Identification of bacterial: Microbiology

Staining techniques in Microbiology

S. NO.Staining techniqueDyeApplication
1. Simple StainingMethylene blue/ basic FuchsinImpart same colors to all bacteria
2.Negative StainingIndia Ink/ NigrosinDemonstration of bacterail capsules
3.Impregnation methodSiverDemonstration of bacterial flagella & Spirochaetes
4.Differential Staining
a. Gram stainingprimary stain: crystal violet;
Gram’s iodine (mordant);
decolorizing agent (ethanol/ acetone);
Secondary counterstain:safranin
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 stainingCarbol fuchsin; 20 % Sulphuric acid (decolorization); Counterstain-methylene blueStaining of Mycobacteria (tubercle & lepra bacilli); acid fastness is due to high lipid content Mycolic acid
c. Alberts stainsAlbert II iodine solutionStaining of Corynebacteria diphtheriae
Table1: Staining technique for bacteria

Following various staining techniques are used for the study of bacteria.

Staining techniques

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

a. Gram staining read more...

b. Acid fast/Ziehl -Nelson staining read more…

c. Albert’s staining read more…

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