MONOCHROME STAINING OR SIMPLE STAINING.
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MONOCHROME STAINING OR SIMPLE STAINING |
Aim:
To observe the morphology and arrangement of
bacterial cells by monochrome staining.
Theory:
Bacteria are very minute in size so can be observed by only using a microscope. In unstained preparation, microorganisms
appear colourless. So by staining bacterial cells, bacteria can be observed as coloured cells. As bacteria appear colourless in unstained preparation, it is
difficult to observe bacteria as such without staining under the microscope.
The chemical composition used to stain the bacteria, such as chemical compounds containing auxochrome and chromophore group is called a stain or dye. We stain the bacteria to make them more readily visible because unstained cells are practically transparent.
Stain may be Acidic (Ex: Eosin, Cangored, Nigrosine, Indiaink) or it may be basic (Ex: Basic fuchsin, Crystal violet, Methylene blue, Safranin). Acidic stains the basic part of a bacterial cell and basic stain, stains the acidic part of a bacterial cell
The chemical composition used to stain the bacteria, such as chemical compounds containing auxochrome and chromophore group is called a stain or dye. We stain the bacteria to make them more readily visible because unstained cells are practically transparent.
Stain may be Acidic (Ex: Eosin, Cangored, Nigrosine, Indiaink) or it may be basic (Ex: Basic fuchsin, Crystal violet, Methylene blue, Safranin). Acidic stains the basic part of a bacterial cell and basic stain, stains the acidic part of a bacterial cell
Mechanism of Staining:
The basic stains have a strong affinity towards the acidic portion of bacterial cells. The surface of the bacterial cells and some cytoplasmic components (Nucleic acid, Ribosomes) have overall acidic characteristics the chromophore group of basic stains it is a positively charged ion so it can easily form an ionic bond with acidic molecules.If a single stain is used for staining of bacteria then the staining method is called “Simple staining or Monochrome staining.”
Requirements:
1) Suspension of bacterial cells.2) Clean grass free slides.
3) Glass marking pencil.
4) Nichrome wire loop
5) Basic stain solution (Crystal violet, basic fuchsin, Methylene blue, Safranin).
6) Immersion oil.
Procedure:
1) Clean grease free glass slide pass through the flame for 2-3 times.2) Mark the help of sterilized nichrome wire loop take loopful suspension aseptically.
3) Prepare smear on the above surface of the marked area.
4) Air dry the smear.
5) Heat and fix the smear by passing it gently 2-3 times through the flame and cool sufficiently.
6) Flood the smear with the given stain and allow to act for 2 min.
7) After 2 minutes, remove the excess of stain and wash the slide properly with water.
8) Air dry the smear and observe under oil immersion lens.
Observation:
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Fig Pink Colour Coccus and Violet colour Bacillus.. |
2) We observed violet coloured rods in separate distribution forms.
Results:
Organisms are rod shaped and cocci shaped their morphology and their arrangement is specific.Conclusion:
1) In monochrome staining only one stained is used2) By using only one stain we can study the morphology and arrangement of organism
3) In this method basic stain is used. It can give colour to bacteria so surface of the bacteria is acidic in nature.
4) As the surface of organism is acidic in nature. Only one stain is used for monochrome staining
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