Endospore staining involves application of a series of dyes. Malachite green stains the endospores and safrinin dyes vegetative cells pink. Here is the endospore stain procedure.
But before discussing the procedure for staining endospores, it is important to understand what these unique structures are and how the power of the endospore was first discovered.
* Heat Resistant Bacteria *
John Tyndall was a 17th century Irish-born English-bred physicist who made many contributions to science, one of which was the discovery that some microbes existed in two forms:
* heat-stable form (endospores)
* heat-sensitive form (active ,living vegetative cells)
Tyndall found that it took either prolonged or intermittent heating to destroy the resistant heat-stable form. The outcome of his research was a method of sterilizing liquid by heating it to boiling point on successive days (“Tyndallization”).
“Tyndallization” is useful for sterilization of growth media in science classes and other situations where autoclaves (instruments that use both heat and pressure to sterilize) are not available for pressure sterilization.
* What Is an Endospore? *
Endospores are produced by very few types of bacteria, most notably the genera Clostridium and Bacillus. These protective structures are made through a process known as sporulation in response to extreme environmental conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of food.
In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but exist in a type of suspended animation, much like the seeds of plants do. When environmental conditions again become favorable, the endospore germinates, returning the bacterium to its normal active and reproducing metabolic state.
* Staining Bacterial Endospores *
Normal water-based techniques, such as the Gram stain, will not stain these tough, resistant structures. In order to stain endspores, malachite green must be forced into the spore with heat, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of Mycobacterium in the Acid-fast Stain.
The protocol for differentially staining endospores and vegetative cells is as follows:
1. Place the heat-fixed bacterial slide over screened water bath and then apply the primary stain malachite green.
2. Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it begins to dry out.
3. Remove the slide from the water bath and rinse the slide with water until water runs clear.
4. Flood slide with the counterstain safrinin for 20 seconds and then rinse.
5. View specimen under oil immersion (magnification of 1000xTM) with a light microscope.
After this staining procedure, the endospores will appear green, having retained the primary stain, malachite green. The vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safrinin.
* Problems Interpreting Endospore Stain *
It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. Endospores are small and typically oval. Large or irregular globs of green on the slide may be artifacts.
Acid-fast cells, such as members of Mycobacterium and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. The uniformly green appearance of endospore stained Acid-fast cells doesn’t mean that they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall.
* Sources *
Bauman, R. (2005) Microbiology.
Park Talaro, K. (2008) Foundations in Microbiology.