Spore Staining


Bacterial endospores formed by the genera Clostridium and Bacillus possess remarkable resistance to physical and chemical agents. Spore does not absorb the stain easily but once stained it does not lose it easily. Heat is applied to force the spores to get a stain. When cooled the will not lose it even when during washing. These remarkable features are due to the presence of dipicolinic acid.

Materials and equipment

 Broth culture of spore-forming bacteria, 3% aqueous solution of Malachite green, safranin, glass slides, inoculation loop, staining rack, hot plate, blotting paper, microscope.

Procedure

  1. Prepare a bacterial smear, air dry, and heat fix.
  2. Cover the smear with a 3% malachite green stain and heat for 1-3 minutes. Avoid boiling and completely drying the stain.
  3. Wash under tap water.
  4. Counterstain with 0.5% aqueous solution of safranin for 30 sec.
  5. Rinse under tap water.
  6. Air dry or blow dry the smear and examine under the oil immersion lens of a microscope.
Note: Vegetative portion of the cell will appear as red while the spores will be visible as green colored. In older preparations, only spores will be visible while vegetative cells will disappear.

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