Explain the requirements of Special Stains?
Special stains are required to make spores clearly visible. Different techniques like negative staining, dorner staining method, schaeffer fulton staining method can be used. The most commonly used one is a schaeffer-fulton method. It is a differential staining technique using 2 different reagents - Malachite green and Safranin.
Malachite green is a primary stain. Spores do not take the stain easily, so heating is applied by steaming for about 5 minutes. This makes the stain penetrate the endospore wall and make vegetative cell, as well as, endospores stain green as you can see in the margin illustration. After primary stain, decolourization is done by tap water. Once the slide is cooled down, washing with tap water is done for about 30 seconds to one minute to remove the excess primary stain. As the stain does not have the strong affinity for vegetative cell, it is removed from the vegetative cell by tap water. So vegetative cells become colourless and spores remain green. Safranin is used as a counter stain. It is absorbed by the vegetative cells, which become colourless during decolourizing step. So after staining by this technique, spores will appear green and vegetative cells red.