Experimentation in practice: Investigating the spontaneous generation of life

by Anthony Carpi, Ph.D., Anne E. Egger, Ph.D.

This material is excerpted from a teaching module on the Visionlearning website, to view this material in context, please visit Research Methods: Experimentation.

Well-controlled experiments generally provide strong evidence of causality, demonstrating whether the manipulation of one variable causes a response in another variable. For example, as early as the 6th century BCE, Anaximander, a Greek philosopher, speculated that life could be formed from a mixture of sea water, mud and sunlight. The idea probably stemmed from the observation of worms, mosquitoes, and other insects “magically” appearing in mudflats and other shallow areas. While the suggestion was challenged on a number of occasions, the idea that living microorganisms could be spontaneously generated from air persisted until the middle of the 18th century. In the 1750s, John Needham, a Scottish clergyman and naturalist, claimed to have proved that spontaneous generation does occur when he showed that microorganisms flourished in certain foods such as soup broth, even after they had been briefly boiled and covered. Several years later, the Italian abbot and biologist Lazzaro Spallanzani, boiled soup broth for over an hour and then placed bowls of this soup in different conditions, sealing some and leaving others exposed to air. Spallanzani found that microorganisms grew in the soup exposed to air but were absent from the sealed soup. He therefore challenged Needham’s conclusions and hypothesized that microorganisms suspended in air settled onto the exposed soup but not the sealed soup, and rejected the idea of spontaneous generation.

Needham countered, arguing that the growth of bacteria in the soup was not due to microbes settling onto the soup from the air, but rather because spontaneous generation required contact with an intangible “life force” in the air itself. He proposed that Spallanzani’s extensive boiling destroyed the “life force” present in the soup, preventing spontaneous generation in the sealed bowls but allowing air to replenish the life force in the open bowls. For several decades, scientists continued to debate the spontaneous generation theory of life, with support for the theory coming from several notable scientists including Félix Pouchet and Henry Bastion. Pouchet, Director of the Rouen Museum of Natural History in France, and Bastion, a well-known British bacteriologist, argued that living organisms could spontaneously arise from chemical processes such as fermentation and putrefaction. The debate became so heated that in 1860, the French Academy of Sciences established the Alhumbert prize of 2,500 francs to the first person who could conclusively resolve the conflict. In 1864, Louis Pasteur achieved that result with a series of well-controlled experiments and in doing so claimed the Alhumbert prize.

Pasteur prepared for his experiments by studying the work of others that came before him. In fact, in April, 1861, Pasteur wrote to Pouchet to obtain a research description that Pouchet had published. In this letter, Pasteur writes:

Paris, 3 April 1861
Dear Colleague,
The difference of our opinions on the famous question of spontaneous generation does not prevent me from esteeming highly your labor and praiseworthy efforts… The sincerity of these sentiments… permits me to have recourse to your obligingness in full confidence. I read with great care everything that you write on the subject that occupies both of us. Now, I cannot obtain a brochure that I understand you have just published.... I would be happy to have a copy of it because I am at present editing the totality of my observations, where naturally I criticize your assertions.
L. Pasteur (Porter, 1961)

Pasteur received the brochure from Pouchet several days later and went on to conduct his own experiments. In these, he repeated Spallanzani’s method of boiling soup broth, but he divided the broth into portions and exposed these portions to different controlled conditions. Some broth was placed in flasks that had straight necks that were open to the air, some broth was placed in sealed flasks that were not open to the air, and some broth was placed into a specially designed set of swan-necked flasks, in which the broth would be open to the air but the air would have to travel a curved path before reaching the broth, thus preventing anything that might be present in the air from simply settling onto the soup (Figure 2). Pasteur then observed the response of the dependent variable (the growth of microorganisms) in response to the independent variable (the design of the flask). Pasteur’s experiments contained both positive controls (samples in the straight necked flasks that he knew would become contaminated with microorganisms) and negative controls (samples in the sealed flasks that he knew would remain sterile). If spontaneous generation did indeed occur upon exposure to air, Pasteur hypothesized, microorganisms would be found in both the swan-neck flasks and the straight-necked flasks, but not in the sealed flasks. Instead, Pasteur found that microorganisms appeared in the straight necked flasks, but not in the sealed flasks or the swan-necked flasks.

Figure 2: Pasteur’s drawings of the flasks he used (Pasteur, 1861). Fig. 25 D, C, and B (top) show various sealed flasks (negative controls); Fig. 26 (bottom right) illustrates a straight-necked flask directly open to the atmosphere (positive control); and Fig. 25 A (bottom left) illustrates the specially designed swan-necked flask (treatment group).

By using controls and replicating his experiment (he used more than one of each type of flask), Pasteur was able to answer many of the questions that still surrounded the issue of spontaneous generation. Pasteur said of his experimental design, “I affirm with the most perfect sincerity that I have never had a single experiment, arranged as I have just explained, which gave me a doubtful result” (Porter, 1961). Pasteur’s work helped refute the theory of spontaneous generation - his experiments showed that air alone was not the cause of bacterial growth in the flask, and his research supported the hypothesis that live microorganisms suspended in air could settle onto the broth in open-necked flasks via gravity.