Characterisation by staining: Endospore staining

Sampling of probiotic bacteria from food samples and isolation on specialised enriched media


Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure capable of surviving for long periods in an unfavorable environment and then giving rise to a new bacterial cell. This structure is called an endospore since it develops within the bacterial cell. Endospores are spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell. Endospore position within the cell is characteristic and may be central, subterminal, or terminal.

Endospores do not stain easily, but, once stained, they strongly resist decolorization. This property is the basis of the Schaeffer-Fulton (Alice B. Schaeffer and MacDonald Fulton were microbiologists at Middlebury College, Vermont, in the 1930s) or Wirtz-Conklin method (Robert Wirtz and Marie E. Conklin were bacteriologists in the early 1900s) of staining endospores. The endospores are stained with malachite green. Heat is used to provide stain penetration. The rest of the cell is then decolorized and counterstained a light red with safranin.


  1. 24- to 48-hour nutrient agar slant cultures of Bacillus subtilis
  2. Clean glass slides
  3. Immersion oil
  4. Wax pencil
  5. Inoculating loop
  6. Hot plate or boiling water bath with staining rack or loop
  7. 5% malachite green solution
  8. Safranin
  9. Bright field microscope


  1. With a wax pencil, place the names of the respective bacteria on the edge of four clean glass slides.
  2. Aseptically transfer one species of bacterium with an inoculating loop to each of the respective slides, air dry (or use a slide warmer), and heat-fix.
  3. Place the slide to be stained on a hot plate or boiling water bath equipped with a staining loop or rack. Cover the smear with paper toweling that has been cut the same size as the microscope slide.
  4. Soak the paper with the malachite green staining solution. Gently heat on the hot plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to steam. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. Do not allow the slide to become dry.
  5. Remove the paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.
  6. Counterstain with safranin for 60 to 90 seconds.
  7. Rinse the slide with water for 30 seconds.
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