Guide To The Endospore Stain – Techniques, Procedures, And Importance

Bacterial endospores can be seen using the endospores stain, a differential stain. Endospores can form in vegetative cells’ terminal, subterminal, and central regions.

Guide To The Endospore Stain - Techniques, Procedures, And Importance

These spores have no metabolic activity and are extremely resistant. It was created as a defense mechanism to help people survive in harsh conditions.

A primary stain, malachite green, is applied to the endospores and vegetative cells, and its penetration through the endospores is aided by heat.

Following that, cells are decolorized, resulting in the removal of stains from only the vegetative cell. Safranin is used to counterstain any cells that have been decolorized. Finally, dark green endospores and pink vegetative cells are obtained. 

What Is An Endospore?

Microorganisms are adaptable to their surroundings; they can sense and adapt to their surroundings. When they are in an environment where their natural source of nutrition has been depleted, they employ a variety of survival strategies.

One such bacterial strategy is the production of endospores, which is usually initiated in situations of nutritional deprivation.

Endospores are seed-like formations produced within bacteria, as the name implies. They are highly resistant, with the goal of ensuring survival and preserving genetic information in the face of environmental stress.

Endospores allow bacteria to survive in conditions that would otherwise kill them, such as high temperatures, pressure, chemical damage, irradiation, and so on.

Endospores of low gram-positive bacteria, in particular, are resistant to these conditions.

Endospore Structure

Endospores have a distinct cell structure. It is surrounded by a proteinaceous outer covering. This coating protects the spore from chemical and enzymatic attack.

It is lined by a inner layer of specialized peptidoglycan that serves as the cortex. It is required for the proper formation of the cortex because it causes dehydration of the spore, which is required to withstand high temperatures.

The germ’s cell wall is located beneath the cortex. This layer is composed of peptidoglycan, which later develops into the bacteria’s cell wall following endospore germination.

There is an inner membrane beneath the cell wall that acts as a permeability barrier, protecting the bacteria from damaging chemicals. The spore’s core is dehydrated and is located in the center.

It has the genetic information of the bacteria, such as dipicolinic acid and DNA which accounts for 10% of the dry weight of the spore, ribosomes, and is essential in endospore dormancy.

Small, Acid Soluble Proteins are also present and are responsible for binding and condensing DNA.

It is also responsible for endospores’ UV-light resistance and shelters them from DNA-damaging chemicals. It also has exosporium, which is an outer layer made up of a glycoprotein.

Endospore Destruction

Under starvation conditions, particularly a lack of nitrogen and carbon sources, a single endospore forms within some bacteria via a process known as sporulation.

When a bacterium detects that environmental conditions are becoming unfavorable, it may initiate the eight-hour endosporulation process.

The DNA is replicated, and a membrane wall called a spore septum forms between it and the rest of the cell.

Endospores are resistant to the majority of agents that would typically kill the vegetative cells from which they formed.

Endospores, unlike persister cells, are the result of a morphological differentiation process activated by environmental nutrient limitation known as starvation; endosporulation is initiated by quorum sensing within the population as it starves.

Endospore Reactivation

When conditions improve, the endospore reactivates, resulting in activation, germination, and outgrowth. Even if an endospore is in a fertile environment, it may not germinate unless it is activated.

This could be caused by heating the endospore. Germination occurs when a dormant endospore begins metabolic activity, breaking hibernation.

It is commonly distinguished by spore coat rupture or absorption, endospore swelling, increased metabolic activity, and loss of resistance to environmental stress.

History Of The Endospore Stain

Endospores were first studied by scientists Cohn and Koch in 1876. Endospores were not able to be stained with common stains such as methylene blue, carbol fuchsin, and safranin.

These scientists, as well as a few others, discovered that spores were dormant and heat resistant. Researchers were looking for alternative methods to improve disease and infection caused by these endospores in the early 1900s.

Dorner published a method for staining endospores in 1922. He discovered a differential staining technique that shows endospores as green and vegetative cells as pinkish red.

Dorner used heat as a step in the process, but it took time, so Schaeffer and Fulton modified his method in 1933.

Schaeffer and Fulton accelerated the heating process by employing a Bunsen burner. Although it was not the most advantageous method, it was far more convenient than Dorner’s method.

This improved method allowed for a faster and easier test while also making the spores more susceptible to the dyes. The Schaeffer- Fulton stain is still used to identify bacteria today.

Endospore Stain Principal

Endospore staining is a differential stain that detects and picks out an endospore from a vegetative cell which is an underdeveloped endospore.

The basic function is to detect the presence or absence of endospores, but some procedures have modified the method by increasing dye concentrations, prolonging heat fixation time, and using ultraviolet radiation.

With improved microscopy technology, some use phase-contrast microscopy, which produces more detailed morphologies of the bacterial endospore.


The Schaeffer-Fulton technique is the most popular used staining technique for endospores in basic laboratories because it is simple and quick to identify the bacteria.

It enlists the use of Malachite green dye which is an alkaline solution with a pH of 11-11.2 and a water-soluble dye with steamed heat, which makes endospore covering soft and lets dye to penetrate into the spore. 

The malachite green dye sticks to the spore mildly and easily washes away if cleaned with water without fixing, which is why the use of steamed heat is important to allow the dye to pierce the endospore.

Water is utilized as a decolorizer to remove the malachite dye from vegetative forms.

Finally, after the green dye has been flushed away by the decolorizing agent, the underdeveloped Furmigates vegetative forms are stained with a counterstain, Safranin reagent, also known as the secondary stain.

Malachite Green dye and Safranin work well in bacteria due to the alkaline nature of the Malachite Green reagents, which are positively charged, and the basophilic cytoplasm of the bacterial cell.

Which creates an magnetism between the bacterial cell and the dye, making it easier to absorb the dye. 

Under the microscope, the vegetative cell forms show as pink stained particles that have taken up the counterstain, whereas the endospores will look like green dotted particles known as ellipses that have taken up the Malachite green dye.


Endospores, unlike vegetative cells, have a permeability barrier. This stops dyes from entering the cell and staining its structures. As a result, the barrier must be destroyed, which is why heat is employed.

The cortex of the endospore is penetrated by heat fixing, allowing the dye to interact with the petodoglycan and produce the desired effects. Heat acts as a mordant in this case.

Following heat fixing, the slide is washed with either tap water or distilled water. Water is used as a decolorizer in this case.

Because malachite green binds so weakly, it is easily washed away. It cannot, however, be easily washed away once it is trapped in the spore wall.

Endospores retain dye after taking it in and are resistant to de-staining. Because they lack the spore wall, vegetative cells lose the stain when washed with water easily.

Following the first clean, a counter stain – safranin is applied. The counter stain’s purpose is to stain the vegetative cells that no longer have the primary stain.

It is worth noting that the primary and secondary stains are of different colors. As a result, they enable the technician to distinguish between different types of cells under the microscope.

The counterstain called safranin is pink in color, whereas the primary stain malachite – is green. Under the microscope, endospores will appear green, while vegetative cells will appear pink/reddish.

Here is a simplified step by step guide to the procedure:

  • Carefully spread the sample onto the center of the microscope slide, making sure the slide is clean before doing so. 
  • Leave the slide to dry naturally in the air before heat fixing the sample.
  • Get some blotting paper and cut it to fit the slide, then put it on the slide and saturate it with the green dye. 
  • Next, carefully heat the slide so that it begins to evaporate. You must make sure that the slide is on a staining rack whilst you do this step. Either boiling water or a Bunsen burner can be used to heat up the slide but avoid excess heat as this can ruin the integrity of the cells. This can then make them shrink and cluster on the slide.
  • Carry on heating the slide but remove it and re-heat it every three to five minutes as you add more green dye to ensure the blotting paper is kept moist. You should only heat it enough so that the slide is steaming. 
  • Take the slide off of the rack and leave it to cool down for a couple of minutes. 
  • Wash the slide thoroughly using either distilled water or tap water. 
  • Add a counter stain to the slide using safranin for around a minute.
  • Wash the slide once again and leave it to air dry. 
  • You can now look at the results on the slide under the microscope. Any debris that is left on the slide will also be stained green so keep this in mind when looking at the results and interpreting what they mean.


Endospore staining is a technique used in biology to differentiate and classify bacteria. On the other hand, it is crucial in medicine and the food industry.

Because they are long-lasting and difficult to destroy, determining whether they are present in canned food and thus avoiding food poisoning is critical to protecting consumers.

The history of the endospore stain dates back to the early 1900s and over time has been altered to be more practical, accurate, and safe. To this day, this staining technique is used to identify bacteria and without it, we would be lost.

Jennifer Dawkins

Leave a Comment

Your email address will not be published. Required fields are marked *