Popping test for endospore staining

Endospores are dormant structures formed in various bacteria under unfavourable conditions. Their size is irregular and highly refractile and heat-resistant. The differentiation between spore and vegetative cell is made possible. Most of the anaerobes and members of the bacilli group of bacteria form spores under unfavourable conditions. The spores surrounded by impervious layers cale spore coats. In the worst situation, the spores come out from the cell and survive independently. When the favourable conditions begin, the spore starts germination and gives rise to a new vegetative cell.

After staining, popping of bacteria is visible which can be observed through oil immersion objective, hence this method is called popping test.


  • Starved culture of a bacterium e.g.  Bacillus megaterium
  • Glass slide and coverslips
  • Potassium permanganate (0.1%)
  • Nitric acid (0.3 N)
  • Microscope


  1. Mount a dried film of spores on a cover slip in the acid oxidizer (e.g. 0.1 % KMnO4 in 0.2 N HNO3) and keep it for 10 – 20 min.
  2. Observe under microscope through oil immersion objective. The consequent popping is visible without staining.


R. C. Dubey – practical microbiology

Gaurav Singh

Editor in Chief Medical Microbiology & Recombinant DNA Technology (RDT) Labs - RDT Labs Magazine

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