Endospore Staining: Principle, Procedure, Results

bacillus cereus_endospore stain

Endospore staining is a differential technique that selectively stains the spores and makes them distinguishable from the vegetative part of the cells. Endospores are produced by a few genera of Gram-positive bacilli, such as Bacillus and Clostridium, in response to adverse environmental conditions. Endospores are highly resistant to environmental conditions such as heat and chemicals (stains and dyes) and therefore require special techniques for staining.

Methods for endospore staining

There are different methods for endospore staining. The most common are

  • Schaeffer-Fulton stain technique
  • Dorner’s methods
  • Modified Zeihl-Nelson’s method
  • Barthelomew-Mittwar’s method
  • Abott method
  • Moller stain technique

Spores can generally be recognized on Gram’s stains (endospores do not stain and appear as refractile, nonstaining bodies). Endospores can also be demonstrated in unstained wet films under a phase-contrast microscope. They appear as large refractile oval or spherical bodies within the mother cell.

Schaeffer-Fulton stain technique

It is the most widely used technique for endospore staining. The technique was first described by Alice B. Schaeffer and MacDonald Fulton in the 1930s. The method utilizes malachite green as the primary stain and safranin as a counterstain.


When a heat-fixed smear is flooded with aqueous malachite green solution (the primary stain) and steamed, the heat assists the stain in penetrating through the spore.  In this technique, heating acts as a mordant. Once the endospore has absorbed the stain, it is resistant to decolorization, but the vegetative cells are easily decolorized with water (leaving the vegetative cells colorless). When counter-stained with safranin, the vegetative cells take the color of safranin and appear red or pink, in contrast to the endospores that appear green.

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 containing endospores should stain pink, while the spores should be seen as green ellipses.
  3. Mature, free endospores should not be associated with vegetative bacteria and should be seen as green ellipses.


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. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required.  Alternatively, the slide may be steamed over a container of boiling water.
  5. 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 once that the temperature is appreciably reduced.
  6. After 5 minutes, 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.

Result & Interpretation

Observe the bacteria under 1000X (oil immersion) total magnification. When viewed under 1000X of a light microscope, vegetative cells appear pink/red, and spores appear green.

Dorner’s Method

Dorner’s method is an alternative method for staining the endospores published by Dorner in 1922. This method utilizes carbol fuchsin as primary stain, acid alcohol as decolorizer, and nigrosin as counterstain. It employed a lengthy heating step but resulted in differential staining of endospores and vegetative cells in the same sample. 

Principle of Dorner’s method for staining endospores

Carbol fuchsin, when applied to a heat-fixed slide and heated, softens the structure of the bacterial spores and the basic fuchsin gets into the spores. When decolorized with acid alcohol, color washes off the vegetative cells, making them colorless.

Since the counterstain nigrosin is negatively charged, bacterial cells don’t easily take up the counterstain. Therefore, vegetative cells appear colorless, endospores stain red, and the background is black.

Procedure for Dorner’s method

  1. Make a smear on a clean glass slide.
  2. Allow the slide to dry (air dry) and then heat fix
  3. Place a blotting paper on the slide (covering the smear) and saturate with carbolfuchsin to steam (for about 5 minutes).
    This should be repeated while adding drops of carbolfuchsin and avoiding overheating (simply heat to steam)
  4. Remove the blotting paper and allow the slide to dry for about a minute
  5. Wash the slide with acid-alcohol for about a minute to decolorize and then rinse with tap water
  6. Add a drop of nigrosine to the smear to form a thin film.
  7. Allow the slide to dry.
  8. Observe under the microscope using oil immersion.

Variation in Dorner’s method

Dorner’s method is further modified by omitting the acid-alcohol decolorizer and using a 7.0% (wt/vol) aqueous solution of the nigrosin. It is performed in a test tube, thus avoiding direct heating procedures.

Procedure of modified Dorner’s method

  1. Mix an aqueous suspension of bacteria with an equal volume of carbol fuchsin in a test tube.
  2. Immerse the tube in a boiling water bath for 10 minutes. Allow cooling for some time.
  3. Mix a loopful of 7% nigrosin on a glass slide with one loopful of the boiled carbol fuchsin-organism suspension and make a thin smear and allow air to dry.
  4. Examine the slide under the oil immersion lens for the presence of endospores.


Vegetative cells are colorless, endospores are red, and the background is black.

Other techniques of endospore staining

Although the principle of endospore staining is the same, variations exist in the choice of primary stain, counterstain, and whether or not decolorizer is used. Some of them are summarized below.

MethodPrimary StainDecolorizerCounterstainInterpretation
Modified Zeihl-Nelson’s methodCarbol Fuschin0.25-0.5% sulphuric acidLeoffler’s methylene blueSpores appear red, bacteria are blue
Dorner methodCarbol FuschinAcid-alcoholNigrosinSpores red Bacteria colorless Background Black
Schaeffer-Fulton StainMalachite GreenWaterSafraninSpores appear green vegetative cells appear pink/red
Bartholomew and mittwer methodMalachite GreenWaterSafraninSpores appear green  vegetative cells appear pink/red
Abbott’s methodMethylene BlueAcid alcoholAniline fuschinSpores appear blue bacteria are red
Moeller’s stainCarbol fuschinAcidified ethanolMethylene blueSpores appear red bacteria are Blue
Modified Moller’s stainKinyoun’s Carbol fuschin2%sulphuric acid and 80% ethanolLoeffler methylene blueSpores appear red bacteria are Blue

References and further reading

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

22 thoughts on “Endospore Staining: Principle, Procedure, Results

  1. Bactria staining which is sour time gram staining all staining chemical n reagent time please tell this

    1. 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.

    1. 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.

  2. 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….

  3. 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 .

    1. 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.

  4. 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?

  5. 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

    1. 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.

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

    Thanking you sir

    1. Dear Ramesh, Please find details about IMVIC Test in this link: https://microbeonline.com/imvic-tests-principle-procedure-and-results/

  7. 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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