Spore staining (Schaeffer-Fulton method): is helpful when other conventional methods won’t work. Some organisms (members of Cloistridium, Desulfotomaculum, and Bacillus) may exist as vegetative cells or spores in order to survive a harsh environment.
Sporogenesis allows an intracellular endospore to develop which is surrounded by layers of spore coats (very tough layers to protect endospore). As the external conditions worsen, the vegetative cell may release the endospore which allows the endospore to become an independent free spore. When external conditions become more favorable, the free spore may revert back (germination) to a vegetative cell.
Spore staining uses 2 reagents
The primary stain is malachite green (with heat). This will color the spores.
The decolorizing agent is water. This will decolorize the vegetative cells.
The counterstain is Safranin which will colorize vegetative cells red.
Clinical significance. Helps to identify members of Cloistridium, Desulfotomaculum, and Bacillus such as Bacillus anthracis (anthrax) and Cloistridia bacteria (tetanus, gas gangrene, food poisoning and pseudomembranous colitis).
Summary of procedure.
- Prepare a smear and heatfix.
- Flood slide with malachite green and place over a beaker of warm water (on a hotplate).
- Steam for 2-3 minutes.
- Cool slide then gently flush with deionized water.
- Flood slide with Safranin for 30 seconds.
- Gently flush slide with deionized water.
- Blot dry with bibulous paper.
Reference
Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.
