Endospore Staining: Principle, Procedure, Results

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.

Principle

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.

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.

Results

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.

References and further reading

• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology
• Hussey, M. A., & Zayaitz, A. (2007, September 29). Endospore Stain Protocol. https://www.asmscience.org/content/education/protocol/protocol.3112