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Endospore Staining Principle, Procedure, Result

There are two methods which are commonly used to stain bacterial endospore such as Schaeffer Fulton and Klein methods.

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The endospore staining technique is a differential staining method. This method helps in the detection of endospore-containing bacteria, thus help in the classification of bacterial cells.

Some bacterial cells have the ability to convert themselves into endospores in extreme environmental conditions. Which helps them to survive in harsh environmental conditions (high temperature, ph, etc ).

In 1922, Dorner first introduce the endospore staining method, which was a very lengthy heating process but resulted in differential staining of endospores and vegetative cells in the same sample. In this process the endospore and free spores appear in green or blue while red dye retain by the vegetative cells.

In 1933, Shaeffer and Fulton modified the Dorner’s endospore staining method and make more faster, but heating with a Bunsen burner was still messy. 

There are two methods which are commonly used to stain bacterial endospore such as Schaeffer Fulton and Klein methods. The main difference between these two methods is the use of dyes. In Schaeffer Fulton method the dye is Malachite Green and Safranin whereas in the Klein method using dye is Carbol Fuchsin and Methylene Blue. 

The staining time of Schaeffer Fulton method is faster as compared to the Klein method, that’s why most microbiologists follow Fulton method.

Purpose of Endospore Staining

The main purpose of endospore staining is to identify the presence of endospore within the bacteria cell, identification of endospore producing bacterial cell, and differentiation between vegetative cells and endospore-forming cells.

Schaeffer-Fulton Method

Endospore Staining Principle

In Schaeffer-Fulton method two stains are used, a primary stain malachite green, and a counterstain Safranin. The malachite green and safranine is an alkaline solution. Because of the alkaline (chromophoric component positively charged) nature, these two stains work well on endospores, they are easily absorbed by the cell, because the bacterial cytoplasm is basophilic in nature.

The malachite green is forced into bacterial cell by applying steam. This primary stain is water-soluble and contains a low affinity for cellular material, that’s why it can be easily decolorized with water.

After that, a counterstain safranin is applied, which will stain the decolorized cells. At the end of endospore staining, the vegetative cells will appear in pink color, and endospores will appear in dark green color.

Reagents required for Endospore Staining

  • Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution). Mixture of 0.5 gm of malachite green, 100 ml of distilled water.
  • Decolorizing agent: Distilled water.
  • Counter Stain: Safranin (mixture of 2.5 grams of safranin O and 100ml of 95% ethanol)

Endospore Staining Procedure with flow chart

Endospore Staining Principle, Procedure, Result
Endospore Staining Principle, Procedure, Result
  1. Take a clean oil-free slide.
  2. Prepare a smear at the center of slide.
  3. Air dry the smear.
  4. Heat fix the slide.
  5. Place a blotting paper over the slide.
  6. Now, saturate the blotting paper with malachite green stain solution.
  7. Place the slide (smear side up) on a  wire gauze on a ring stand.
  8. Heat the slide, until it started to evaporate. The heating can be done by Bunsen burner of boiling water bath.
  9. Remove the heat and again reheat it as needed to keep the slide steaming for about 3-5 minutes. Make sure the moisture of paper maintains, add a few drops of malachite green to keep it moist. Avoid overheating the slide. Don’t add too much stain.
  10. After 5-6 minutes remove the slide from the rack and allow it to cool.
  11. Remove the paper and keep the slide at room temperature for 2 minutes.
  12. Wash the slide by running tap water to remove the malachite green from the both side of slide.
  13. Flood the slide with counterstain, Safranin and wait for 2-3 minutes.
  14. Rinse both side of the slide.
  15. Allow the slide to dry for a few minutes.
  16. Now slide is ready to observe under the microscope.
endospore staining flow chart
endospore staining flow chart

Endospore Staining Results

Endospore: Will appear in green color.

Vegetative Cells: Will appear in brownish-red to pink color.

Endospore Staining by Dorner’s Method


  • Counterstain: Carbolfuchsin stain (it can be easily prepared by 0.3 gm of basic fuchsin, 10 ml of ethanol, 95% (vol/vol), 5 ml of phenol, heat-melted crystals, 95 ml of distilled water).
  • Decolorizer: Decolorizing solvent (acid-alcohol). It can be easily prepared by 97 ml of ethanol, 95% (vol/vol), 3 ml of hydrochloric acid (concentrated)
  • Counterstain: Nigrosin solution. It can be easily prepared by 10 gm of nigrosin, 100 ml of distilled water.


  1. Perform the 1 – 5 steps from the above procedure.
  2. Saturate the blotting paper with primary stain, carbolfuschin.
  3. Now perform the 7-11 steps from the above procedure.
  4. Decolorize the slide by adding acid-alcohol solution on it and then wash it with running tap water.
  5. Placed a drop of nigrosine on one end of a slide and create a thin film of a stain all over the smear with the help of another clean slide.
  6. Allow the slide to dry for a few minutes.
  7. Now slide is ready to observe under the microscope.


Endospore: Will appear in red color, against dark background.

Vegetative Cells: Will appear as colorless.

Endospore Staining Video


  1. https://www.microscopemaster.com/endospore-stain.html
  2. https://en.wikipedia.org/wiki/Endospore_staining
  3. https://iopscience.iop.org/article/10.1088/1742-6596/812/1/012066/pdf
  4. https://microbiologyinfo.com/endospore-staining-principle-reagents-procedure-and-result/
  5. https://www.asmscience.org/content/education/protocol/protocol.3112
  6. https://microbeonline.com/endospore-staining-principle-procedure-results/



MN Editors. (April 2, 2021).Endospore Staining Principle, Procedure, Result. Retrieved from https://microbiologynote.com/endospore-staining-principle-procedure-result/


MN Editors. "Endospore Staining Principle, Procedure, Result." Microbiology Note, Microbiologynote.com, April 2, 2021.


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