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.

What is a bacterial endospore?

Certain pathogenic bacterial genera, such as Clostridium, create endospores or spores in order to survive in hostile environmental conditions. Endospores can occur either within the bacterial cell or as free spores.

Bacteria can continue in this sporulated state until suitable conditions allow them to germinate and revert to a vegetative state.

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.

Principle of Endospore Staining

The primary dye Malachite green is a somewhat weakly binding dye to the cell wall and spore wall. In fact, if thoroughly rinsed with water, the dye is readily removed from the cell wall, but not from the spore wall once it has become immobilised.

The spores are infused with malachite green by heating the bacterial emulsion. This process of heating discolours the vegetative cells and endospores. The water decolorizes the vegetative cells, but the endospores and free spores do not release the stain. On the slide, the safranin counterstain is utilised to colour the vegetative cells. Endospores will be malachite green and seem green (sometimes slightly bluish), while vegetative cells will be brownish-red or pinkish.

Schaeffer-Fulton Method of Endospore Staining

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

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 Results

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

Endospore Staining by Dorner’s Method

Requirement

• 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.

Procedure

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.

Result

• Endospore: Will appear in red color, against dark background.
• Vegetative Cells: Will appear as colorless.

Other Methods of endospore staining

Although the idea of endospore staining is consistent, there are differences in the main stain, counterstain, and use of decolorizer. Several are summarised below.

Method	Primary Stain	Decolorizer	Counterstain	Interpretation
Modified Zeihl-Nelson’s method	Carbol Fuschin	0.25-0.5% sulphuric acid	Leoffler’s methylene blue	Spores appear red, bacteria are blue
Dorner method	Carbol Fuschin	Acid-alcohol	Nigrosin	Spores red Bacteria colorless Background Black
Schaeffer-Fulton Stain	Malachite Green	Water	Safranin	Spores appear green vegetative cells appear pink/red
Bartholomew and mittwer method	Malachite Green	Water	Safranin	Spores appear green  vegetative cells appear pink/red
Abbott’s method	Methylene Blue	Acid alcohol	Aniline fuschin	Spores appear blue bacteria are red
Moeller’s stain	Carbol fuschin	Acidified ethanol	Methylene blue	Spores appear red bacteria are Blue
Modified Moller’s stain	Kinyoun’s Carbol fuschin	2%sulphuric acid and 80% ethanol	Loeffler methylene blue	Spores appear red bacteria are Blue

Applications of the Endospore stain

The endospore stain is a type of differential staining technique used in microbiology to identify and distinguish bacterial endospores from other types of bacteria. The main applications of the endospore stain are:

1. Identification of endospores: The endospore stain allows for the specific detection and visualization of endospores, which are highly resistant structures produced by some types of bacteria.
2. Microbial classification: Endospore staining can be used as a diagnostic tool to differentiate between different types of bacteria based on the presence or absence of endospores.
3. Bacterial viability: Endospore staining can also be used to determine the viability of bacterial populations, as dead endospores will not take up the stain and will appear unstained or lighter in color.
4. Food and water quality control: The endospore stain can be used in food and water testing to identify the presence of endospore-forming bacteria, which can indicate contamination or spoilage.
5. Environmental monitoring: The endospore stain can be used to monitor bacterial populations in soil, water, and other environmental samples to determine the presence of endospore-forming bacteria.
6. Bacterial research: The endospore stain can be used in bacterial research to study the formation, structure, and resistance of endospores, as well as to investigate the mechanisms involved in endospore formation and germination.

Advantages of endospore staining

The endospore stain is a widely used staining technique in microbiology, and it has several advantages, including:

1. Specificity: The endospore stain is highly specific and allows for the specific detection and visualization of endospores, which are resistant structures produced by some types of bacteria.
2. Differentiation: Endospore staining can differentiate between endospore-forming and non-endospore-forming bacteria, which can aid in bacterial classification and identification.
3. Viability determination: Endospore staining can be used to determine the viability of bacterial populations, as dead endospores will not take up the stain and will appear unstained or lighter in color.
4. Ease of use: The endospore stain is a simple and straightforward staining technique that can be easily performed in a laboratory setting.
5. Cost-effective: Endospore staining is relatively low cost compared to other staining techniques, and it can be performed using commonly available reagents.
6. Resistant to decolorization: Endospores are highly resistant to decolorization and heat, and the endospore stain is similarly resistant, which allows for long-term preservation and analysis of endospore-stained samples.
7. Versatility: The endospore stain can be used in a variety of settings, including food and water quality control, environmental monitoring, and bacterial research.

Comments and tips

• Some labs do not use paper towels as described in the Schaefer-Fulton procedure (an ill-fitting piece of paper may burn or leak)
• The age of the culture will affect sporulation. Young cultures (less than or a day old) may have only vegetative cells, whereas older cultures (5 to 7 days old) are excellent for good sporulation.
• Heat fixing should be done with minimal flaming as excess heat will destroy the integrity of the cells, causing them to shrink and to aggregate together on the slide.
• It should be noted that any debris on the slide can also take up and hold the malachite green stain and so caution should be taken when interpreting slides.

Endospore Staining Video

FAQ

References

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/