Endospore Staining- Types, Principle, Procedure And Interpretation

Endospore Staining Definition


The vegetative phase of a bacterial cell is referred to as a “typical bacterial cell.” When given favourable environmental circumstances, including the supply of nutrients, temperature, water, and oxygen, it may multiply without restriction. However, certain bacterial species are able to create a protective layer to shield themselves from harsh climatic circumstances, including a nutrient deficiency, an insufficient supply of oxygen or carbon dioxide, or a shortage of water and moisture, and easily adapt to these circumstances. The term “endospore” refers to this unique structure.

A group of bacteria from the Firmicute family develop an endospore, which is a non-vegetative structure. They have unique traits that enable them to maintain their stability and endure challenging circumstances for extended periods of time.

These have a dormant stage, a hard exterior, and they are spores, which are non-reproductive forms and have excellent resistance to high temperatures, radiation, and chemicals like disinfectants and acids. Because they may live because of this, it is difficult to stain them with ordinary dyes; instead, a special stain is used that combines a particular dye with heat and steam. The Endospore stain, commonly referred to as the spore stain, is the name of this staining method. It is mostly used to find and confirm the existence of bacterial vegetative forms and endospores in a cell.

A number of Clostridium and Bacillus species are among these endospore-forming bacteria. Through attacking humans, these soil-dwelling microorganisms cause major clinical consequences. For instance, Clostridium tetani, for instance, causes tetanus, whereas Clostridium botulinum generates the paralysing toxin botulin. Bacillus cereus produces food poisoning, as well as Bacillus anthracis produces anthrax in both animals and humans.

Depending on the reagents employed, several endospore staining procedures are categorised;

  1. Schaeffer Fulton StainMalachite Green dye and safranin were employed in the Schaeffer Fulton Stain.
  2. Dorner method of Endospore staining is done using the Dorner technique, which employs Carbolfuchsin stain, acid alcohol, and nigrosin solution.

Objectives of endospore staining

  • in order to check for endospores.
  • to recognise microorganisms that produce endospores.
  • To distinguish between the endospore and vegetative types


The goal of endospore staining, a differential stain, is to find, recognise, and distinguish an endospore from the vegetative cell (an underdeveloped endospore). Although the basic function of the role is to determine if an endospore is present or not, other techniques have changed the method by boosting dye concentrations, lengthening the heat-fixing process, and using UV radiation.

Some people use phase-contrast microscopy, which is quick and generates more precise morphologies of the bacterial endospore thanks to better microscopy technology.

1. Schaeffer Fulton Stain

The Schaeffer-Fulton method of endospore staining is the easiest one to employ in basic laboratories, since it makes it simple and quick to identify the bacteria. Malachite green dye, a water-soluble dye with an alkaline pH range of 11–12, is used in combination with steam heat to soften the endospore coating and allow dye penetration into the spore.

The application of steaming heat is crucial to enable the dye to permeate the endospore, since malachite green only weakly binds to the spore and readily washes away if rinsed with water without fixing. The decolorizing agent, water, is used to wash the malachite dye from vegetative forms. Last but not least, once the decolorizing agent has washed the malachite dye away, the undeveloped vegetative forms of the Furmigates are stained with a counterstain known as Safranin reagent, commonly known as the secondary stain (Water).

Safranin and malachite green dye are effective against bacteria because the cytoplasm of the bacteria is basophilic, which attracts the positively charged, alkaline malachite green reagents. This attraction makes it simpler for the dye to be absorbed by the bacteria. The vegetative cell structures that absorb the counterstain are visible when viewing the cells under a microscope as pinkish-red stains, while the endospores, which have taken up the Malachite green dye, are visible as green dotted particles (ellipses).


  • Green malachite dye
  • Water (Decoloriser)
  • Safranin


Equipment: Bunsen burner, glass slide, and inoculation loop

Making a microscope slide (adapted for all other endospore staining techniques).

  • Alcohol should be used to clean the glass slide (with the visible circles) of any stains.
  • In each circle, add two tiny droplets of water using a sterile inoculation loop.
  • Aseptically open the tube carrying the bacteria culture, burn the top, and extract a loopful of the culture. Rekindle the fire and close the tube.
  • In the drop on the slide, smear the bacterial culture.
  • When it is entirely dry, air dry.
  • By passing the slide three to four times over the blue flame with the smearing facing up, you heat-fix it. NB: Don’t ignite the bacteria-containing side.
  • Allow it to cool before beginning to stain.

Staining procedure

  • Cover the streaks with a sheet of absorbent paper.
  • Put the slide atop a staining rack with a heating beaker or water bath.
  • After filling the absorbent paper with malachite green, steam it for three to five minutes.
  • Toss the discoloured absorbent paper in the trash, and give it a couple of minutes to cool.
  • Clean the slide carefully by tilting it so that the water runs over the discoloured areas. This will be done to remove any excess dye on the two sides of the slide, as well as any excess dye that has stained all the vegetative forms in the heat-fixed smear.
  • Add safranin for one minute as a counterstain.
  • To remove the safranin reagent, thoroughly wash the slide on both sides with water.
  • Ensure that the bottom of the slide is dry prior to placing it on the microscope stage to be viewed with the oil immersion lens at 1000x for maximum magnification.


The malachite green dye should stain the endospores green, whereas the vegetative forms would acquire the pink/red stain from safranin.

Evaluation of outcomes

The endospores absorb the green pigment of malachite green, whereas the vegetative forms absorb the pink/red pigment of the counterstain (safranin).

This is because while smearing and heat fixing, the steam’s heat aids the malachite green dye to penetrate into the endospore, and the dye is hard to eliminate during the water washing.

And as the vegetative forms’ delicate outer layer makes the dye readily wash away, they pick up the final stain, which is the counterstain, making them seem pink-red.

2. Dorner method for staining


The stain carbolfuchsin.

A color-removing agent (acid-alcohol)

Counterstain (Nigrosin solution)


(Adapting the aforementioned process to produce a microscopic slide)

Staining procedure

Cover the smearing with a piece of absorbent paper.

Fill it completely with carbol-fuschin, then heat-fix it by steaming it for 5–10 minutes over a beaker of boiling water while adding additional colour to the smear.

The absorbent paper should be removed and treated with acid-alcohol for 1 minute before being rinsed with tap water and dried by tapping.

As a counterstain, apply a thin layer of nigrosin reagent.

Look at the slide with a 1,000x oil immersion lens to check for endospores.


Endospores are red, whereas vegetative cells are colourless.

Applications of the Endospore stain

  • For the purpose of detecting Firmicute groups of bacteria, such as Bacillus spp. and Clostridium spp.
  • To identify endospore-producing bacteria in samples
  • Distinguish them from bacteria that are vegetative or non-spore-producing

Advantages of endospore staining:

Because it is a differential stain, you can recognise particular bacteria that produce endospores

In addition to indicating the existence of bacteria that produce endospores, it also enables the detection and recognition of vegetative forms in bacterial cultures.


It is only able to identify the existence of endospore-forming bacteria.


The Klein method of endospore staining is an additional, less popular method of endospore staining.

The use of dyes—Malachite Green dye is used in the Schahuffer Fulton stain, whereas Methylene blue solution is used in the Klein method—distinguishes these two staining methods from one another.


  • Oktari A., Supriatin Y., Kamal M., Syafrullah H., The bacterial Endospore Stain on Schaufferton using methylene Blue Solution; Journal of Physics,2017.
  • Marisse A. H., Anna Z., Endospore Stain Protocol; American Society of Microbiology. 2012.

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