In the 19th century, a physicist, named John Tyndall discovered endospores. Endospores are highly resistant, non-reproductive, metabolically dormant structures produced by bacteria commonly found in soil and water. Spore formation (sporulation) occurs in bacteria in approximately 6 to 8 hours after being exposed to adverse environmental conditions.Mature endospores are released from normally-growing cells that are called vegetative cells. When spores are exposed to favorable conditions that support their growth, they can germinate into a vegetative cell within 90 minutes.

Spore-forming Bacteria- Spores are formed by only two genera of Gram-positive rods derived from the Firmicute phylum
Bacillus spp.
Clostridium spp.

Bacterial spores are highly resistant to

  • High temperature
  •  Starvation
  •  Dehydration
  •  Ultraviolet and gamma radiation
  •  Chemicals disinfectant
  •  Lysozymes
  •  Antibiotics

Structure of Bacterial Spore

An endospore is structurally and chemically more complex than the vegetative cell. It contains more layers than vegetative cells. The arrangement of spore layers is as follows:

Exosporium- The spore is sometimes surrounded by a thin covering which overlies the spore coat.
Spore coat – is resistant to many toxic molecules. It acts like a sieve that excludes large toxic molecules like lysozyme. Spore coat may also contain enzymes that are involved in germination.
Cortex – consists of peptidoglycan and present beneath spore coat.
Core wall – lies beneath the cortex and surrounds the core of the endospore.
Core –contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs (small acid-soluble spore proteins). SASPs tightly bind and condense the DNA, and protect the spore DNA from UV radiation, heat, and DNA-damaging chemicals.
Within the core, endospore consists of calcium dipicolinate, which is thought to stabilize the DNA. The resistance of Bacterial spore may be mediated by dipicolinic acid and calcium may aid in resistance to heat and oxidizing agents.

Endospore formation
Under unfavorable conditions especially the lack of carbon and nitrogen sources, a bacterium may start the process of endosporulation, which takes about eight hours. The genetic material of the cell is replicated and a membrane wall known as a spore septum begins to form between it and the rest of the cell. Plasma-membrane surrounds this wall and forms a septum called forespore septum. During this time, Calcium dipicolinate is incorporated into the forespore.
Next, the peptidoglycan cortex formation is started between the two layers and the bacterium adds a spore coat to the outside of the forespore. In the final stages of endospore formation, the newly forming endospore is dehydrated and allowed to mature. The cortex contains an inner membrane known as the core. The inner membrane that surrounds this core leads to the endospore’s resistance against UV light and harsh chemicals that would normally destroy microbes.

Endospore formation and cycle Location
The location of spores varies in different species. Endospores may be present:
• in the middle of the bacterium (central) e.g.Bacillus cereus
• at the end of the bacterium (terminal) e.g. Clostridium tetani, the pathogen that causes the disease tetanus.
• and near the end of the bacteria (subterminal)

The shape of spores also varies in different species. Endospores may be spherical or oval. Spore shape may also be of diagnostic use’

Endospore Staining
Due to the impermeability of the endospore wall to dyes and stains, it is difficult to visualize endospore under the light microscope. In 1922, Dorner published a method for staining endospores. In this method of staining the vegetative cells appear colorless, endospores red, and the background appears black.In 1933, Shaeffer and Fulton modified Dorner’s method of endospore.

Endospore Staining by Dorner’s Method


Carbolfuchsin stain
• basic fuchsin. 0.3gm
• 10 ml of 95% (vol/vol)ethanol
• 5 ml of phenol
• 95 ml of dH2O
Dissolve the basic fuchsin in the ethanol and add the phenol dissolved in the water. Mix and let stand for several days. Filter before use.

Decolorizing solvent (acid-alcohol)
• 97 ml of 95% ethanol
• Conc. HCL, 3ml

Counterstain (Nigrosin solution)
• 10 gm of nigrosine
• 100 ml of dH2O

1. Take a clean grease free slide and make smear.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper
3. Saturate the blotting paper with carbolfuchsin and steam for 5 to 10 minutes, keeping the paper moist and adding more dye as required.
4. Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute; rinse with tap water and blot dry.
5. Take a drop of nigrosine on one end of a slide and make a thin film of a stain all over the smear with the help of another slide.
6. Allow the film of Nigrosin to air dry.
Vegetative cells appear colorless, endospores red, and the background appears black under microscopes.

Shaeffer and Fulton staining
The endospore stain is a differential stain which selectively stains bacterial endospores. The main purpose of endospore staining is to differentiate bacterial spores from non-spore formers and other vegetative cells. In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for the cellular material, so vegetative cells may be decolorized with water. Safranin is then applied to counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will be dark green.

Procedure of Endospore Staining

1. Take a clean grease free slide and make smear.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper.
3. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required.
4. Wash the slide in tap water.
5. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry.
6. Examine the slide under the microscope for the presence of endospores.

Endospores appear bright green and vegetative cells are brownish red to pink under the microscope.

Moeller staining
Another staining technique for endospores is the Moeller stain. That allows the endospore to show up as red, while the rest of the cell stains blue
Moeller staining involves the use of a steamed dye reagent in order to increase the stainability of endospores;
carbolfuchsin is the primary stain used in this method. Endospores are stained red, while the counterstain methylene blue stains the vegetative bacteria blue.
• Carbolfuchsin is applied to a heat-fixed slide.
• The slide is heated over a bunsen burner or suspended over a hot water bath, covered with a paper towel, and steamed for 3 minutes.
• slide is rinsed with acidified ethanol
• Counter-stained with Methylene blue.