The following activity will teach students how to complete an endospore stain and differentiate between vegetative cells and endospores. Read the following procedure carefully before starting the activity.
Materials
2 microscope slides
Wax pencil
Bacillus cereus (24-hour) and Bacillus cereus (5-day)
Label the edge of the microscope slide with 1 person's initials and the bacterial species. You may abbreviate the name of the bacteria so it fits on the slide. Try to write small.
On the bottom of the slide, you may draw a circle in the middle of the slide with sharpie a or wax pencil so you know where to make your smear. This is optional.
Sterilize your inoculation loop and use it to pick up a small amount of DI water. Place the water on top of the slide, in the middle or, in the circle if you drew one.
Sterilize your loop again and pick up a small bacterial colony from the stock plate.
Gently, mix the bacteria into the water drop on the slide. You want to spread the water and bacteria out to about the size of a coin.
Let the water and bacteria smear completely air dry. The slide should look dry and "crusty" before moving to Step 2.
Step 2: Heat Fixation
Once the bacterial smear is completely dry, use a clothespin to clip the end of your slide.
Then, place your slide directly on a hot plate for 30 seconds.
After 30 seconds, place your slide onto the slide stand in your staining tray. Don't touch the slide, it will be hot. Don't place the slide on the cold table, the glass slide could crack.
Once your slide is completely cool, move on to Step 3.
Step 3: Spore Staining
Primary Staining
Take your heat-fixed slide to the fume hood.
Once the water is boiling, place your slide on the slide rack above the boiling water.
Cover the smear area of your slide with a square piece of bibulous paper.
Carefully apply the malachite green stain to the bibulous paper.
Steam with the stain on the slide for 5 minutes while continuously applying more stain so the bibulous paper never dries out.
After 5 minutes remove the paper with forceps and discard it in the trash bin.
Using a clothespin take your slide back to your lab station and rinse with water.
Decolorizer, Wash Step
Hold the slide at a 45° angle and wash the slide with a gentle DI water rinse.
Counterstain
Add a few drops of safranin to the smear.
Leave the stain for 1 minute.
Hold the slide at a 45° angle and rinse with DI water.
Place the slide between 2 sheets of bibulous paper and gently pat dry. Do not wipe the slide to dry it.
Microscopy
When the slide is dry, observe your specimen using oil immersion.
Repeat Steps 1 through 3 for the remaining bacterial species.
When both slides are done, clean your microscope and place it back in the cabinet.
Rinse your staining tray in a sink and put all materials back in their labeled locations.
Results
Species
Vegetative Cells (+/-)
Endospores (+/-)
Spore Location
Bacterial Morphology and Arrangement
Bacillus cereus (24-hour)
Bacillus cereus (5-day)
General Questions
1. Fill in the blanks: ___________________ are the metabolically active cells that stain ___________________ during the spore stain procedure. ___________________ are mainly metabolically inactive and stain ___________________.
2. Why might a species of bacteria form endospores? What is the advantage of forming spores for bacteria?
3. What genera of bacteria are known to have pathogenic (i.e., disease-causing) species and are spore-forming genera?
4. What is the purpose of the steam in the spore stain?
5. Which step serves as the differential step? Explain your answer.
6. What role do broad-spectrum antibiotics play in the development of C. difficile infections, and how do they affect the normal gut flora?
7. Why is the ability of the endospore stain to identify C. difficile endospores important for infection control in healthcare settings?
