Procedure of Simple Staining: To study size, shape, arrangement, and properties and differentiate specific groups of microorganisms, biological stains are used. Stain is an organic compound containing a benzene ring with chromophore and auxochrome group. Different staining techniques are used for the visualization, differentiation, and separation of bacteria in terms of morphological characteristics and cellular structures.
Procedure of Simple Staining
In simple staining, the bacterial smear is stained with a single stain e.g. methylene blue, crystal violet, carbol fuchsin safranin, etc. Basic stains with a positively charged chromogen are used. Bacterial nucleic acids 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 and arrangement of bacterial cells.
The surface of a bacterial cell has acidic characteristics because of a large number of carboxyl groups located on the cell surface due to acidic amino acids. Therefore, when the ionization of carboxyl groups takes place, it imparts a negative charge to the cell surface.
In nature, H+ is replaced by another positively charged ion. e.g. Na+ or K+ and H+ bond with oxygen to form water. Thus, the surface of an unstained bacterial cell is represented as shown below.
Basic dyes are commonly used for monochrome staining. These dyes are available as a salt of acids. e.g. methylene blue chloride. When methylene blue rehydrates, it ionizes to form methylene blue and chloride ions. The positively charged ions have the coloring property.
In addition to methylene blue for staining, the exchange of MB+ with Na+ on the bacterial cell surface takes place, resulting in ionic bond formation between MB+ and the cell surface. Thus, when the coloring agent forms an ionic bond with cell or cell components, it results in the staining of the cell. The most commonly used basic stains are methylene blue (2 to 3 minutes), crystal violet (1 to 2 minutes), and carbol fuchsin (15 to 30 seconds).
Make sure you also check our other amazing Article on : Preservation of Cultures