Staining Bacterial Endospores (Schaeffer-Fulton Method)


Basic Techniques of Biotechnologies
  Differential Stains
    Gram Stain
    Acid-Fast Stain
    Special Stains
      Staining Bacterial Endospores (Schaeffer-Fulton Method)
      Flagella and Capsule Stains

Purpose To learn a technique for staining bacterial endospores
Materials 3- to 5-day-old agar slant culture of Bacillus subtilis
24-hour-old slant culture of Staphylococcus epidermidis
Malachite green solution
Safranin solution
Diamond glass-marking pencil
Slide rack
500-ml beaker
Tripod with asbestos mat

  1. Place a drop of water on a slide. Emulsify a small amount of each of the slant cultures in the same drop of water.
  2. Ring and code the slide with the diamond marking pencil.
  3. Stain the slide by the endospore stain:
    1. Place the slides on a slide rack extended across a beaker of boiling water held on a tripod (an electric burner may be used instead of a Bunsen burner). An asbestos mat should protect the beaker from the Bunsen flame beneath (fig. 7.2).
    2. Flood slide with malachite green and allow to steam gently for 5 to 10 minutes. The stain itself should not boil; if it does, reduce the heat. If the stain appears to be evaporating and drying too rapidly, add a little more. Keep the slide flooded.
    3. Allow the slide to cool slightly, then use forceps to drain the slide over a sink or staining tray and rinse with water for about 30 seconds until no more green washes out.
    4. Counterstain the preparation with safranin for 30 seconds, then rinse again with tap water. Blot or air-dry the slide.
    5. Examine the smear under the oil-immersion objective and record your observations under Results.
A simple method for applying heat when staining smears, using the endospore stain technique.
Figure 7.2 A simple method for applying heat when staining smears, using the endospore stain technique.

  1. In the circle, make a sketch of your microscopic observations. Note the difference between the staining properties of the staphylococci and the endospores. Use colored pencils if they are available. Indicate the color of the spores in contrast to the vegetative cells.

    What is the location of the endospore within the bacterial cell (e.g., terminal, subterminal, central)? __________________