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Renin Angiotensin Mechanism.

Identification of bacteria using staining techniques: Introduction, Simple Staining.

 

Introduction:

  • To study size, shape, arrangement of bacterias and their differentiation biological stains are used.

  • Staining is a supplementary method that gives divergence to the microscopic image for better vision under the microscope.

  • Stain can be defined as a chemical reagent or dye that imparts color change to the specimen.

Objectives of Staining:

  • Enables us to see the organism better: 

    • As microorganisms are a very minute creature as well as transparent, so it makes the specimen easy to identify.

  • Helps to differentiate organisms: 

    • Some microbes retain the colour of stain, and some don’t, which helps to distinguish between two different groups of organisms.

  • To identify a particular structure: 

    • For further study of microorganisms, it is also important to study the various internal and external structures of organisms like flagella, capsule, nucleus, spores etc.

Mechanism of Staining:

  • Stain generally consists of “Chromogen” and “Auxochrome”.+

  • Chromogen: When the benzene ring reacts with the chromophore group, then the compound which is formed is called chromogen. 

  • The chromophore is a coloured compound that contains the unsaturated group.

  • Auxochrome: These are the specific groups that impart a positive and negative charge to the chromogen and are capable of ionising it.

  •  After this, the ionised stain binds to the cell with opposite charges.

  • Therefore, when the auxochrome group is present in the chromogen “A dye or stain is formed”.

  • Then this colouring agent that stains the biomolecular constituents of a specimen such as protein, as well as cellular parts that have to be dyed, depends upon the electrical charge found on the chromogen portion that imparts colour to the specimen.

Protocol of Staining:

  • Staining generally involves three steps:

  1. Preparation of smear

  2. Fixation of smear

  3. Staining of the specimen

Types of Staining:

  1. Simple Staining:

    1. Direct Staining

    2. Indirect Staining

  2. Differential Staining:

    1. Gram Staining.

    2. Acid Fast Staining.

  3. Special Staining:

    1. Capsule staining

    2. Endospore Staining.

    3. Flagella Staining.

Simple Staining:

  • It determines the cell shape, size and arrangement of the microorganism. 

  • Simple method to perform. 

  • To perform this staining, it requires the use of a single stain only. 

  • These are of two types,

    • Direct Staining

    • Indirect Staining.

Direct Staining:

  • A single basic stain is used.

  • e.g. Methylene Blue, Crystal Violet, Carbol Fuschin, Safranin etc.

  • These stains have a “Positive Charge” on their structures.

  • Principle:

    • Cell wall of bacteria contains a lot of -COOH groups from amino acids which on dissociation gives negatively charged -COO- ions.

    • -COOH----------> -COO-  + H+ .

    • The positively charged stain gets combined to the cell components with negative charge imparting color to the structure of the specimen.

  • Stains the cell against a transparent background.

Indirect Staining:

  • Also called “Negative Staining”.

  • A SIngle acidic stain is used.

  • e.g. Eosin, Nigrosine, india ink, congo red.

  • These stains have a “Negative Charge” on their structures.

  • Principle:

    • As these stains have negative charge on their structure they don't react with the cell structures they just get deposited around the cell and hence form a colored background highlighting the transparent cells.

  • Stains the background highlighting the transparent cells.

  • This technique is also useful for demonstration of bacterial capsules.

  • Does not require fixation, as the chemical does not interact with the cell there is no harm to the bacterial cell.
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