Exercise 13: Endospore Stain 🧫

Be prepared to identify a 24hour culture from a week old culture and be able to explain how you can tell

24 hour culture: Have more spores. Week old culture: Have more cells.

Endospores

Bacterial spore that can survive harsh conditions.

Sporulation

Endospore formation

Know why endospores are so resistant to destruction

It allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress. Endospores resist extremes of heat, drying, freezing, radiation, and chemicals that would kill vegetative cells

Vegetative cell

Metabolically active form of bacterium in which reproduction can occur. The normally-growing cell that forms the endospore is called a vegetative cell.

Be able to name the genus of the two groups that are known to produce endospores

Produced by Bacillus, Clostridia.

Know the purpose and procedure of an endospore stain (cold technique)

Purpose: A differential stain used to visualize bacterial emdospores. 1. Prepare smears of organisms to be tested for presence of endospores on a clean microscope slide and air dry it. 2. Heat fix the smear. 3. Place a small piece of blotting paper over the smear and place the slide on a wire gauze on a ring stand. 4. Heat the slide gently till it starts to evaporate. 5. Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-5 minutes. As the paper begins to dry add a drop or two of *malachite green* to keep it moist. # DO NOT OVERHEAT. 6. After 5 minutes carefully remove the slide from the rack using a clothespin. 7. Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes. 8. Rinse the slide thoroughly with tap water (to wash the malachite green from both sides of the microscope slide). 9. Stain the smear with safranin for 2 minutes. 10. Rinse both side of the slide to remove the secondary stain and blot the slide/ air dry. 11. Observe the bacteria under oil immersion.

Be prepared to name a process as either sporulation or germination.

Sporulation: Induced by environmental conditions Germination: When environmental conditions become favorable; The free spore germinates to a vegetative cell by the rupture of spores. *These are means of survival.