1.3: The Myth of Spontaneous Generation

Learning Objectives

• Describe spontaneous generation theory and state that this is a theory that was once widely accepted and is an incorrect / false idea.
• Describe biogenesis theory and state that this theory is currently widely accepted.
• Apply the term "turbid" to microbiological cultures and the implications of turbidity.
• Describe Spallanzani's experiment that began to disprove spontaneous generation of microorganisms and why the scientific community did not reject spontaneous generation theory following his experiment.
• Interpret the meaning of Spallanzani's experimental results disproving spontaneous generation of microorganisms.
• Describe Pasteur's experiment that definitively disproved spontaneous generation of microorganisms and why the scientific community accepted the results of this experiment.
• Interpret the meaning of Pasteur's experimental results disproving spontaneous generation of microorganisms.
• Describe the achievements of Tyndall related to sterilization of media.
• Define endospore.
• Explain why endospores prevent sterilization of microbiological media by boiling only.
• Recreate and interpret Spallanzani's experiment disproving spontaneous generation of microorganisms.
• Recreate and interpret Pasteur's experiment disproving spontaneous generation of microorganisms.

 

The Myth of Spontaneous Generation

Spontaneous generation theory is a myth. It is a false idea that was once a widely accepted belief in the scientific community. Its ideas were so strong that it took multiple experiments by several different scientists to disprove the theory and start to shift the mindset about how living things arise.

Spontaneous generation theory says living things can arise or come from non-living things. In the microbial world, it was believed the microorganisms could be created from a fluid such as a beef broth. This is because a beef broth left out would inevitably begin growing microorganisms that scientists observed using their microscopes. These microbes did not come from the broth however as the scientists of the time believed. In reality, the microorganisms were either in the broth already (in small amounts) or landed in the broth from the air. Once even one cell was present in the beef broth, the broth could be used by the cells as a source of nutrients they used to be able to grow, divide, and produce a robust population of microorganisms that could then be seen with the microscope.

 

Figure 1: (Left) Spontaneous generation is an incorrect/false theory where scientists of the past believed that a liquid broth could create microbial cells from the broth itself (life coming from non-living matter). (Right) In reality, cells come from other cells (cell theory) or life comes from life (theory of biogenesis). A microbial cell divides to produce other microbial cells. The theory of biogenesis is the correct/true theory currently accepted today.

 

Instead of the theory of Spontaneous Generation, today the theory of biogenesis is currently accepted by the scientific community. This theory says that life comes from life. In other words, living things do not arise from non-living matter. A related theory that is also currently accepted is Cell Theory and it says that cells come from other cells (among other things).

 

Historical Experiments Disproving Spontaneous Generation of Microorganisms

Spallanzani's Experiment Disproving Spontaneous Generation Theory of Microorganisms

Lazzaro Spallanzani was an Italian priest who re-examined the spontaneous generation of microorganisms (e.g. bacteria) using a nutrient-rich broth such as a meat broth. He designed and conducted a famous experiment that began to question the validity of spontaneous generation theory.

A nutrient broth that is not sterile will eventually show microbial growth by becoming cloudy unless it is sterilized and microbes are prevented from entering the broth (usually by sealing the container). Cells present in the broth will use the broth as nutrients, divide, and produce a robust population of microorganisms. The broth can be examined using the microscope so that microorganisms growing there can be observed. A broth that begins clear that contains one or more cells (or is exposed to the environment where cells can enter the broth through the air), given time for microbial growth, will become turbid (cloudy or thick).

Figure 2: Comparison of sterile microbiology medium (left) that is a clear liquid and a turbid medium (right) that is a cloudy liquid. The sterile medium lacks live microorganisms. The turbid broth contains a dense population of microorganisms.

 

Spallanzani boiled nutrient broth to kill the microorganisms (sterilization). He compared covered and uncovered boiled nutrient broths to see if the broths would become turbid (cloudy), indicating microbial growth. What Spallanzani observed was the uncovered boiled broth became turbid over time since microorganisms were able to enter the broth from the air. The covered boiled broth however did not become turbid since microorganisms could not enter the sterile broth. This result indicated microorganisms were in the air and would contaminate a broth if exposed.

 

Figure 3: Diagram showing Spallanzani's experimental setup and results. (Top) An uncovered flask containing clear broth is sterilized by boiling. After a period of time (hours/days) the flask is re-examined and found to be turbid. This turbidity is microbial growth in the uncovered flask. Microbes from the air were able to land in the sterile broth and begin growing and dividing to produce a microbial population/community that appears turbid. (Bottom) A covered flask containing clear broth is sterilized by boiling. After a period of time (hours/days) the flask is re-examined and found to be clear. This clear appearance in the sealed flask is an indication that medium remained sterile. Since the sterile medium was sealed, microbes could not land in the medium and begin growing and dividing. As a result, the medium remained sterile.

The scientific community, having believed that spontaneous generation was a fact of nature, still resisted the possibility that spontaneous generation is not possible for microorganisms. The argument was that the sealed flask in Spallanzani's experiment was closed off from an oxygen (O₂). Perhaps microorganisms could not grow in the broth because there was not enough O₂ present in the flask? As a result, spontaneous generation theory persisted.

 

Pasteur's Swan-Neck Experiment Disproving Spontaneous Generation Theory of Microorganisms

Louis Pasteur designed and conducted an experiment that provided strong evidence disproving spontaneous generation of microorganisms. Pasteur used a flask with a swan-neck that could prevent microbes, including those attached to dust particles in the air, from reaching the sterile nutrient broth while enabling O₂ to pass into the flask. Since Spallanzani's experiment was scrutinized because O₂ could not reach the nutrient broth. The fact that Pasteur developed a flask that allowed O₂ to the broth and not microbes in the air created the conditions the scientific community needed to accept that spontaneous generation of microorganisms does not occur (microbes do not come from non-living matter).

 

Figure 4: Animation showing Pastures' swan-neck experiment. Two flasks are compared to establish the validity of opposing scientific theories: spontaneous generation theory and biogenesis theory. Two swan-neck flasks are prepared with nutrient broth. Both are sterilized by boiling. One flask the swan-neck is unaltered (experimental) and the other flask the swan-neck is broken off (control). The flask with the swan neck remains clear and sterile. The flask with the broken swan-neck became turbid. This indicated that cells must be introduced into a nutrient broth before microbial growth will occur.

 

In Pasteur's experiment, a nutrient broth is sterilized in a flask with a swan-neck tube attached. The swan-neck enabled O2 to reach the broth. The low dip in the swan neck traps dust and microbes and prevents them from reaching the broth. After sterilization, the flask with the swan-neck stayed sterile indefinitely, despite being open to the air. This design effectively trapped microbe-carrying dust particles that could contaminate a sterile growth medium (i.e. introduce microorganisms to the sterile growth medium). If the swan-neck tube was broken off, the result is a direct path for microbes in the air and attached to dust particles to enter the growth medium. Given time, a sterilized flask with the swan-neck broken off became turbid (cloudy), indicating microbial growth. These results indicated that microorganisms come from other microorganisms and that microorganisms do not come from non-living broth. This result conclusively settled the dispute about spontaneous generation: spontaneous generation theory was incorrect and biogenesis theory is correct.

 

Tyndall's Discovery and Endospores

Pasteur’s swan-necked experiment was then challenged when John Tyndall, an Irish scientist, repeated Pasteur’s experiment and found some boiled growth media remained sterile while others did not, despite very long boiling times. Through a series of experiments, Tyndall found that nutrient broth can contain heat-resistant microbes (now known as endospores – discovered by Ferdinand Cohn the same year as Tyndall’s work). Endospores can be produced from certain bacterial species (e.g. Bacillus sp. and Clostridium sp.) when growth/nutrient conditions are poor. Endospores are analagous to a survival bunker where bacterial cells transfer their DNA into a protective structure to survive the harsh conditions. Endospores can change back into normal bacterial cells (called vegetative cells [these are active bacterial cells whereas endospores are an inactive form]) if/when environmental conditions improve and are better for the microbes to grow.

 

Figure 5: (A) Endospores form from bacterial cells. Diagram shows the process how bacterial cells experiencing poor growth conditions can form endospores in the following steps: 1. DNA is replicated, 2. cellular division of cytoplasmic membrane, 3. prespore formation begins, 4. cortex forms, 5. spore coat formation begins, 6. maturation of exosporium formation, 7. mother cell releases mature spore. (B) Microscopic image prepared with an endospore stain shows bacterial cells (pink) with forming spore inside of them (green). (C) Microscopic image showing endospores.

 

Tyndall developed a process that he found completely sterilized growth media, including killing those containing heat-resistant microbes (endospores). This process is a lengthy series of steps involving repeatedly heating then resting the medium multiple times. This repeated heating and resting process insures sterilization of a medium.

 

Figure 6: An autoclave. Autoclaves provide a rapid means of sterilization using pressure and head and they come in different shapes and sizes. This is an example of a free-standing autoclave with media bottles inside ready to be sterilized.

 

More recently, it has been found endospores can also be killed when medium is heated under pressure. The typical method of sterilization used in laboratories today uses an autoclave, a device that places materials to be sterilized under pressure while heating. What used to take a few days to sterilize medium with Tyndall's process now can be sterilized in about an hour.

 

Re-creation of Lazzaro Spallanzani’s Spontaneous Generation Experiment

In this laboratory activity you will re-create Spallanzani's experiment. debunk the myth of spontaneous generation... again!

Laboratory Instructions

1. Write your group name, experiment name, and the date on two pieces of tape and place on the outside of two test tubes (put tape about half way down on the test tube so you can add a cap onto one of the test tubes).
2. Use a graduated cylinder to measure TSB.
3. Put 8 mL of TSB into each of the two test tubes.
4. Put a cap onto one of the test tubes and leave the other test tube uncapped.
5. Put test tubes in a central location indicated by your instructor. Your test tubes will be autoclaved by your instructor to sterilize them (kill all microbes) and then placed onto a bench.
6. Create a hypothesis with your group:
   1. Do you expect that the covered, sterilized test tube will show microbial growth? Why or why not?
   2. Do you expect that the uncovered, sterilized test tube will show microbial growth? Why or why not?
   3. If you said that microbial growth will occur in one of the test tubes, where did the microbes come from?
7. You will examine your results next class. If the solution is turbid (foggy), that indicates microbial growth. If the solution is clear, that indicates no microbial growth.
8. Results:

1. 1. Covered, sterilized tube:
   2. Uncovered, sterilized tube:

9. Conclusions. What do the results you listed above mean about spontaneous generation? Consider your hypotheses above to help you answer.

 

Re-creation of Louis Pasteur’s Spontaneous Generation Experiment

In this laboratory activity you will re-create Pasteur's experiment. Debunk the myth of spontaneous generation... again!

Laboratory Instructions

1. Write your group name, experiment name, and the date on two pieces of tape and place on the outside of two 125 mL flasks.
2. Use a graduated cylinder to measure TSB.
3. Add 50 mL of TSB to each of the 125 mL flasks.
4. You may or may not be asked to bend glass tubing into a swan-neck shape using a Bunsen burner.
5. Put one swan-neck glass tube into one stopper and a straight glass tube into another stopper.
6. Place the two stoppers securely into the 125 mL flasks.
7. Put the flasks in a central location indicated by your instructor. Your flasks will be autoclaved by your instructor to sterilize them (kill all microbes) and then placed onto a bench.
8. Create a hypothesis with your group:
   1. Do you expect that the sterilized flask with the swan-neck glass tube will show microbial growth? Why or why not?
   2. Do you expect that the sterilized flask with the straight glass tube will show microbial growth? Why or why not?
   3. If you said that microbial growth will occur in one of the test tubes, where did the microbes come from?
   4. Why were scientists more convinced that spontaneous generation was incorrect by Pasteur's experiment and less convinced by Spallanzani's experiment?
9. You will examine your results next class. If the solution is turbid (foggy), that indicates microbial growth. If the solution is clear, that indicates no microbial growth.
10. Results:

1. 1. Sterilized flask with swan-neck tube:
   2. Sterilized flask with straight tube:

11. Conclusions. What do the results you listed above mean about spontaneous generation? Consider your hypotheses above to help you answer.

 

Attributions

• Chapter Image: Swan-necked flask used by Pasteur. Wellcome M0012521.jpg by Wellcome Images is licensed under CC BY 4.0
• 202004 flask yellow.svg by DataBase Center for Life Science (DBCLS) is licensed under CC BY 4.0
• Average prokaryote cell- unlabled.svg by LadyofHats is in the public domain
• BHI media.png by Ajpolino is licensed under CC BY-SA 4.0
• Endospore Bazillus.jpg by Geoman3 is licensed under CC BY-SA 3.0
• Endospore Formation.png by Farah, Sophia, Alex is licensed under CC BY-SA 4.0
• K. rhizophila - 28h.jpg by Alexandre.Cz is licensed under CC BY-SA 3.0
• Menu Navigation2.png by Antoine03 is licensed under CC BY-SA 4.0
• OSC Microbio 02 04 Endospores.jpg by CNX OpenStax is licensed under CC BY 4.0
• Pasteur's experiment testing spontaneous generation and biogenesis.gif by hebiologyprimer is listened under CC BY-SA 4.0
• Systec H-Series Autoclaves.jpg by Foto Studio Wiegand is licensed under CC BY-SA 4.0