7: Respiration and Fermentation

Lab Safety

1. Wear safety goggles and gloves during all procedures.
2. Handle bromothymol blue and methylene blue solutions with care; avoid skin or eye contact.
3. Clean up spills immediately and dispose of biological and chemical waste properly.
4. Wash hands thoroughly after completing the lab.

Suggested Student Roles and Group Size

For groups of 2–3 students:

• Experiment Operator: Conducts tests and ensures accurate timing.
• Data Recorder: Records all measurements and ensures worksheet completion.
• Materials Manager (optional): Prepares solutions and cleans up materials.

Overall Timeline:

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. Seed Preparation

• Two days before the experiment: Place 15 peas into a sealable container and cover with several inches of nonchlorinated water. Soak overnight.
• One day before the experiment: Drain the water from the soaked peas, place them on a damp paper towel, and store them in a plastic bag in the dark overnight.
• Keep the remaining 5 peas dry (no soaking required).

2. Experiment Setup

• Label 20 microcentrifuge tubes using a permanent marker:
  • Tubes #1–6: Soaked peas
  • Tubes #7–11: Dry peas
  • Tubes #12–16: red plastic beads

3. Indicator Solution

• Put on gloves. Using a pipette or dropper, add 0.5 mL of bromothymol blue solution (BTB) to each microcentrifuge tube.

4. Seal Tubes with Cotton

• You can skip this step if your peas do not fall to the bottom of the tube and make contact with the pH indicator.
• Place a small plug of nonabsorbent cotton or foam lightly packed into each tube. Ensure the cotton allows airflow between the pea or bead and the BTB solution. The goal is to have a place for the pea to rest without coming into contact with the BTB.

1. Add Materials

• For Tubes #1–6: Add one soaked pea to each tube.
• For Tubes #7–11: Add one dry pea to each tube.
• For Tubes #12–16: Add one red plastic bead to each tube.

2. Record Initial Observations

• Observe and record the initial color of the bromothymol blue solution in all 20 tubes. Note any variation.
• Important note: only make observations about the liquid in the bottom of the tube, not the cotton.

3. Incubation

• Place all the tubes in a rack or tray. Store them in a safe location, where they will not get bumped, at room temperature.
• After 1 hour, observe and record the color of the BTB solution in each tube.
• After 2 hours, observe and record the color of the BTB solution again.

4. Clean Up

• At the end of the experiment, pour the used BTB solution into the designated waste container as instructed.
• Rinse and wash all microcentrifuge tubes thoroughly or dispose of them in the appropriate container.

Data Table 1.

Use the table below to record your observations.

Questions for Review

1. What is the color of the indicator at at

• Neutral pH?
• Basic pH?
• Acidic pH?

2. What was the purpose of Tubes 12-16?
   
3. What specifically was produced as a result of cellular respiration that changed the color of the indicator?

4. How is carbon dioxide an indicator that cellular respiration is taking place in these peas?

5. Germination is the process by which a dormant seed begins to sprout and grow into a seedling. What are some possible metabolic processes that are required for seed germination?

6. During respiration, a seed metabolizes sugars. What is the source of the sugar metabolized by the seed?

7. What variables do you think may affect the respiration rate of the seeds?

8. The equation for cellular respiration is:

\[\ce{C6H12O6 + 6O2 → 6CO2 + 6H2O + 32 ATP}\]

The energy released from the complete oxidation of glucose under standard conditions is 686 kcal/mol. The energy released from the hydrolysis of ATP to ADP and inorganic phosphate under standard conditions is 7.3 kcal/mol. Using the equation for cellular respiration above, calculate the efficiency of respiration (i.e. the percentage of chemical energy in glucose that is transferred to ATP). *For help with answering this question, refer to Concept 9.4 (Campbell Textbook).

Optional Activity or Demonstration

This part of the lab investigates aerobic cellular respiration by Saccharomyces cerevisiae, also referred to as “baker’s yeast” and “brewer’s yeast.” Yeast is a unicellular fungus that can convert glucose into carbon dioxide and ATP when oxygen is present. Methylene blue dye can be used as an indicator for aerobic respiration in yeast. Aerobic respiration releases hydrogen ions and electrons that are picked up by the methylene blue dye, gradually turning the dye colorless. This redox reaction can be observed when viewing a wet mount of yeast and methylene blue under the compound light microscope. The mitochondria of yeast cells undergoing aerobic respiration will appear as a clear area surrounded by a ring of light blue cytoplasm. If cellular respiration is not taking place, the mitochondria will absorb the blue dye and will not turn colorless.

Materials:

• Distilled water
• Transfer pipette
• Methylene blue dye (in dropper bottle)
• Compound light microscope
• Microscope slide and cover slip

Procedure:

1. Prepare yeast suspension: Add 7 grams yeast to 50 ml warm tap water. Stir to mix and keep in 37℃ incubator. (Your instructor may have already prepared this before lab)
2. The instructor or one group should add 10mL of the warm yeast suspension to a small beaker or test tube. Add 1-2 drops of methylene blue dye to this sample. Return the remaining yeast suspension to the incubator.
3. Place a drop of the dyed yeast suspension on a clean microscope slide with a transfer pipette and place a cover slip on the microscope slide over the yeast suspension.
4. Observe the yeast using the scanning objective lens. Use the coarse adjustment knob to focus on the yeast cells. Switch to the low-power objective lens and then to the high-power objective lens.
5. In the circle below, draw or insert a picture of several yeast cells undergoing aerobic respiration and several yeast cells not undergoing aerobic respiration. Label the cytoplasm and nucleus if visible.

Roles for 6 Students:

1. Experiment Operators (2 students):

• Prepare test tubes and saccharometers, add solutions, and ensure no errors during setup.

2. Timekeeper/Data Recorder (2 students):

• Manage the timer and record CO2 column heights at each time point.

3. Materials Managers (2 students):

• Gather materials, assist with setup, and clean up after the experiment.

Materials:

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

Procedure:

1. SETUP AND LABELING Label both your test tubes and saccharometers 1-4 with tape (don’t write directly on the saccharometer glass). This careful labeling is crucial because you'll be tracking different experimental conditions in each tube.
2. PREPARE GLUCOSE SolutionS Using your transfer pipette, add the specified amounts of glucose and distilled water from Table 2 to each test tube. Be precise with these measurements, as they create the different concentrations we'll be testing.
3. ADD YEAST Solution This step requires quick, careful work. Add the yeast solution amounts listed in Table 1 to each test tube. Work efficiently because fermentation begins immediately when yeast contacts glucose. Each tube will have a different concentration to test how this affects the fermentation rate.
   1. NOTE: all solutions should be combined and mixed in test tubes before being added to the saccharometers.
4. TRANSFER TO SACCHAROMETERS Pour each mixture into its matching labeled saccharometer. The saccharometer's design will allow us to measure the CO2 produced by fermentation. Keep them on your tray as a safety measure - fermentation can sometimes be vigorous enough to cause overflow.
   1. If available, the saccharomers can be placed in a warm incubation chamber for the duration of the experiment. This can greatly increase the rate of fermentation for the short 20-minute experimental period.
5. REMOVE AIR BUBBLES Gently tilt each saccharometer to release any trapped air bubbles in the vertical arms. This step is essential for accurate measurements - trapped bubbles could make our readings inaccurate.
6. MEASURE AND RECORD Start your timer and record the initial height of the air column in millimeters. This is your "time zero" measurement and it should be as close to 0mm as possible. Continue measuring every 2 minutes for 20 minutes total. You'll see the air column grow as fermentation produces more CO2.
7. CLEANUP PROCEDURES After completing all measurements, carefully transport the saccharometers one at a time to the sink. Empty the contents, wash them thoroughly, and return them to the plastic tray, storing them on their sides to prevent damage.

The data you collect will show how quickly yeast ferments glucose under different conditions. The height of the CO2 column directly relates to the fermentation rate - a taller column means more fermentation activity. This helps us understand how substrate concentration affects the rate of this important biological process.

Remember to keep your data table neat and organized - you'll use these measurements to graph the relationship between time and CO2 production for each concentration.