3rd lab spore stain
TRANSCRIPT
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)