Staining techniques in Microbiology
|S. NO.||Staining technique||Dye||Application|
|1.||Simple Staining||Methylene blue/ basic Fuchsin||Impart same colors to all bacteria|
|2.||Negative Staining||India Ink/ Nigrosin||Demonstration of bacterail capsules|
|3.||Impregnation method||Siver||Demonstration of bacterial flagella & Spirochaetes|
|a. Gram staining||primary stain: crystal violet;|
Gram’s iodine (mordant);
decolorizing agent (ethanol/ acetone);
|To find out gram-positive bacteria (resist decolorization & retain the color of primary stain i.ee violet)& gram-negative bacteria (decolorized by acetone/ethanol & therefore take counterstain i.e. safranin & appear Red)|
|b. Acid fast staining||Carbol fuchsin; 20 % Sulphuric acid (decolorization); Counterstain-methylene blue||Staining of Mycobacteria (tubercle & lepra bacilli); acid fastness is due to high lipid content Mycolic acid|
|c. Alberts stains||Albert II iodine solution||Staining of Corynebacteria diphtheriae|
Following various staining techniques are used for the study of bacteria.
A. Unstained/wet preparation
These preparations are used to study bacterial motility (Example hanging drop preparation) & for demonstration of spirochaetes ( dark ground microscopy).
B. Staining preparations for bacteria
Due to lack of contrast, the structure of bacteria cannot be seen under a microscope. Hence to produce Colour contrast, staining methods were developed.
In the staining technique, bacterial culture is fixed on the slide with heat by flaming underneath slide. Fixed smear is stained by various following staining technique.
- 1. Simple staining,
- 2. Negative staining
- 3. Impregnation method
- 4. Differential staining
1. Simple staining
Simple stain imparts same colour to all the bacteria in smear
Example with Thailand blue and basic fuchsin dye.
2. Negative staining
In this technique, background gets stained and unstained bacteria stand out in contrast under microscope.
Example: negative staining is used to demonstrate bacterial capsule (since capsule do not take stain)
3. Impregnation method
The very thin structure of bacteria is thickened by the impregnation of silver to make them visible under a microscope. Example demonstration of bacterial flagella and spirochaetes.
4. Differential staining
As name indicates these technique imparts different colour to different bacteria or bacterial structure. Differential stain technique includes the following technique
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