Endospore Staining

Content:

  • About
  • Principle
  • Requirements
  • Procedure
  • Observation and result

About:

  • During unfavourable condition some bacteria goes in its dormant structure that is metabolically inactive and unable to reproduce and grow.
  • They develop structure called endospore which protects the cell from lethal agents like heat, radiation and chemicals.
  • In clinical procedure, pathogen identification include the describing the shape and position of endospores present in the bacterial cell.

Principle:

  • This method of staining is a differential staining which is used to detect, identify and differentiate endospore from vegetative cell.
  • The role of the method is to detect presence or absence of endospore some modification is done by increasing concentration of dye, increasing heat fixing duration and application of ultraviolet radiation.

Requirements and Reagents:

  • Fresh culture of B. cereus or B. subtilis and Staphylococcus aureus
  • Malachite green (5% aqueous)
  • Safarnin
  • Staining tray
  • Glass slide
  • Blotting paper
  • Microscope

Procedure:

  • Take separate clean slides and make smear of B.subtilis and S. aureus.
  • Air dry and heat fix.
  • Add few drops of malachite green on the smear.
  • Heat the slide under steam and add stain to avoid dryness.
  • Wash the slide with water running slowly on the slide.
  • Add few drops of counter stain i.e. safranin for 30 seconds.
  • Wash out the smear with slowly running distilled water.
  • Dry the slide with blotting paper.

Observation:

  • Observe the slide under oil-immersion microscope. From the microscopic field represent the size and position of the endospore. Write the colour of the spore and vegetative cell.

Result:

  • In B.cereus the endospore stain green and in vegetative cell stain red. Vegetative cell are rod shaped and S. aureus are spherical in shape.

Example:

TypeExample
PositiveBacillus anthracis, C. botulinum, Clostridium perfringens
NegativeSalmonella spp, E. coli

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