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.
