Endospore Staining-Principle, Reagents, Procedure And Result Interpretation

By Prof Mariam M Mirambo


Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria.Within bacteria, endospores are protective structures used to survive extreme conditions, but this protective nature makes them difficult to stain using normal techniques such as simple staining and Gram staining. Special techniques for endospore staining include the Schaeffer–Fulton stain and the Moeller stain.

Principle Of Endospore Staining

The primary dye for endospore staining is malachite green. It takes a long time for the spores to stain due to their density, so time acts as the mordant when performing this differential stain; the slide with the bacterium should be soaked in malachite green for at least 30 minutes and then rinsed off with water which acts as the decolorizer. A counterstain to differentiate the vegetative cells is commonly 0.5% safranin. In the end, a proper smear would show the endospore as a green dot within either a red or pink-colored cell.Terbium can also used to detect endospores, as it acts as an assay of dipicolinic acid based on photoluminescence.

Types of endospores that may be identified include free endospores, central endospores, central and swollen endospores, and subterminal endospores. Mycobacterium is one obstacle that is faced with this type of staining because it will still stain green even though it does not produce any endospores. This is due to its waxy cell wall which retains the malachite green dye even after the decolorizing process. A different type of staining called acid-fast stain will have to be done in order to get further information about this particular type of bacterium.


Method 1: Schaeffer Fulton Stain

Reagents And Materials Required

  • Glass Slides
  • Specimen/Bacterial culture
  • Tissue paper
  • Inoculating loop
  • Spirit lamp/Bunsen burner
  • Staining tray
  • Microscope
  • Wash Bottle
  • Tap water/ Distilled water
  • Safranine (2.5% aqueous solution)
  • Malachite green (5% aqueous solution)

The 0.5% aqueous solution of Malachite green is prepared as follows:

  • 5 gm of malachite green.
  • 100 ml of distilled water

Mix the 0.5 gm of Malachite green powder in the 100 ml of Distilled water.

Prepare the 2.5% aqueous solution of safranine as follows:

  • 5 gm of safranine O
  • 100 ml of 95% ethanol

Mix the 2.5 gm of Safranine O powder in 100 ml of 95% Ethanol.

Procedure Of Endospore Staining

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

Interpretation of results

The Cells containing endospores appears as the red colored rod-shaped structure along with an intracellular spherical or elliptical green colored structure. It represents the red colored vegetative bacilli with green colored endospores (intracellular spores).The non-sporing bacterial cell appears red in color but no intracellular green color structures present which makes it easier to distinguish the sporing & non-sporing cells.

The vegetative forms stain pink/red because they take up the counterstain (Safranin) while the endospores take up the green from the Malachite green.This is because, during smearing and heat fixing, the malachite green penetrates into the endospore with the help of the heat from the steam, and during the water-rinse, the dye is not easily washed away. And for the vegetative forms, the dye is easily washed away because of their fragile outer covering, hence they take up the last stain which is the counterstain, hence they appear pink-red.

Examples of endospore stain positive organisms

  • Clostridium perfringens
  • C. tetani
  • C. botulinum
  • Bacillus cereus
  • Bacillus anthracis
  • Sporosarcina spp
  • Desulfotomaculum spp
  • Sporolactobacillus spp

Examples of endospore stain negative organisms

  • E. coli
  • Salmonella spp 

Method 2: Dorner’s Method


  • Carbolfuchsin stain (0.3 gm of basic fuchsin,10 ml of ethanol, 95% (vol/vol), 5 ml of phenol, heat-melted crystals and 95 ml of distilled water).
  • A decolorizing agent (acid-alcohol) {97 ml of ethanol, 95% (vol/vol), 3 ml of hydrochloric acid (concentrated)}.
  • A counterstain (nigrosin solution) {10 gm of nigrosin and100 ml of distilled water}.


  1. Take a clean grease free slide and make smear using sterile technique.
  2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide.
  3. Saturate the blotting paper with carbolfuchsin and steam for 5 to 10 minutes, keeping the paper moist and adding more dye as required. Alternatively, the slides may be steamed over a container of boiling water.
  4. Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute; rinse with tap water and blot dry.
  5. Further take a drop of nigrosine on one end of a slide and make a thin film of a stain all over the smear with the help of other slide.
  6. Allow the film of Nigrosin to air dry.
  7. After air drying observe the slide under oil immersion.


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

Uses of the Endospore stain

  • For detection of Firmicute groups of bacteria i.e Clostridium spp and Bacillus spp
  • For identification of endospore producing bacteria in samples
  • for differentiation of spore-producing bacterial from vegetative forms of bacteria

Further References

  1. http://www.austincc.edu/microbugz/endospore_stain.php
  2. https://en.wikipedia.org/wiki/Endospore_staining
  3. https://microbeonline.com/endospore-staining-principle-procedure-results/
  4. https://microbiologyinfo.com/endospore-staining-principle-reagents-procedure-and-result/
  5. https://www.microscopemaster.com/endospore-stain.html
  6. http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/endospore_stain.htm
  7. http://www.asmscience.org/content/education/protocol/protocol.3112
  8. https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-specimens/
  9. https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/differential-staining-techniques/
  10. https://www.scienceprofonline.com/microbiology/endospore-bacteria-stain-procedure.html