Simple Staining
* Simple staining implies the use of only a single stain, which is usually sufficient to reveal the morphological features of most microbial cells, including relative size, shape and characteristic arrangements for groups of cells.
* This method uses only single stain that does not differentiate between different types of organisms.
* Simple stains are used to stain whole cells or specific cellular component.
* Direct (Positive) staining : stain OBJECT
* Indirect (Negative) staining : stain BACKGROUND
a. Monochrome staining
Principle -
In Monochrome staining, the bacterial smear is stained with a single reagent, which produces a distinctive contrast between the organisms and its background.
Mechanism -
Basic stains with a positively charged chromogen are preferred because bacterial nucleic acid and certain cell wall components carry a negative charge hat strongly attracts and blinds to the cationic chromogen.
To purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells.
Stains -
The most commonly used basic stains are methylene blue, crystal violent, safranin and carbol fuchsin etc.
Procedure -
i) Preparation of a smear and heat fixing
* Using as sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to clean grease free microscope slide or transfer an isolated colony from a culture plate to a slide with a sterile water drop.
* Disperse the bacteria on the loop in the drop of water on the slide and make a thin and even smear on slide.
* Allow the smear to dry thoroughly .
* Hear-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.
ii) Staining
* Cover the smear with methylene blue and allow the dye to remain in the smear for approximately one minute (staining time is not critical here: somewhere between 30 seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in it, the darker will be the stain).
* Using distilled water wash bottle, gentle wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.
* Wash off any stain that got on the bottom of slide as well.
* Wipe the back of the slide and blot the stained surface with a clean filter paper.
* Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.
* Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply immersion oil directly to the smear and focus the smear under oil with the 100X objective.
i. Preparation of a smear and heat fixing
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ii) staining
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Observation -
* The bacterial cells usually stain uniformly and the colour of the cell depends on the types of dye used.
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b. Negative staining
* The main purpose of negative staining is to study the Morphological shape, size and arrangement of the bacteria cells that is difficult to stain e.g. Spirilla.
* It can also be used to stain cells that are too delicate to be heat-fixed.
* As heat fixation is not required in negative staining, the cells are not subjected to the distorting effect of chemicals and heat, due to which their natural size and shape can be seen.
* It is also used to prepare biological samples for electron microscopy.
* It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron- scattering power.
Principle -
Negative staining is based upon the principle that there is no reaction between the stain and the specimen. This is accomplished when the stain is used at a pH at which the interaction between stain and biological materials is negligible.
Mechanism -
* The acidic stain, with its negatively charged chromogen is used in negative staining.
* Since the surface of most bacterial cells is negatively charged, the cell surface repels the acidic stain.
* The glass of slide i.e. background will stain, but the bacterial cells will not.
* The bacteria will show up as clear spot against a dark background.
Stains -
Acidic stains such as 10% Nigrosin, 2% Congo red, India ink etc.
Procedure -
* Place a very small drop (more than a loop full, less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.
* Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.
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* Use another slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.
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* Push the slide away from the drop of suspended organisms to form a thin smear Air dry.
* Note: Do not heat fix the slide.
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* Focus a thin area under oil immersion and observe the unstained cells surrounded by the grey stain.
Observation -
Dark background with a light specimen.
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