Spontaneous Generation


Where do microbes come from? Do they come from non-living materials or can they only come from pre-existing microorganisms? This demonstration will provide insight into the answers to these questions.


  • Sterilization

  • Spontaneous generation


Nutrient broth, 4 g*
Paraffin wax*
Water, distilled, 500 mL
Autoclave or pressure cooker
Beaker, 500-mL
Erlenmeyer flasks, 250-mL, 7
Hot plate
Rubber stoppers, one-hole, #6, 3*
Rubber stoppers, solid, #6, 2*
S-shaped tube*
Stirring rod
Straight glass tube*
U-shaped tube*
*Materials included in kit.

Safety Precautions

Be sure to follow directions carefully when using an autoclave or pressure cooker. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.


Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All flasks should be autoclaved before disposing of the media contents. Open the sealed stoppers and place the flasks in the autoclave or pressure cooker. Once autoclaved, the flask contents can be flushed down the drain with volumes of water.

Prelab Preparation

  1. Make 500 mL of nutrient broth solution by dissolving 4 g of powdered nutrient broth in 500 mL of distilled water. If necessary, heat slightly to dissolve completely. The broth should be a clear golden color and not cloudy.
  2. Label seven Erlenmeyer flasks 1 through 7.
  3. Pour approximately 70 mL of the nutrient broth into each of the seven labeled flasks.
  4. Treat each of the flasks as pictured in Figure 1 and with these additional notes:

Flask 1: Plug with a no-hole stopper and seal with paraffin wax. To do this melt the wax in a beaker at low temperature on a hot plate. Spread the wax to form a seal where the stopper and flask meet.
Flask 2 and 3: Bring the nutrient broth in these flasks to a slow boil using a hot plate. Leave Flask 2 open to the air. Stopper and seal Flask 3 with paraffin wax like Flask 1.
Flasks 4, 5, 6 and 7: Place appropriate stoppers and glass tubes in these flasks as shown in Figure 1. Place all four flasks into an autoclave (or pressure cooker) and sterilize. When using a pressure cooker, bring the cooker to 15 lbs. of pressure for 20 minutes. After sterilization, release pressure very slowly so that the broth is not drawn into the tubes. (Allow a pressure cooker to cool completely before releasing pressure.) Seal Flasks 5, 6 and 7 as shown while maintaining an upright position.

  1. After the flasks have cooled they are ready for student observations.


  1. Distribute an Origin of Microbes Worksheet to each student.
  2. Use the Discussion section information to introduce the experiment to students. Do not indicate any possible results.
  3. Explain in detail (and have students record in column 1) how each flask was prepared.
  4. Have students date and record the initial condition of the broth in the flasks. Then have students predict what they think will happen over a period of time. Ask them to clarify their hypotheses and possible results as they make their predictions.
  5. Place the flasks in a safe place where they might sit for a long time undisturbed. (A locked display cabinet might be ideal.)
  6. Have students observe the flasks over the next several weeks or months. When they note any microbial growth (nutrient broth turns cloudy and/or growth occurs on broth surface) have them record the data on their worksheets along with dates.
  7. When the “ideal” results have been achieved, record all results and have a complete discussion about the origin of microbes in this demonstration. It is most likely that Flasks 1–5 will become contaminated. How long will Flasks 6 and 7 stay uncontaminated? (Some of Pasteur’s original flasks are still not contaminated in the museum in France!)

Student Worksheet PDF


Correlation to Next Generation Science Standards (NGSS)

Science & Engineering Practices

Planning and carrying out investigations

Disciplinary Core Ideas

MS-LS1.A: Structure and Function
HS-LS1.A: Structure and Function

Crosscutting Concepts

Scale, proportion, and quantity

Performance Expectations

MS-LS1-1. Conduct an investigation to provide evidence that living things are made of cells; either one cell or many different numbers and types of cells


The flasks in this demonstration represent in summary the classic experiments of Lazzaro Spallanzani (Flasks 1–3) and Louis Pasteur (Flasks 4–7). Both scientists were trying to prove whether microbes could arise “spontaneously” from food or other media or could they only arise from other microorganisms.

The experiments are historically interesting because in Spallanzani’s day (1729–1799) it was assumed that boiling kills all microbes. (Today, of course, we know that this is not true.) The results in Flask 3, thus, become very significant. In Spallanzani’s time it was used as evidence in support of spontaneous generation. Since it was assumed that all microbes were killed with boiling, the only explanation for the contamination of Flask 3 seemed to be spontaneous generation from the broth!

Flasks 4–7 represent, in summary, Louis Pasteur’s experiments (1822–1895). The flask setups and the results reveal very interesting characteristics of microbes. Autoclaving does kill all microbes and thus “sterilized” media will remain free of microorganisms if the microbes cannot come in direct contact with the media. In Flasks 4 and 5 microbes will eventually be brought in contact with the media as microbes fall into the flasks under the force of gravity pulling them straight down. These flasks will become cloudy with time.

Microbes fall straight down via gravity and/or are carried by air currents. Though some microbes are mobile they cannot move very long distances of their own accord. Without forced air currents, Flasks 6 and 7 will remain sterile (void of microbial growth) indefinitely. Falling microbes in Flask 7 get trapped in the bottom of the S-tube and do not get into the media. So even though Flasks 6 and 7 are “open” to the air, they will not easily become contaminated, even over very long periods of time. (Even hundreds of years!) If Flask 7 is turned over so that some of the media runs into the bottom of the S-tube and then returned to its upright position (this can be done at the end of the experiment) it will become contaminated very quickly.

Next Generation Science Standards and NGSS are registered trademarks of Achieve. Neither Achieve nor the lead states and partners that developed the Next Generation Science Standards were involved in the production of this product, and do not endorse it.