The Endospore Staining Technique


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endospore staining
A stained preparation of Bacillus subtilis showing endospores as green and the vegetative cells as pink. (credit: modification of work by American Society for Microbiology)

The Endospore Staining (OpenStax Microbiology)

Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain (malachite green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin. The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible.

Endospore-staining techniques are important for identifying Bacillus and Clostridium, two genera of endosporeproducing bacteria that contain clinically significant species. Among others, B. anthracis (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible for the typically hospital-acquired infection known as “C. diff.”

Related Topic: Preparing Specimens for Light Microscopy


Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at:


Other Related Research:

Research Article: A Monoclonal Antibody That Tracks Endospore Formation in the Microsporidium Nosema bombycis

Date Published: March 26, 2015 Publisher: Public Library of Science Author(s): Yanhong Li, Meiling Tao, Fuping Ma, Guoqing Pan, Zeyang Zhou, Zhengli Wu, Erjun Ling. Abstract: Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such … Continue reading

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Endospore Staining- Principle, Reagents, Procedure and Result