MicrobeLibrary - Spore Stain of Bacillus subtilis

This is a preparation of Bacillus subtilis, stained to demonstrate endospores. After growth on Nutrient agar at 37 degrees Celsius for 24 hours, the organisms were removed from the plate and suspended in a drop of water on a glass slide. The water was allowed to air dry and then the bacteria were heat fixed to the slide by passing it through the flame of a Bunsen burner 3-4 times. The slide was allowed to cool. The endospores were stained with 5 percent Malachite green by flooding the slide with stain and placing a flame under the slide until the stain solution started to produce steam. The warmed Malachite green solution was left on the cells for 5 minutes. Continuous steaming (heating) of the slide was not necessary. The slide was rinsed with tap water and the cells were counterstained for 30 seconds with 0.25 percent Safranin O (the same counterstain used in the Gram reaction). After completing the staining procedure, if the endospores are not green, modifying the technique to include continuous steaming with Malachite green for 5 minutes may be necessary. The average cell size was 3-5 microns.