2nd Lab

SPORE STAIN

• Endospores are a dormant stage of some bacterium that

allows it to survive conditions that would normally kill

bacteria such as extreme drought or heat

• Endospores provide resistance against:

• drying

• Low nutrient conditions

• Radiation

• High temperatures and various chemical disinfectants

Bacterial Endospores

Steps of sporulation include:

bacterium's DNA chromosome replicated (is copied)

cell's plasma membrane pinches off between the

replicated chromosomes, forming the forespore

a second membrane encloses the forespore, with

calcium and dipicolinic acid forming a cortex

between the inner and outer membrane

an external spore coat encloses the endospore

endospore is released once the vegetative cell that

generated it dies and disintegrates

https://www.youtube.com/watch?v=UHsqFjP1dZg&li

st=PL17FCBBBA999CCCC8&index=2

THE SHAPE OF THE SPORE IS AN

IDENTIFYING CHARACTERISTIC

• Swelled vs. Not swelled

spore

Bacterial cell spore

Bacterial cell

spore

THE LOCATION OF THE SPORE

• Central, Sub-Terminal, and Terminal spores

STAIN PROCEDURE

SCHAEFFER-FULTON METHOD1. Prepare a smear. Air Dry. Heat fix

2. Put the slide on steam rack

3. Flood the smear with Malachite Green stain

4. Steam slide for 10 minutes (every minute, add a few more drops of Malachite Green stain)

5. Allow slide to cool (after the 10 min. steam process)

6. Drain slide and rinse for 30 seconds with DI water (discard filter paper)

7. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute)

8. Drain the slide and rinse with DI water

9. Blot Dry

10. Use oil immersion objective to view

Spore Staining procedure

ENDOSPORE STAIN EXAMPLE

SPORES: GREEN

CELL: RED OR PINK

Each student will make a smear and Endospore stain of:

Bacillus subtilis

Uses Nigrosin (negative stain)

In this stain, red endospores are in

colorless vegetative cells.

Endospore staining

(Dorner Method)