells that not receive vigorous
physical or chemical treatments. Hence natural size and shape of
microorganisms can be seen by this method.
 It is possible to observe bacteria that are difficult to stain Eg. Spirila.

10. Firstly take glass slide, Cover slip, Loop & Culture Media and Microscope
All Equipments are washed using Ethanol and drying under flame sterilization
Inoculum loop streak on culture media and bacteria attached to Loop
Streak on glass slide
Now add some drops of Indicator or dyes on surface of slide ( Eosin)
Allow to dry slide & then wash the slide under tap water for removing excess stain
Put the Coverslip on stain & Placed on the Microscope
Now see the bacteria under microscope and observed the size& shape of bacteria
The acidic stain does not penetrate the cell because of the negative charge on the
surface of bacteria hence unstained cells are easily observed against the colored
background.

11. Gram Staining/ Differential Staining
 The gram staining is one of the most important and widely used for the
differentiation and identification of bacteria.
 It is invented by Hans Christian gram in 1884.
 It is not reveals the size and shape of bacteria but also used to differentiate
into Gram positive and Gram negative cells i.e. means identified Gram
positive bacteria and Gram negative Bacteria.
 In these technique use of two or more dyes or stain at a time.
 In this process the bacterial smear is subjected to staining reagent such as
Crystal violet, Grams Iodine, Alcohol and Safranin.
Requirements
1. Glass slide, Cover slip, Watch glass, Nichrome wire loop
2. Culture Media( In which bacteria is present)
3. Stains or Dyes: Crystal violet, Grams Iodine, Safranin
4. Chemicals: Alcohol and Acetone

12. Important Terms
1. Crystal Violet: Basic stain
2. Acetone and Alcohol : Decolorizer ( 95%)
3. Safranin : Counter stain ( Polar dye) (0.25%)
4. Mordant Grams Iodine: Fix the crystal violet dye

13. Firstly take glass slide, Cover slip, Loop & Culture Media and Microscope
All Equipments washed With Ethanol &Drying under flame for complete sterilization
Now take Inoculum loop streak on culture media and bacteria attached to Loop
Streak on glass slide and fix the Smear
Add drop of Crystal violet dye (Primary stain) and allow to dry & after 1 min wash slide
Then add Grams Iodine solution which fix the crystal violet dye with cell wall
After 1 min wash the slide all Bacteria Dark Violet & Purple Color
Wash with Ethanol or Acetone (95%)
Dark Violet Bacteria
( Retain the stain )
Colorless Bacteria
( loss the stain)
Add Safranin(0.25%) & stain for 1 min
Dark Violet cell Color
Gram Positive Bacteria
Pink cell Color
Gram Negative Bacteria

15. Examples of Gram Positive and Gram Negative cell
 Gram Positive Bacteria
Cocci: Staphylococcus, Streptococcus, Enterococcus
Bacilli : Bacillus anthracis, Clostridium tetani, Actinomyces, Nocardia
 Gram Negative Bacteria
Cocci: Neisseria, Moraxell
Bacilli : Escherichia Coli, Salmonella Typhi, Shigella, Yersinia, Pseudomonas,

16. Acid Fast Staining
 Acid fast staining is also known as Ziehl Neilson staining discovered by the
Paul Ehrlich in 1883.
 It is Differential staining (use of two or more dyes)
 It is used for those which is wax like and impermeable cell wall(Does have
cell wall)
 The microorganism does no identified by gram staining so used acid fast
staining because it does not have cell wall.
 It is categorized into Acid fast stain and Non acid fast stain.
 Gram resistant ( waxy like or waxy cell walls) Non motile, Pleomorphic rod,
related to Actinomyces.
Requirements:
1. Glass slide, Coverslip, Watch glass and Nichrome wire loop.
2. Culture Media( In which bacteria is present)
3. Carbolfuchsin stain
4. Deionized water and Alcohol ( Acid alcohol contains 3%HCL & 95% Ethanol)
5. Methylene Blue ( Counter stain )

17. Firstly take glass slide, Cover slip, Loop & Culture Media and Microscope
All Equipments are washed using Ethanol and drying under flame sterilization
Inoculum loop streak on culture media and bacteria attached to Loop
Streak on glass slide and smear it
Now add some drops Carbol Fuchsin on surface of Bacteria
Allow to dry slide & then wash the slide with using Ethanol for Decolorisation
Put the Coverslip on stain & Placed on the Microscope
Now Red color to bacteria ( Acid fast Stain Bacteria)
Non colorized bacteria ( Non-Acid fast Stain Bacteria)
Add methylene Blue
Show Non acid fast stain
If bacteria give
Red or Pink Color
Acid fast stain
If bacteria give
Blue Color
Non Acid fast stain

18. Ex. Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium Leprae, Mycobacterium africanum