Staining Types and Sample Smear Preparation,While studying the bacteria, turning is the most important phenomena. Stains are also called chromogens. The bacterial cells (or other specimen) are stained in order to make it easy to be studied under a microscope.
Followings are the types of stain that are used commonly.
- Simple staining,
- Negative staining,
- Differential staining.
- Simple staining:
- Simple staining:
Simple staining involves the staining of of specimens with a chromogen or stain. After staining the cell is washed and then it can be observed under a microscope. The purpose of washing is that only the organelles which we want to study under the microscope get the stain while the remaining stain is washed out from the cell this making it easy for observation.
- Negative staining:
The negative stains are also called acidic stain. The acidic strength do not stain living cells, instead they stick to the non living cells. The basic stains given colour to the living cells, so normally these are used. Acidic stains are used in a few cases. For staining the cell with acidic stains, the cell or bacteria should be fresh and young and not old because old bacteria or cell will die after getting the stain. Therefore it’s observation will be impossible. In acidic staining only negative stains are used.
- Differential staining:
Differential staining is used to differentiate between gram positive and gram negative bacteria. More than one stains are used in case of differential staining. Here two results are obtained from the same condition; that is the conditions are kept the same for both cases on the same slide, chemicals and everything.
Clepaiala pneumonia cause pneumonia. It has two strains, one is encapsulated and the other is not. So both are taken on the same slide, using same chemicals, steps, procedures and all other conditions are kept the same for both. At the end two types of results are obtained; Bacillus a thracis- which has spores and Bacillus pumillus- which has no spores. To test such types of specimen this straining is used.
Preparing sample smear:
In negative stains we have;
- Primary stains (crystal violet),
- Secondary stains (safranine)
- Mordant (iodine)
- Decolourises (70% ethanol and 70% alcohol).
The chemicals mentioned (in the brackets) can vary but these are the standards. Differential staining is selected on the basis of tissue’s nature to be stained.
Take a water drop on the slide with toothpick and add bacterial colony to it. Thus a bacterial smear is made. Then air dry it, to avoid shrinking and to bring the cell to normal condition.
Then, pass this sample over the flame to fix it. Thus, it will not get washed away with washing, this step is called heat fixation.
Now stain the smear (primary staining) and wash the extra stain with the help of wash bottle. In the next step, secondary staining is carried out.
The mordant is used to enhance the colouration. Give the smear 1 to 2 minutes with the mordant.
It will enhance any stain that is used prior to its addition. Now, decolorize the sample using decolorizers. Treat it for about 3 to 4 minutes.
The smear should be very very thin or else the stain will appear as cells (smear is very minute and nearly impossible to see. Lighter the smear, better will be the results), and the bacterial colony will have lost its surface. The result will be, cellular appearance of the stain.
Stain retention reason:
- Lipids in gram positive and gram negative bacteria; lipids are more in gram negative bacteria and less in gram positive bacteria.
- Polypeptidoglycane; it is less in gram negative bacteria and more in gram positive bacteria. The peptidoglycane resist the colouration, it is more in gram positive bacteria so gram positive bacteria can not be stained.
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