Endospore Stain Showing Vegetative Cells in Pink and Endospores in Green, Viewed  Under Oil Immersion @ 1000xTM

What Is a Bacterial Endospore?

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Endospores are hardy, defensive structures that enable some bacteria to survive harmful environmental conditions, such as starvation, high temperatures, desiccation, chemical disinfectants and extremes in pH. Each endospore is produced by a vegetative cell, an active bacterial cell that undergoes metabolism, divides and goes about the daily business of being alive.

Which Bacteria Can form Endospores?
The ability to form endospores, a process called sporulation, is a rare talent. 
Article Summary: Some bacteria are able to produce tough, dormant structures called endospores which allow them to survive when stressed.
Bacterial Endospores & Vegetative Cells Class Notes
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Prokaryotic Cell, Mariana Ruiz

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Staining Bacterial Endospores
Water-based differential staining techniques, such as the Gram stain, will not impart color to endospores. In order to stain these tough structures, the endospore stain is used. 

During the endospore etain protocol, a bacterial smear of an endospore-producing bacteria, such as Bacillus, is prepared. Then the dye malachite green is forced into the spore with heat from a water bath, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of  Mycobacterium in the acid-fast stain
After the primary stain of malachite green is used, the slide is rinsed and the red counterstain safranin is used to impart color to the vegetative bacterial cell. In the end endospores are dyed a blue-green color and vegetative cells are red.

Sources and Resources
  • Bauman, R. (2012) Microbiology With Diseases by Body System, third edition. Benjamin Cummings.
  • Tortora, G., Funke, B. & Case, C. (2013) Microbiology, An Introduction. Pearson.
Endospore Stain Showing Green Endospores and Red Vegitative Cells
Endospore stained bacterial sample viewed
 @ 1000xTM. Vegetative, rod-shaped cells appear red and oval endospores blue-green. 
Click here for more endospore related photos.
Examples of How Different Types of Bacteria Form Endospores
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SPO VIDEO: How to Do an 
Endospore Stain
Page last updated: 5/2014

Bacteria that can do this neat trick are few, but include the notable genera Clostridium and Bacillus. See the Wikipedia article on endospores for a list of endospore-forming bacterial genera.

How are Endospores Formed?
When a vegetative cell of an endospore-forming bacteria detects that essential nutrients are running out it begins to sporulate, a process that takes about 8-10 hours and results in the formation of one endospore.

Steps of sporulation include:
  • cell's plasma membrane pinches off between the replicated chromosomes, forming the forespore
  • a second membrane encloses the forespore, with calcium and dipicolinic acid forming a cortex between the inner and outer membrane
  • an external spore coat encloses the endospore
  • endospore is released once the vegetative cell that generated it dies and disintegrates

As an endospore, the bacterium is in a dormant, inert state, in which it does not metabolize food (eat) or reproduce, but rather exists in a type of suspended animation, kind of like the seed of a plant. When environmental conditions again become favorable, the endospore germinates, resulting in a fresh, new bacterium, identical to the one that formed the spore.

How Were Endospores Discovered?
John Tyndall, a 17th century European physicist, made many contributions to science, including the discovery that some microbes existed in two forms:
  • heat-stable form (endospores)
  • heat-sensitive form (active, living vegetative cells)

Tyndall found that it took either prolonged or intermittent heating to destroy the resistant heat-stable form. The outcome of this research was a method of sterilizing liquid by heating it to boiling point on successive days, now referred to as Tyndallization.

Tyndallization is still useful for sterilization of growth media in science classes and other situations where modern, expensive autoclaves (instruments that use both heat and pressure to sterilize) are not available for sterilization.

Instructor's Corner



Photographic guides to differential stains 
now available!
1. Gram
2. Acid-fast
3. Endopsore