Spore Stain: Introduction, Principle, Requirements, Procedure, Result Interpretation and Keynotes

Spore Stain: Introduction, Principle, Requirements, Procedure, Result Interpretation and Keynotes

Introduction of Spore Stain

When conditions for vegetative growth are not favorable, especially when carbon and nitrogen become unavailable spore formation occurs Spore-forming species are represented in most classes, including the Bacilli, the Clostridia, the Erysipelotrichi are able to survive by forming resistant endospores. Spore formation involves a change in enzyme activity and morphology. The spore may be positioned at the end (terminal) of the bacterium or centrally (median). It may be round, oval, or elongate. Endospores being dense and thick-walled, are able to withstand dehydration, heat, cold, and the action of disinfectants. A spore is unable to multiply but when conditions for vegetative growth return, it is able to produce a bacterial cell that is capable of reproducing. Spores of Bacillus species are dormant and extremely resistant to many environmental stresses including, heat, desiccation, radiation, and a variety of toxic chemicals. As a consequence, spores can survive for extremely long periods, certainly hundreds of years and perhaps much longer. Spore resistance is due to a variety of factors, including the outer spore coats and the relative impermeability of the spore’s inner membrane. There are also novel features of the spore’s central region or core, the site of spore DNA, which plays a major role in spore resistance. The spore stain applies to show endospores in the stained preparation that does not only assist the organism is sporulated or not but also to differentiate between vegetative cells and endospores. The most commonly used spore staining is Schaeffer-Fulton Method.  Dorner method is an alternative method that utilizes nigrosin as the counterstain.

Principle of Spore Stain

Malachite green is a primary stain that stains vegetative cells easily but not spores due to their impervious coats, which will not take the primary stain easily. For further penetration, heat is applied. In this preparation, both the vegetative cell and spore will appear green. Washing with tap water removes the primary stain of vegetative cells (colorless) but leaving the spores stained. Safranin is the counterstain that stains vegetative cells and that will absorb the counterstain and appear red. The spores retain the green of the primary stain.

Requirements for Spore Staining

  • Test organisms
  • Control strains (Negative strain is Escherichia coli and positive strain  is Bacillus  subtilis)
  • A clean and grease-free glass slide
  • Bunsen burner
  • Inoculating loop
  • Primary stain
  • Decolourizer ( tap water)
  • Counterstain
  • Heat resistant beaker
  • Hot plate/ laboratory accessible preparation
  • Tissue paper
  • Compound Microscope
  • Cedarwood oil


Procedure of Spore Stain


  • Take a clean, and grease-free slide for making a smear.
  • Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
  • With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times
  • Cover smear with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. Note: Do not allow the stain to evaporate; refill the stain as needed. Check the stain from boiling by adjusting the hot plate temperature.
  • Remove slides from the hot plate, cool, and wash under running tap water.
  • Cover the smear with a counterstain, safranin for 30 seconds.
  • Rinse with tap water.
  • Leave for air drying or if urgent blot dry with bibulous paper and observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Result Interpretation of Spore Stain

Endospores: Endospores/ free spores are bright green.

Vegetative Cells: They are brownish red to pink.

Negative control: Absence of spores

Positive Control: Presence of green spores

Test organisms: Sporulated (green) and non-sporulated color of the counterstain

Keynotes on Spore Stain

  1. Endospores can survive without nutrients.
  2. An endospore is a resting, tough, and non-reproductive structure and is formed normally due to a lack of nutrients, and generally occurs in gram-positive bacteria. spores remaining viable over 10,000 years, and revival of spores millions of years old has been maintained.
  3. The endospore is made of DNA, ribosomes, and large amounts of dipicolinic acid. Dipicolinic acid is a spore-specific chemical that emerges to assist in the ability of endospores to maintain dormancy and thus save from ultraviolet radiation, desiccation, high temperature, extreme freezing, and chemical disinfectants.
  4. Endospore forming bacteria is Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum species, Sporolactobacillus species, Sporosarcina species, Coxiella burnetii whereas negative bacteria are E. coli, Klebsiella, Salmonella, etc.
  5.  Schaeffer-Fulton is the most common staining method of spores.

Further Readings

  1. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacterial-spore
  2. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  3. District Laboratory Practice in Tropical Countries – Part-2-   Monica Cheesebrough-   2nd Edn Update
  4. https://micro.cornell.edu/sites/micro.cornell.edu/files/shared/documents/Hutchison2014.pdf
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1482921/
  6. https://en.wikipedia.org/wiki/Endospore
  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC277547/
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