Why is Heat Used in Endospore Staining

A differential staining process is used in staining endospores. The malachite green is the primary stain that stains both vegetative cells and endospores within the sample. Then, the use of heat helps to penetrate the primary stain to the endospore. After decolorization, the counterstain safranin is used to stain the background. 

Two pathogenic genera known as Bacillus and Clostridium produce metabolically inactive endospores to resist adverse environmental conditions. Since endospores cause a number of lethal diseases, the identification of them is quite important in clinical samples.

Key Areas Covered

1. What are Endospores
    – Definition, Facts
2. What is Endospore Staining
    – Techniques of Endospore Staining
3. Why is Heat Used in Endospore Staining
     – Use of  Heat

Key Terms: Endospores, Heat, Malachite Green, Pores, Vegetative Cells

Why is Heat Used in Endospore Staining - Infographic

What are Endospores

An endospore is a resistant structure produced by some pathogenic bacterial genera such as Bacillus and Clostridium in order to survive under unfavorable environmental conditions. These unfavorable conditions can be heat, desiccation, radiation or chemicals. The process of production of endospores from vegetative cells is called sporulation. It may take 8-10 hours to complete the process. Endospores may reside inside the bacterial cell or may exist as free spores. Endospore formation is shown in figure 1.

Why is Heat Used in Endospore Staining_Figure 1

Figure 1: Endospore Formation

Endospores are metabolically inactive or dormant. They contain a small amount of cytoplasm and DNA covered by a protective outer covering. The cell wall consists of dipicolinic acid, giving heat resistant properties to the endospore. Endospores germinate to produce new organisms when the environmental conditions become favorable. The treatment of moist heat at 121 °C for 15 minutes may destroy bacterial endospores. 

What is Endospore Staining

Endospore staining is a differential process of staining in which different structures of the specimen are stained in different colors. It allows the identification of endospores from vegetative cells. The standard endospore-staining process is Schaeffer-Dulton technique. The primary stain in this technique is malachite green, and the counterstain is safranin.

Schaeffer-Dulton Technique – Procedure

1. After smearing on a microscope slide, allow the bacterial sample to saturate with the malachite green solution.
2. Then, gently heat the slide for 3-5 minutes until the dye starts to evaporate.
3. Allow the slide to cool and wash it with water for decolorization.
4. Finally, after counterstaining, rinse the slide.
5. Air-dry the slide and observe it under a microscope.

Here, endospores are stained in green with malachite green and vegetative cells are stained in pink color with safranin. Stained endospores by Schaeffer-Dulton technique is shown in figure 2.

Why is Heat Used in Endospore Staining_Figure 2

Figure 2: Endospores (green) and Vgetative Cells (pink)

An alternative technique of endospore staining can be identified known as Doner’s method. Endospores will be stained in red in this method.

Why is Heat Used in Endospore Staining

The keratin covering of endospores resists staining. Therefore, the primary stain has to be forced into the endospore. The use of heat is to enhance the penetration of the primary stain into the endospore. The slide with malachite green can be heated up to 3-5 minutes. The heating time is directly proportional to the amounts of dyes penetrated into the wall of endospores. Longer heating times make pores on the endospore wall, facilitating the penetration of more dyes.  


Endospores are the reproductive cells of most pathogenic bacteria. Therefore, the identification of endospores in a sample is critical in diagnostics.


1. “Endospore Stain – Understanding Definitions, Techniques and Procedures.” MicroscopeMaster, .

Image Courtesy:

1. “Endospore Formation” By Farah, Sophia, Alex – Own work via
2. “Bacillus subtilis Spore” By Y tambe (original uploader) – Own work via

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

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