Endospore Staining: Principle, Procedure and Results

Last updated on June 4th, 2020

When vegetative cells of certain bacteria such as Bacillus spp and Clostridium spp are subjected to environmental stresses such as nutrient deprivation, they produce metabolically inactive or dormant form-endospore. Formation of endospores circumvent the problems associated with environmental stress and helps them to survive.

During unfavorable conditions (especially when carbon and nitrogen become unavailable) spore-forming bacilli form endospores. The endospores may be spherical, subspherical or oval in shape, they may differ in their location within the cell (i.e., central, terminal, or subterminal), and they may or may not swell the cell.

  1. Central endospores are located within the middle of the vegetative cell.  
  2. Terminal endospores are located at the end of the vegetative cell.  
  3. Sub-terminal endospores are located between the middle and the end of the cell.

The size, shape, and location endospores are particularly useful for identifying Clostridium, Bacillus, and related species.

Spore location

Find more information about Endospore Structure and Importance Here

Most endospore-forming bacteria are found in soil or aquatic environments.  However, some species of Bacillus and Clostridium have medical significance.  Clostridium perfringens, C. botulinum (a potential agent of bioterrorism), and C. tetani are the causative agents of gas gangrene, botulism, and tetanus, respectively.  Bacillus anthracis and Bacillus cereus are the causative agents of anthrax and self-limiting food poisoning, respectively.

Spores can generally be discerned on Gram’s stains (endospores do not stain and appear as refractile, nonstaining bodies), but occasionally specific spore stains may be helpful.

Principle of Spore Staining

A differential staining technique (the Schaeffer-Fulton method) is used to distinguish between the vegetative cells and the endospores.  A primary stain (malachite green) is used to stain the endospores.  Because endospores resist staining, the malachite green will be forced into (i.e, malachite green permeate the spore wall) the endospores by heating.  In this technique, heating acts as a mordant.

There is no need of using any decolorizer in this spore staining as the primary dye malachite green bind relatively weakly to the cell wall and spore wall. In fact, If washed well with water the dye comes right out of cell wall however not from spore wall once the dye is locked in. Water is used to decolorize the vegetative cells. 

Note: In Gram Staining and AFB Staining we use alcohol or acid-alcohol or acid as a decolorizer but in spore staining, water is sufficient to be used as decolorizer because:

  • malachite green dye is water-soluble and does not adhere well to the cell wall
  • vegetative cells have been disrupted by heat,
    because of these reasons, the malachite green rinses easily from the vegetative cells. 
bacillus cereus_endospore stain

As the endospores are resistant to staining, the endospores are equally resistant to de-staining and will retain the primary dye while the vegetative cells will lose the stain.  The addition of a counterstain or secondary stain (safranin) is used to stain the decolorized vegetative cells.

When visualized under microscopy the cells should have three characteristics:

  1. the vegetative cells should appear pink/red (i.e. the color of counterstain),
  2. the vegetative cells that contain endospores should stain pink while the spores should be seen as green ellipses within the cells.
  3. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses.

Procedure of endospore stain

Spore staining procedure
Spore staining procedure
  1. Prepare smears of organisms to be tested for the presence of endospores on a clean microscope slide and air dry it.
  2. Heat fix the smear.
  3. Place a small piece of blotting paper  (absorbent paper) over the smear and place the slide (smear side up) on a  wire gauze on a ring stand.
  4. Heat the slide gently till it starts to evaporate (either by putting the slide on a staining rack that has been placed over a boiling water bath or via bunsen burner).
  5. Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-5 minutes.  As the paper begins to dry add a drop or two of malachite green to keep it moist, but don’t add so much at one time that the temperature is appreciably reduced.
  6. After 5 minutes carefully remove the slide from the rack using a clothespin
  7. Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes.
  8. Rinse the slide thoroughly with tap water (to wash the malachite green from both sides of the microscope slide).
  9. Stain the smear with safranin for 2 minutes.
  10. Rinse both sides of the slide to remove the secondary stain and blot the slide/ air dry.

Observe the bacteria under 1000X (oil immersion) total magnification. 


The vegetative cells will appear pink/red  and the spores will appear green.

Spore staining characteristics of selected Gram-positive bacilli


Spore staining characteristics

Clostridium septicum

Thin gram-positive bacilli with numerous ovoid or citron-shaped, subterminal spores are present.

Clostridium perfringens

Relatively large “boxcar”-shaped gram-positive bacilli are seen, spores are usually present but the demonstration of spores is frequently difficult.

Clostridium tetani

Gram-positive bacilli with the presence of round, terminal spores.

Clostridium sordellii

Gram-positive bacilli with clumps of free spores and bacteria distended with ovoid subterminal spores.

Clostridium tertium

Thin gram-positive bacilli with subterminal spores

Bacillus cereus

Oval spores may be observed centrally or subterminally and the cells are not swollen in the areas where the spore is located.

Bacillus anthracis

B.anthracis appear as large gram-positive bacilli, with the individual cells having squared-off or slightly concave ends. Often, the organisms are arranged in chains that have the appearance of bamboo. The spore stain shows terminal/subterminal spores that do not swell the vegetative cell

References and further reading

About Acharya Tankeshwar 466 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


    • Dear Maira
      Thank you so much for visiting my blog and for your inspirational comment. Can you please clarify about this section of your comment “do write time and reagents”.

      I will be looking forward to hear from you. Wishing you great time ahead.

    • Dear Dnyanad
      Thank you so much for your question. It’s a very good question so I have incorporated the answer in the blog post as it may help other students as well. The reasons are; Malachite green is water soluble, vegetative cells have been disrupted by heat.

  1. why do anaerobic bacterias die in the presence of oxygen where as aerobic bacteria do not? what is the significance factor that causes this to be so?
    thanks for the willingness to help….

  2. Sir ,

    What is the need of paper towel or blotting paper which is used to stain the sample with Malachite Green ? ( as already malachite green binds relatively weak to the cell and spore wall , so why is it not given a direct contact to the sample , instead of using paper towel ? )

    Kindly please reply .
    Regards .

    • Dear Ankit, we use paper towel to give more contact time between the dye and the bacterial cells. If paper towel is not used, the dye evaporates quickly.

  3. Dear Sir!
    Thank you for your blog.
    Tell me please, what concentration of malachite green and safranin do you use?
    Are 1% water solutions suitable?

  4. Sir… this blog is very helpful but there is a doubt that the slide should observed under 100x but in blog it’s written that 1000x

    • Thank you Bhagyashree Das for your inspiring comment. Regarding the magnification, the total magnification achieved when using a 100x oil immersion lens with 10X binocular eyepeice is 1000x.

  5. hi sir
    thank you for your protocol for endospore staining
    i need IMVIC test protocol and media composition.

    Thanking you sir

  6. sir i am currently studying in bsc microbiology 4th year, and as per our curriculum we have to do a project on any topic for three months.so please can you help me in this like where should i apply for my project or if you have your own lab,can you help me for this.

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