Spore Staining

 Aim

To perform endospore staining and to visualize endospores

Principle

During unfavorable conditions or environmental stresses such as nutrient deprivation, certain bacteria such as Bacillus spp and Clostridium spp form endospores.  The endospore helps them to survive the adverse conditions. They are formed within different areas of the cell, central, subterminal, or terminal.   Most endospore forming bacteria are found in soil or aquatic environments.  Some species have medical significance.  Clostridium perfringens, C. botulinum and C. tetani are the causative agents of gas gangrene, botulism and tetanus, respectively and Bacillus anthracis and Bacillus cereus cause anthrax and food poisoning.

The Schaeffer-Fulton method, a differential staining technique is used to perform spore staining.  A primary stain (malachite green) is used to stain the endospores. Since endospores resist staining, heating is done as a mordant to force the malachite green into the endospores. Washing step will remove the stain from vegetative cells, but not from the endospore and the vegetative cells will uptake the counterstain or secondary stain, safranin.

Materials required

1. Cultures - 48 hour old Bacillus species culture

2. Reagents - Malachite green, safranin

3. Equipments - Bunsen burner, inoculating needle, staining tray, microscope

Procedure

1.  The bacterial smear was prepared and heat fixed in a clean glass slide

2. The smear was flooded in Malachite green and the slide was heated gently till it starts to evaporate for about 3-5 minutes. 

3. After 5 minutes the slide was allowed to cool to room temperature.

4. Malachite green was washed off gently using slow running tap water

5.  The smear was flooded with safranin for 2 minutes.

6. The slide was washed off gently using slow running tap water

7. The smear was air dried and observed under oil immersion objective

Observation

The vegetative cells appeared pink/red, in the color of counter stain and the vegetative cells having endospores appeared as red cells having green endospores within.  Mature, free endospores appeared in green colour.

Results

The vegetative cells appeared in pink/red colour and the spores were green in colour.