Identification of bacterial: Microbiology

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
4.	Differential Staining
	a. Gram staining	primary 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 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

Table1: Staining technique for bacteria

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

Identification of bacterial: Microbiology