In this experiment, you will measure the reaction rate of anaerobic respiration (ethanol fermentation).  Review the chemical equations below, then answer the questions.

Glucose    →    Pyruvate + ATP

Glycolysis

Pyruvate    →     Ethanol + CO₂

Fermentation

1. How much (net) ATP is produced by this process? __________________________
2. Where inside the cell does this process take place? __________________________
3. Define the term ANAEROBIC: __________________________________________
4. Identify the two major steps of anaerobic respiration:____________________________________

To create an anaerobic environment, you will assemble your tubes into

RESPIROMETERS.  Respirometers isolate the chemical reactants from the atmospheric oxygen.  Holding a small test tube upright, slide the larger test tube over the top of the small test tube and then using a finger, push the small test tube up into the larger test tube and invert the apparatus.  This creates a “tube within a tube” trapping the experimental contents inside the small tube and cutting off the supply of oxygen.  Practice creating respirometers by filling the small tubes to the top with water.

As yeast begin the anaerobic fermentation process inside a respirometer, the carbon dioxide gas produced will remain trapped in the small tube.  Small bubbles will accumulate at the top of the tube.  This will push the fluid level down inside the small tube.  This change in the fluid level can be measured and will provide data about the rate of anaerobic respiration.  The larger the change in fluid level, the higher the reaction rate.

You will be using graduated pipettes (1 mL and 5 mL) to measure the volume of the experimental solutions.  Your instructor will demonstrate how to accurately measure and pipette the fluids.  You may practice using pipettes with water. When pipetting fluids, keep the tip away from the bottom of the container, otherwise you form a seal, leading to air bubble formation.

Be careful to prevent cross-contamination between containers.  When transferring fluids to a container that already contains a liquid, do not submerge the pipet tip into the solution, especially if you plan to use the pipet again.

The fluids in the pipets can be measured in either direction as illustrated in the figure.

Protective Equipment

Goggles & Gloves

Procedure

In this experiment, the effect of various compounds on yeast fermentation will be studied.  A yeast solution will be added to the tube contents listed below and then a respirometer will be prepared.  Following incubation in a water bath, the change in fluid level due to CO₂ production will be determined.

Experimental compounds:

Examine the compounds listed below.  Using the information, predict the rate of anaerobic respiration (thus the amount of fermentation) that will be produced in each tube and write the prediction in Table 1.

• Glucose – a monosaccharide commonly used as a source of energy for respiration.
• Pyruvate – a product of glycolysis, a fermentation reactant molecule.
• Magnesium sulfate (MgSO₄) – provides a cofactor that activates some enzymes used in glycolysis.
• Sodium fluoride (NaF) – a solution that inhibits some enzymes used in glycolysis.

Table 1.  Experimental Predictions

Laboratory Procedure

1. Label 6 small test tubes. Label 6 large test tubes to correlate with the small tubes.

2. Using the graduated pipettes, dispense the experimental solutions into each small test tube. See Table 2 for a list of contents and volumes to add to each tube.  
3. Add all solutions (Glucose, H₂O, NaF, MgSO₄, and Pyruvate) to all designated tubes first. All small tubes should have the same volume at this point in the procedure.
4. Then, working quickly to ensure all tubes start fermenting at approximately the same time, top all tubes with the shaken yeast suspension. Refer to the lab PowerPoint to view how the contents are added to the small test tubes.

5. Create the respirometer by sliding a large tube over a small tube and flipping quickly. Measure the initial bubble height (mm) located in the small tube.  
6. Place all tubes in the 37°C water bath at the same time for 40 minutes.
7. After 40 minutes, retrieve the test tubes. Measure the final height of the bubble.  The initial bubble height and final bubble height of each tube can be found in the PowerPoint associated with this lab.
8. Calculate the net change in bubble height and record the number (mm) in Table 2.

Table 2.  Experimental Tube Contents and Results

9. Which tube is the positive control? _______
10. Which tube is the negative control? _______
11. Identify the independent variables in this experiment: __________________________________
12. Identify the dependent variable in this experiment: ____________________________________
13. Why does Tube 5 produce CO₂ even though an inhibitor of glycolysis was present?

18. Explain the results seen in Tubes 3 and 4. (Why are the CO₂ values different?)
19. Did MgSO₄ promote respiration? _______   If not, what are some possible reasons?
20. What is the difference between “respiration” and “breathing”? Explain how they are related.
21. Does cellular respiration occur simultaneously with photosynthesis in plants? Explain your answer.
22. Do plant cells or animal cells conduct the process of cellular respiration? Explain your answer.

Clean up

• Wash all tubes and use alcohol wipes to remove all colored marks on the tubes
• Invert washed tubes in the test tube rack to dry. Place test tube rack on the drying pad on the lab counter.
• Wash all glass pipettes.

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