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Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them highly resistant to chemicals. Endospores do not contain ATP which indicates how dormant they are. Endospores contain a tough outer coating made up of keratin which protects them from nucleic DNA as well as other adaptations. Endospores are able to regerminate into vegetative cells.This protective nature makes them difficult to stain using normal techniques such as simple staining and Gram staining. Special techniques for endospore staining include the Schaeffer–Fulton stain and the Moeller stain.
Endospores are able to last for decades in multiple hard conditions, such as drying, and freezing since the DNA inside the endospore is able to survive. Most bacteria are unable to form endospores but there are a few examples. Bacillus anthraces, which causes anthrax as well as Clostridium titani, which is a spore that causes lock jaw and rigid paralysis. Clostridium botulinum is found in foods that have not been canned properly. Clostridium botulinum is sometimes sold as botox and prevents nerve transmission.Types of endospores that may be identified include free endospores, central endospores( middle of the cell), subterminal( between the end and middle of the cell), and terminal ( end of the cell) endospores. There can also be a combination of terminal or subterminal. Endospores can be differentiated based on shape either, spherical or elliptical (oval) and size relative to the cell, weather they cause the cell to look swollen or not
The primary dye for endospore staining is malachite green. It takes a long time for the spores to stain due to their density, so time acts as the mordant when performing this differential stain; the slide with the bacterium should be soaked in malachite green for at least 30 minutes and then rinsed off with water which acts as the decolorizer. A counterstain to differentiate the vegetative cells is commonly 0.5% safranin. In the end, a proper smear would show the endospore as a green dot within either a red or pink-colored cell. Mycobacterium is one obstacle that is faced with this type of staining because it will still stain green even though it does not produce any endospores. This is due to its waxy cell wall which retains the malachite green dye even after the decolorizing process. A different type of staining called acid-fast stain will have to be done in order to get further information about this particular type of bacterium.
In the Schaeffer-Fulton method, a primary stain containing malachite green is forced into the spore by steaming the bacteria. Malachite green can be left on the slide for 15 minutes or more to stain the spores. Malachite green is water soluable, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin.
Terbium can also used to detect endospores, as it acts as an assay of dipicolinic acid based on photoluminescence.