Endospore staining

Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample[1], which can be useful for classifying bacteria.[2] Within bacteria, endospores are protective structures used to survive extreme conditions, but 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.

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.

Terbium can also used to detect endospores, as it acts as an assay of dipicolinic acid based on photoluminescence.[3]

Types of endospores that may be identified include free endospores, central endospores, central and swollen endospores, and subterminal endospores. 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.

References

Microbiology An Introduction Tenth Edition; Tortora Funke Case
Hussey, Marise; Zayaitz, Anne (2011-09-29). "Endospore Stain Protocol". American Society for Microbiology. Archived from the original on 2012-06-01. Retrieved 2012-03-06.
Rosen, D. L.; Sharpless, C.; McGown, L. B. (1997). "Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence". Analytical Chemistry. 69 (6): 1082–1085. doi:10.1021/ac960939w.

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[1] Microbiology An Introduction Tenth Edition; Tortora Funke Case

[Hussey-2] Hussey, Marise; Zayaitz, Anne (2011-09-29). "Endospore Stain Protocol". American Society for Microbiology. Archived from the original on 2012-06-01. Retrieved 2012-03-06.

[3] Rosen, D. L.; Sharpless, C.; McGown, L. B. (1997). "Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence". Analytical Chemistry. 69 (6): 1082–1085. doi:10.1021/ac960939w.