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Differential Staining: Capsule and Spore Staining

Similar to Gram stain, acid fast stain, and flagellar stain, capsule and spore stain are used to differentiate between microbes.


Purpose: Our immune system contains neutrophils and macrophages that fight against foreign bodies or antigens. Capsules, which contains mucoid polysaccharides or polypeptides, protects bacterial cells against our immune system (macrophage and neutrophils). Capsules are also resistant to stains, hence capsule stain techniques are staining around the cell and not the cell wall or membrane directly (different from Gram and Acid fast stains).

Acidic or Negative Stain – used to stain the background

  • Nigrosin
  • Congo Red
  • India Ink

In this first step, there is NO heat fixing because it will cause the cells to shrink, which will give a false reading of a white halo around the cells.  This may cause a misrepresentation of a capsule.

Cytoplasm, basic stain

  • Maneval’s
  • Carbol fuschin

The unstained, halo, seen between the stain in cells are the capsule.

Spore or Endospore Staining

All spores are Gram +

Purpose of forming an endospore:

– dormant stage of a cell allowing it to survive in harsh environments

  • protects the microbe in a nutrient deficient environment
  • protects the microbe from dessication (drying up)

How does the spore beneficial for the bacterial cell?

Spores are resistant to heat and chemicals because of the protein covering of the cell, called keratin.

How does Keratin affect the spore staining and what needs to be done?

Keratin also prevents the stain from entering the spore, hence steaming is performed to force the primary stain, malachite green, into the spore. This is known as bacterial emulsion in the endospore staining method of Schaeffer-Fulton.

Common spore forming species?

Bacillus and Clostridium. Most Bacillus is found in soil, freshwater, or marine saprophytes.  However, there are other pathogens like, Bacillus anthracis – causing anthrax

Clostridium are found in soil, aquatics saprophytes, or in the human intestines.

Four well known ones are:

  • C. tetani (tetanus)
  • C. botulinum (botulism)
  • C. perfringens (gas gangrene)
  • C. difficile (pseudomembranonous colitis)

Older or Newer Cultures of Bacillus will show higher spore formation?

Older ones! Due to lacking in nutrients and competitive living environment, it is the older cultures of this bacteria that will show more spores!

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