Some Bacteria such as Bacillus and Clostridium have the ability to produce endospore. Endospores are resistant to heat, drying, radiation and various chemical disinfectants.
Endospores formation (sporulation) :
It occurs through a complex series of event. One endospore is produced within each vegetative bacterium. Once the endospore is formed, the vegetative portion of the bacterium is degraded and dormant endospore is released.
Endospore contains :
- DNA, ribosome, cytoplasmic membrane, cell wall and then everything else that are essential for bacterial life.
- These cell constituents are then surrounded by a series of protective coats including a cortex, a spore coat, and sometimes an exosporium. The dehydrated state of the endospore also contributes to its resistance.
- Endospores are able to survive for a long period of time until environmental conditions become favourable for growth. The endospore then germinates producing a single vegetative bacterium.
- Endospores are difficult to stain, due to its resistant nature to heat and chemicals. Strong dyes and vigorous staining conditions are required. However once stained, endospores are hard to decolorize. Since few bacterial genera produce endospores, the endospore stain is good diagnostic test for species of Bacillus and Clostridium.
Procedure :
- Smear preparation and heat fixing of smear.
- Place a small piece of paper towel square over the smear and saturate with
MALACHITE GREEN for 30-60 seconds.
- Heat gently by passing the Bunsen burner under the slide for 5 minutes. Heat the slide until steam can be seen rising from the surface. Remove slide the heat until steaming stops; then gently reheat. As the dye evaporate, continually add more.
- Allow slide to cool and wash the excess stain. Remove the paper towel square off the slide with a forceps and wash off excess stain with didtilled water.
- Blot the slide dry and counterstain for 1 minute with 0.5% SAFRANIN.
- Wash off the excess safranin with water, blot dry, and observe using oilimmersion microscopy.
Results :
The vegetative cells will appear red and the spores will appear green.
