Endospores Laboratory Activity
When essential nutrients are depleted from an environment, certain Gram-positive bacteria, such as
those of the genera Clostridium and Bacillus, form specialized “dormant” cells called endospores. Some
organisms from these 2 genera are significant in the medical field since they are known to cause disease.
Species in the genus Clostridium are known to be the causative agent for diseases such as gangrene, tetanus, and
botulism. Species in the genus Bacillus cause anthrax and food poisoning. Endospores are unique to bacteria
and they are highly durable with thick walls and additional layers to help protect them from varying
environmental factors. The endospore is formed internal to the bacterial cell membrane. A similar structure to
the endospore can be formed by the Gram-negative organism Coxiella burnetii, it is not however a true
endospore. Only Gram-positive organisms are known to form endospores.
The process of endospore formation within a vegetative cell takes several hours and is known as
sporulation (Figure 1). Vegetative cells of endospore-forming bacteria begin sporulation when a key nutrient,
such as carbon or nitrogen, becomes scarce or absent. In the lab the key nutrients become limited when cell
replication reaches the end of an exponential growth phase and enters into the stationary phase meaning the
number of dying cells equals the number of replicating cells. Once formed endospores can remain dormant for
thousands of years. An endospore returns to its vegetative state by a process called germination. Germination is
triggered by physical or chemical damage to the endospores coat. The endospore’s enzymes then break down
the extra layers surrounding the endospore, water enters, and metabolism resumes. Since one vegetative cell
forms a single endospore, sporulation in bacteria is not a means of reproduction meaning this process does not
increase the number of cells. Bacterial endospores differ from spores formed by eukaryotic fungi and algae,
which detach from the parent and develop into another organism and, therefore, represents reproduction.
Figure 1. The process of sporulation outlined.
When released into an environment, endospores can survive extreme heat, lack of water, exposure to
toxic chemicals (disinfectants), and radiation. For example, a 7500 year old endospore of Thermoactinomyces
vulgaris from the freezing muds of Elk Lake Minnesota germinated after being warmed and placed in nutrient
media, and 25-40 million year old endospores found in the gut of a stingless bee entombed in amber were found
in the Dominican Republic. After being placed in nutrient media these 25-40 million year old endospores
reportedly germinated!
Endospores are important from a clinical viewpoint and in the food industry because they are resistant to
processes that normally kill vegetative cells. Such processes include heating, freezing, desiccation (drying), use
of chemicals, and UV radiation. Most cells are killed when temperatures reach above 70 degrees Celsius,
endospores however can survive boiling water for several hours or more. Endospores of thermophilic (heatloving) bacteria can survive in boiling temperatures for up to 19 hours. Endospore-forming bacteria are a
problem in the food industry because they are likely to survive under-processing, and, if conditions for growth
occur, some species produce toxins and disease. Special methods for controlling endospores will be discussed
later in the semester.
In this demonstration Bacillus subtillus and Escherichia coli will be tested for the ability to resist the
effects heating and freezing.
Materials: 12 Tryptic Soy Agar plates, inoculating loop, hot plate
Procedure 1 HEAT:
1. A boiling water bath will be placed at your lab bench. Cultures with B. subtillus and E. coli will be
provided.
2. 2 TSA plates will be needed for a time 0 streak plate procedure.
3. Place both culture tubes into the boiling water. Offset the time you place them in the water bath to allow
for streak plating
4. 2 TSA plates will be streaked at T 0, T 1 minute, T 5 minutes, T 10 minutes, and T 15 minutes. For a
total of 10 plates.
Procedure 2 FREEZING:
1. Cultures of B. subtillus and E. coli have been frozen in TSB. Allow cultures to fully thaw and then
streak them on separate TSA plates.
Name:___________________
Endospore Activity Report
Week 1
A hypothesis is a proposed explanation for a natural phenomenon. A good hypothesis should be written to limit
variables for testing.
Form a hypothesis for each of the 2 experiments:
Heat:
Freezing:
Week 2
Record your observations below for each organism at each time point following incubation (use + for little
growth, ++ for medium growth, and +++ for heavy growth).
B. subtillis
Time 0
Time 1 minute
Time 2 minutes
Time 3 minutes
Time 5 minutes
Amount of Growth
E. coli
Time 0
Time 1 minute
Time 2 minutes
Time 3 minutes
Time 5 minutes
Amount of Growth
1. What can you conclude about E. coli’s ability to form an endospore?
2. What can you conclude about B. subtillis’ ability to form an endospore?
3. Did you accept or reject your hypothesis?
4. Which broad group of microorganisms is capable of forming endospores (Gram + or Gram -)?
5. Which genera of bacteria are capable of forming endospores?
6. Why do certain microorganisms form endospores?
7. How does the ability to form an endospore contribute to the ability of a microbe to cause disease?
8. Why is the ability of an organism to form an endospore significant for people who can fruits and vegetables
at home?
9. Use your book. Give the scientific name of the specific organism that causes botulism and list the signs and
symptoms of the disease.
10. Why is the ability of an organism to form an endospore significant for bioterrorism?