A vegetative cell (bacteria) is a normal bacterium that grows and multiplies in favorable environmental conditions. Here, favorable conditions simply mean that there is sufficient nutrients, optimal temperature range and moisture etc that support the growth and multiplication of the cells allowing them to thrive.
As a result of poor/extreme environmental conditions, vegetative cells undergo a process referred to as sporulation to form endospores.
Once such bacteria as Bacillus spp and Clostridium spp find themselves in unfavorable conditions, sporulation begins with the cells packing everything they need to survive through these extreme conditions.
Unlike the vegetative cells, endospores are dormant (inactive) and highly resistant to such extreme conditions as very high temperatures, chemical changes, enzymatic actions and desiccation among others. This allows the endospores to protect and preserve the genetic material of the cell and allow it to thrive once the cell finds itself in favorable conditions again.
* Sporulation is the process through which endospores are formed. The entire process takes 8 to 10 hours. Whereas endospores reside inside the original cell, free spores (spores) exist outside the cell on their own.
* Endospores are likely to be found in soil and aquatic environments.
Essentially, endospore stain is a differential stain. As such, it allows for the differentiation of structures and thus the characterization of a cell based on its physical and chemical nature. In this case, endospore stain as a differential staining technique is largely used for the purposes of distinguishing between vegetative cells and endospores.
For endoscopes, there are two major staining techniques used. These include:
This is the most common technique for endospore staining.
Steps in the Procedure
Unlike the vegetative cells, endospores possess a permeability barrier. This prevents dyes from entering to stain the structures of the cell. For this reason, the barrier has to be destroyed, which is why heat is used. Through heat fixing, the cortex of the endospore is penetrated allowing for the dye to interact with the petodoglycan and thus produce desired effects. In this case, heat acts as a mordant.
* a mordant is a substance that is used alongside the stain/dye to fix it in a given material
After heat fixing, the slide is washed using tap water or distilled water. Here, water is used as a decolorizer. Because malachite green binds relatively weakly, it can be washed off easily. However, it cannot be washed off easily once it is locked in the spore wall.
Once they take in the dye, endospores retain the dye and will be resistant to de-staining. However, vegetative cells will easily lose the stain when washed with water because they lack the spore wall. After the initial washing, a counter stain (safranin) is used. The purpose of the counter stain is to stain the vegetative cells that lost the primary stain.
Here, it is worth noting that the primary stain and the secondary stain are of different colors. As such, they allow the technician to differentiate different types of cells under the microscope. Whereas the counterstain (safranin) is pink/reddish in color, the primary stain (malachite green) is green in color. Therefore, endospores will appear green in color while the vegetative cells will pink/reddish in color under the microscope.
* While different stains are used in this technique, the purpose of each step taken here is similar to the one described above.
When viewed under the microscope, endospores will be red in colour.
In biology, endospore staining is used for the purposes of differentiating and classifying bacteria. On the other hand, it is also very important in medicine and the food industry.
Because they are tough and hard to destroy it is very important to determine whether they are present in canned food and thus avoid food poisoning to protect consumers.
Related and Interesting articles:
Gram Staining - Purpose, Procedure and Preparation
Capsule Stain - Definitions, Methods and Procedures
Jackie Reynolds, Richland College, BIOL 2421. Fall 2011,
Marise A. Hussey, Anne Zayaitz. 2007. Endospore stain protocol.