Staining Bacterial Endospores (Schaeffer-Fulton Method)

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 Slides Diamond glass-marking pencil Slide rack 500-ml beaker Tripod with asbestos mat Forceps

Procedures

Place a drop of water on a slide. Emulsify a small amount of each of the slant cultures in the same drop of water.
Ring and code the slide with the diamond marking pencil.
Stain the slide by the endospore stain:

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).
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.
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.
Counterstain the preparation with safranin for 30 seconds, then rinse again with tap water. Blot or air-dry the slide.
Examine the smear under the oil-immersion objective and record your observations under Results.

Figure 7.2 A simple method for applying heat when staining smears, using the endospore stain technique.

Results

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)? __________________

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