Staining-the basic concept

Staining

Microorganism are very minute, generally they are colorless and transparent so it is very difficult to focus through microscope. The use of special types of microscopy such as phase contrast and dark field techniques is design to improve visualization; however these techniques require special microscopes and training. In common purpose we use light microscope for direct observation of bacteria, to observe through this we have to increase the contrast of the organisms against their background is necessary, to do this microorganism should be colored against their background i.e  application of staining techniques.

Staining: It is the process of coloring of microbial cells and their parts with the application of a dye or dyes to a fixed smear.

Stains or dyes: Yhey are coloring compounds, chemically they are salt as they contain acidic or basic part of stain. Microbiologists mostly use aniline or synthetic dyes. Each dye molecule has two functional groups, the auxochrome and chromophore.

•       Auxochrome: It ionizes and gives the molecules the ability to react with substrate.

•       Chromophore: It is site of unsaturation and absorbs specific wavelength of light. The color of the solution obtained is that of the  unabsorbed light which transmitted through it.

The synthetic dyes be classified as Acidic, Basic and Neutral dyes depending on whether the coloring bearing compound is a cation and an anion.

•       Acidic dyes: Anionic dyes, which react with substrate group which ionize to produce positive charge such as corboxyl, phenolic or sulfydryl etc. Anionic dyes are usually found as sodium. Eg: Nigrosin, Congo red, Eosin, Acid fuchsin etc.

•       Basic dyes: Cationic dyes, which react with substrate group which ionize to produce negative charge such as ammonium ions. Anionic dyes are usually found as chlorides or sulfates. Eg: Methylene Blue, Crysal Violet, Safranin etc.

•       On the basis of number of dyes and staining techniques used, staining are of following types;

•       1:   Simple staining: In this staining only single dye is used. Morphology of bacteria, yeast and mold are observed. Generally, positively charged chromophores are used in this staining. Methylene blue, Basic fuchsin, Crystal violet etc are used.

•       2:            Negative Staining: It is the techniques where the background is stained leaving the bacterial cell unstained due to electrostatic repelling between same charged cell and stain used. Heat fix is avoided so the accurate size, shape and arrangement can be measured and studied. Bacteria which are hard to stain like Spirochaetes, Mycobacterium and Nocardia are used in this techniques. Capsule staining is one of this techniques. Stains nigrosin, congored, eosin, acid fuchsin are used.

•       3:            Differential Staining: In this techniques more than one dye is used. It is applied in categorization of bacteria into two groups. It stains specific structure of cell eg: flagella, spore, capsule etc. Gram staining categorizes bacteria into two groups i.e gram positive and gram negative.