Spontaneous Generation
Recreation of Pasteur’s Experiment


Background — Spontaneous Generation

In this experiment, you will be recreating Pasteur’s famous experiment that disproved spontaneous generation and investigating the conditions under which bacteria will/will not grow.

Today, we take many things in science for granted. Many experiments have been performed and much knowledge has been accumulated that people didn’t always know. For centuries, people based their beliefs on their interpretations of what they saw going on in the world around them without testing their ideas to determine the validity of these theories — in other words, they didn’t use the scientific method to arrive at answers to their questions. Rather, their conclusions were based on untested observations.

Among these ideas, for centuries, since at least the time of Aristotle (4th Century BC), people (including scientists) believed that simple living organisms could come into being by spontaneous generation. This was the idea that non-living objects can give rise to living organisms. It was common “knowledge” that simple organisms like worms, beetles, frogs, amd salamanders could come from dust, mud, etc., and food left out, quickly “swarmed” with life. For example:


Observation: Every year in the spring, the Nile River flooded areas of Egypt along the river, leaving behind nutrient-rich mud that enabled the people to grow that year’s crop of food. However, along with the muddy soil, large numbers of frogs appeared that weren’t around in drier times.
Conclusion: It was perfectly obvious to people back then that muddy soil gave rise to the frogs.

Observation: In many parts of Europe, medieval farmers stored grain in barns with thatched roofs (like Shakespeare’s house). As a roof aged, it was not uncommon for it to start leaking. This could lead to spoiled or moldy grain, and of course there were lots of mice around.
Conclusion: It was obvious to them that the mice came from the moldy grain.

Observation: In the cities, there were no sewers, no garbage trucks, no electricity, and no refrigeration. Sewage flowed in the gutters along the streets, and the sidewalks were raised above the streets to give people a place to walk. In the intersections, raised stepping stones were strategically placed to allow pedestrians to cross the intersection, yet were spaced such that carriage wheels could pass between them. In the morning, the contents of the chamber pots were tossed out the nearest window. Food was purchased and prepared on a daily basis, and when people were done eating a meal, the bones and left-overs were tossed out the window, too. A chivalrous gentleman always walked closest to the street when escorting a woman, so if a horse and carriage came by and splashed up the filth flowing in the gutters, it would land on him, and not the lady’s expensive silk gown (many of these gowns were so ornately embroidered that they were not easily washable, and neither washing machines nor dry cleaners existed). Many cities also had major rat problems. People back then may or may not have not connected the presence of rats with the spread of Bubonic Plague (Black Death, a dreaded and fatal disease), but they were probably bothered by the rats chewing on things and by the rat fleas biting them (just as cat/dog owners, even now, are bitten by the offspring of their pet’s fleas). People may not have realized that the Plague was spread by the bites of those fleas, but I imagine they knew that if only they could get rid of the rats, the pesky fleas would soon disappear, too — hence the story of the Pied Piper of Hamelin, Germany, leading all the rats out of town.
Conclusion: Obviously, all the sewage and garbage turned into the rats.

Observation: Since there were no refrigerators, the mandatory, daily trip to the butcher shop, especially in summer, meant battling the flies around the carcasses. Typically, carcasses were “hung by their heels,” and customers selected which chunk the butcher would carve off for them.
Conclusion: Obviously, the rotting meat that had been hanging in the sun all day was the source of the flies.


From this came a number of interesting recipes, such as:

Recipe for bees:
Kill a young bull, and bury it in an upright position so that its horns protrude from the ground. After a month, a swarm of bees will fly out of the corpse.
Jan Baptista van Helmont’s recipe for mice:
Place a dirty shirt or some rags in an open pot or barrel containing a few grains of wheat or some wheat bran, and in 21 days, mice will appear. There will be adult males and females present, and they will be capable of mating and reproducing more mice.

Disproving Spontaneous Generation

Fly Experiment In 1668, Francesco Redi, an Italian physician, did an experiment with flies and wide-mouth jars containing meat. This was a true scientific experiment — many people say this was the first real experiment — containing the following elements:

  1. Observation: There are flies around meat carcasses at the butcher shop.
  2. Question: Where do the flies come from? Does rotting meat turn into or produce the flies?
  3. Hypothesis: Rotten meat does not turn into flies. Only flies can make more flies.
  4. Prediction: If meat cannot turn into flies, then rotting meat in a sealed (fly-proof) container should not produce flies or maggots.
  5. Testing: Wide-mouth jars each containing a piece of meat were subjected to several variations of “openness” while all other variables were kept the same.
  6. Data: Presence or absence of flies and maggots observed in each jar was recorded. In the control group of jars, flies were seen entering the jars. Later, maggots, then more flies were seen on the meat. In the gauze-covered jars, no flies were seen in the jars, but were observed around and on the gauze, and later a few maggots were seen on the meat. In the sealed jars, no maggots or flies were ever seen on the meat.
  7. Conclusion(s): Only flies can make more flies. In the uncovered jars, flies entered and laid eggs on the meat. Maggots hatched from these eggs and grew into more adult flies. Adult flies laid eggs on the gauze on the gauze-covered jars. These eggs or the maggots from them dropped through the gauze onto the meat. In the sealed jars, no flies, maggots, nor eggs could enter, thus none were seen in those jars. Maggots arose only where flies were able to lay eggs. This experiment disproved the idea of spontaneous generation for larger organisms.

After this experiment, people were willing to acknowledge that “larger” organisms didn’t arise by spontaneous generation, but had to have parents. With the development and refinement of the microscope in the 1600s, people began seeing all sorts of new life forms such as yeast and other fungi, bacteria, and various protists. No one knew from where these organisms came, but people figured out they were associated with things like spoiled broth. This seemed to add new evidence to the idea of spontaneous generation — it seemed perfectly logical that these minute organisms should arise spontaneously. When Jean Baptiste Lamarck proposed his theory of evolution, to reconcile his ideas with Aristotle’s Scala naturae, he proposed that as creatures strive for greater perfection, thus move up the “ladder,” new organisms arise by spontaneous generation to fill the vacated places on the lower rungs.

In 1745 to 1748, John Needham, a Scottish clergyman and naturalist showed that microorganisms flourished in various soups that had been exposed to the air. He claimed that there was a “life force” present in the molecules of all inorganic matter, including air and the oxygen in it, that could cause spontaneous generation to occur, thus accounting for the presence of bacteria in his soups. He even briefly boiled some of his soup and poured it into “clean” flasks with cork lids, and microorganisms still grew there.

A few years later (1765 to 1767), Lazzaro Spallanzani, an Italian abbot and biologist, tried several variations on Needham’s soup experiments. First, he boiled soup for one hour, then sealed the glass flasks that contained it by melting the mouths of the flasks shut. Soup in those flasks stayed sterile. He then boiled another batch of soup for only a few minutes before sealing the flasks, and found that microorganisms grew in that soup. In a third batch, soup was boiled for an hour, but the flasks were sealed with real-cork corks (which, thus, were loose-fitting enough to let some air in), and microorganisms grew in that soup. Spallanzani concluded that while one hour of boiling would sterilize the soup, only a few minutes of boiling was not enough to kill any bacteria initially present, and the microorganisms in the flasks of spoiled soup had entered from the air.

This initiated a heated argument between Needham and Spallanzani over sterilization (boiled broth in closed vs. open containers) as a way of refuting spontaneous generation. Needham claimed that Spallanzani’s “over-extensive” boiling used to sterilize the containers had killed the “life force.” He felt that bacteria could not develop (by spontaneous generation) in the sealed containers because the life force could not get in, but in the open container, the broth rotted because it had access to fresh air, hence the life force inherent in its molecules, which contained and replenished the life force needed to trigger spontaneous generation. In the minimally-boiled flasks, he felt the boiling was not severe enough to destroy the life force, so bacteria were still able to develop.

By 1860, the debate had become so heated that the Paris Academy of Sciences offered a prize for any experiments that would help resolve this conflict. The prize was claimed in 1864 by Louis Pasteur, as he published the results of an experiment he did to disproved spontaneous generation in these microscopic organisms.

  1. Observation(s): From Needham’s and Spallanzani’s experiments, it was known that soup that was exposed to the air spoiled — bacteria grew in it. Containers of soup that had been boiled for one hour, and then were sealed, remained sterile. Boiling for only a few minutes was not enough to sterilize the soup. Pasteur had previously demonstrated that the dust collected by drawing air through a cotton ball contained large numbers of bacteria, hence he knew that bacteria were present in the air and could be filtered out by using a cotton ball. He also knew that bacteria would settle out on the walls of a long, bent, glass tube as air was passed through it.
  2. Question: Is there indeed a “life force” present in air (or oxygen) that can cause bacteria to develop by spontaneous generation? Is there a means of allowing air to enter a container, thus any life force, if such does exist, but not the bacteria that are present in that air?
  3. Hypothesis: There is no such life force in air, and a container of sterilized broth will remain sterile, even if exposed to the air, as long as bacteria cannot enter the flask.
  4. Prediction: If there is no life force, broth in swan-neck flasks should remain sterile, even if exposed to air, because any bacteria in the air will settle on the walls of the initial portion of the neck. Broth in flasks plugged with cotton should remain sterile because the cotton is able to filter bacteria out of the air.
  5. Testing: Pasteur boiled broth in various-shaped flasks to sterilize it, then let it cool. As the broth and air in the containers cooled, fresh room air was drawn into the containers. None of the flasks were sealed — all were exposed to the outside air in one way or another.
  6. Data: Broth in flasks with necks opening straight up spoiled (as evidenced by a bad odor, cloudiness in previously clear broth, and microscopic examination of the broth confirming the presence of bacteria), while broth in swan-neck flasks did not, even though fresh air could get it. Broth in flasks with cotton plugs did not spoil, even though air could get through the cotton. If the neck of a swan-neck flask was broken off short, allowing bacteria to enter, then the broth became contaminated.
  7. Conclusion(s): There is no such life force in air, and organisms do not arise by spontaneous generation in this manner. To quote Louis Pasteur, “Life is a germ, and a germ is Life. Never will the doctrine of spontaneous generation recover from the mortal blow of this simple experiment.”

One very important point to note here is that Pasteur did not seek to find an answer to the broad question, “Has spontaneous generation ever occurred?” Rather, as any good scientist, he limited his scope to a very narrow piece of the picture: “Is it possible for spontaneous generation to occur given the specific conditions under which Needham (and others) claims it will occur,” i.e. the “life force?” Interestingly, in 1936, when Alexander Ivanovich Oparin, a Russian scientist, published The Origins of Life, in which he described hypothetical conditions which he felt would have been necessary for life to first come into existence on early Earth, some scientists found it difficult to acknowledge that under the very different conditions which Oparin was proposing for early Earth, some form of “spontaneous generation” might indeed have taken place.


Bibliography

Alcamo, I. Edward. 1997. Fundamentals of Microbiology, 5th Ed. Benjamin Cummings Publ. Co., Menlo Park, CA. (pp. 7-9)

Curtis, Helena. 1983. Biology, 4th Ed. Worth Publ. NY. (pp. 77-78, 238)

Lewis, Ricki. 1992. Life. Wm.C. Brown. Dubuque, IA. (p. 59)

Schroeder, Gerald L. 1990. Genesis and the Big Bang. Bantam Books. NY. (pp. 107-110)

The Slow Death of Spontaneous Generation (1668-1859)


Recreation of Pasteur’s Experiment

Materials Needed

A note on the type of broth used: Salt is used as a preservative (think of some cured ham that’s so salty it can be left at room temperature without spoiling). If the external environment around a cell (including bacterial cells) is very salty, water from within the cell will flow out of the cell to the more-salty, exterior environment. That dehydrates the cell, usually killing it. In this experiment, we want to show the difference in bacterial growth following various sterilization treatments or storage methods, without having a lot of salt present in the broth that might interfere with or inhibit the growth of bacteria. Thus, it is best to use low-salt broth.

Safety Considerations

Procedure

Note that these are general directions. Students are encouraged to design their own experiments. Glass tubing may be bent into any desired shape or left straight. Tubing may or may not be inserted, and/or a no-hole stopper may be used. Optionally, the end of a tube may be plugged with a bit of cotton. Beef or chicken broth (or other?) may be used. Broth may be unboiled or may be boiled for varying lengths of time, either in a “double boiler” in a pan of water or over a Bunsen burner. Stoppers may be inserted before or after boiling, and/or broth may be boiled in another container and poured into a “clean” flask. Broth could be stored at different temperatures (refrigerator, room temperature, incubator). PLEASE CLEARLY MARK ALL FLASKS AS TO CONTENTS, TREATMENT, AND OWNER(S).

  1. Students should work in teams of 2 to 3 people. Each team should perform the following steps.
    2. Soup and Flask
    Broth and Flask
  2. Decide what combinations of lid, tubing, openness, boiling or not, boiling time, boiling in flask vs pouring in later, presence/absence of cotton “plug,” etc. you wish to test. Mark Erlenmeyer flasks accordingly. The two suggestions listed here would be most like the equipment that Pasteur used.
    1. flask with stopper and glass tube going straight up
    2. flask with stopper and glass tube bent in S-curve
  3. Place about 50 mL of broth in each Erlenmeyer flask.
  4. Place appropriate lids on flasks.

    5. Heating Flask
    Boiling the Broth     Four Flasks Set Up
    Four Flasks Set Up
  5. Boil broth in flasks with appropriate lids on them for 30 min., then let cool. Optionally, you may decide to boil some broth separately, let it cool a bit, then pour it into a “clean” flask. For comparison, you may also decide to not boil the broth in one flask. You could also compare boiling for 5 min vs boiling for 20 or 30 min. Note: if you’re boiling the broth in a flask for 30 min, it must be a very slow boil so all your broth doesn’t evaporate — watch your flask as it boils so it doesn’t boil dry and burn!

    6. Colonies Growing
    Colonies Growing in Broth
  6. For the next several lab periods, observe the flasks and record any changes in color, turbidity, smell, etc. Caution: if you remove the lid from (“open”) a flask to examine it, and leave the lid off for any significant amount of time or set the lid down on the desk, etc., any of those actions may introduce bacteria into a flask that might otherwise have been sterile, thereby changing the outcome of your experiment. To avoid possible contamination, it’s better to observe your flasks without removing the lids.
    Remember to record what you did and how you set up your experiment (draw pictures, too). Take notes on any changes in appearance or smell of your flask(s), and compare data with other students in your lab section.

    7. Clean-Up
    Clean Up When You’re Done!
  7. When you are finished observing your flasks, CLEAN UP! Make sure to thoroughly wash all glassware. Do NOT attempt to remove glass tubing from stoppers — another class can use them in the future.


Other Things to Include in Your Notebook

Make sure you have all of the following in your lab notebook:


Copyright © 1999 by D. B. Fankhauser and J. Stein Carter. All rights reserved.
Based on printed protocol Copyright © 1999 D. B. Fankhauser and J. L. Stein Carter.
This page has been accessed Counter times since 14 Mar 2001.