2.5.1: Endospore Stain

Endospores are metabolically inactive structures produced by members of the genera Bacillus and Clostridium. These structures serve to protect a copy of the genetic material in harsh environments and are resistant to extremes in temperature, radiation, and chemicals. Endospores are produced via a process called sporogenesis (sporulation), in which a vegetative cell (sporangium), a metabolic active cell, works to construct several protective layers around a copy of the cell’s genetic material, producing what is known as the endospore.

Endospores are clinically important because several disease-causing microbes can produce these protective structures. Some examples of endospore-forming pathogens include Bacillus anthracis (anthrax), Clostridium tetani (tetanus), Clostridium botulinum (botulism), Clostridium perfringens (gas gangrene), and Clostridium difficile (pseudomembranous colitis).

Endospores are resistant to staining and therefore if present appear as clear structures inside a cell after the Gram stain. In order to visualize endospores, we must use the endospore stain. The Shaeffer-Fulton method will be utilized in this exercise to stain and visualize endospores. This method begins with the addition of malachite green stain applied to the sample in the presence of steam heat. The heat serves to drive the stain through the thick protective layers of the endospore. The slide is removed from the heat and allowed to cool, thereby trapping the stain in the endospore. Malachite green does not have a great affinity for cellular structures, and therefore does not bind readily to the vegetative cell (sporangium) and can easily be washed out of the cell.

This decolorization is accomplished using water. The water is unable to penetrate the protective coat of the endospore, thus the endospore remains green, while the vegetative cell becomes colorless as the water washes the stain out. The counter-stain safranin is then applied to the sample. Safranin easily penetrates the cell wall of the vegetative cell staining it pink, while it is unable to penetrate the endospore coat.

Therefore, at the end of the staining procedure, vegetative cells will appear pink, while endospores appear green.

Endospores will have a spherical or elliptical appearance when observed. They may also be located at different areas of the vegetative cell depending on the species. There are three classifications based on the location of the endospore within the vegetative cell; central, terminal, and subterminal. Central endospores are located at the middle of the bacillus shaped cell, while terminal are located at the very end of the bacillus, and subterminal endospores is an intermediate to the central and terminal endospores. While still in the vegetative cell the endospore may or may not result in a bulging of the vegetative cell.

Bacillus subtilis stained by malachite green and safranin. Endospores in green and the bacteria in red. (x1000)
Source: Y tambe, Wikimedia Commons, CC BY 3.0