Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prior to beginning the lab, four holes must be made in each fermentation tube’s cap. Punch the holes with a heavy-duty pin or needle in a diamond pattern around the indentation in the middle of the cap. Use a piece of paper towel or rubber gloves to provide more grip while inserting and removing the pin or needle
2. To ensure good results, before each class freshly prepare each solution as follows:

• • 7% suspension of yeast: Add one package (7g) to 100 mL of tap water in a 150-mL beaker. Water temperature should be no colder than about 22 °C.
  • 1% solution of glucose: Add one gram of glucose/dextrose to 100 mL of tap water in a beaker.

3. Determine the optimal time to read the glucose test strip. Dip the test strip into the 1% glucose solution. Determine how many seconds it takes before the test strip indicates 90 mmol of glucose which is the same as 1%. This time is the time students should use before reading the test strip.

Safety Precautions

Although materials in this activity are considered nonhazardous, wear safety glasses or goggles whenever working with chemicals, heat or glassware in the laboratory. Wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

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 used solutions from this lab (i.e., yeast–glucose mixture, colored beaker water) may be disposed of down the drain with large amounts of water according to Flinn Suggested Disposal Method #26b. Unused solutions of glucose may be stored for short periods of time in a refrigerator, if other classes are going to be doing the same lab.

Lab Hints

• Enough materials are provided in this kit for 32 students working in groups of 3–4 students. This laboratory activity can reasonably be completed in one 50-minute class period if the glucose solution and yeast suspension are pre-made. The pre-laboratory activity should be completed at least one day before the lab and could be assigned as homework. Graphing and data analysis can be completed the day following the lab.
• Unless you have a small class, this lab should be done with a minimum of three persons per group. Not only will each person have a responsibility such as—timer/data recorder, glucose tester, tube marker—but lab materials can be conserved.
• Collecting as much data as possible during a lab increases the ability to observe trends and analyze the data. Therefore, you may want to assign certain student groups to increase the reaction rate for this lab by:

1. 1. Pre-warming the glucose and/or yeast suspensions above room temperature.
   2. Placing the small 50 mL beaker inside a large beaker of warm (37–40 °C) water. If this is done; measurements must be taken every 3 minutes resulting in 10 readings in 30 minutes.

• Reading the markings on the side of the fermentation tubes is a critical part of this lab. For this reason, we have provided fermentation tube reader cards. Hold the card against the side of the tube and the marks on the tube can be easily converted to mL.
• More precise pH measurements of the solution in the beaker containing the resazurin can be made by giving each group pH paper.
• The glucose test strips will be useful only to give approximations of glucose concentrations. If the color of a test strip falls between the colors shown on the color chart provided in the kit, an estimated amount of glucose (in mmol/L) may need to be made.
• Since a Post-LabQuestion asks whether or not a control was used, the control(s) may be set up on a tabletop for students to observe.

Teacher Tips

• For long-term usage, make copies of the fermentation tube reader cards page, then laminate and cut them out for students to use.

• To reuse the fermentation tubes, thoroughly rinse them with tap and distilled water, clean off the marks on the outside with alcohol and store for future use.

Further Extensions

• At what point does temperature negatively affect the production of CO₂? This experiment may be extended to study the effects of temperature on respiration/fermentation.

1. Take the temperature inside a refrigerator, place a fermentation tube and beaker inside and remove it every 5 minutes to take measurements.
2. Place several tubes and beakers into gradually increasing water temperatures.

• Glucose concentration may also be changed and compared to the “normal” concentration used in the lab. Does increasing or decreasing the concentration affect the amount of CO₂ produced? In what way?
• Would bubbling air into the yeast/glucose solution lead to an increase in the amount of CO₂ produced and decrease the amount of alcohol produced? The “Pasteur Effect,” as it is now called, may be investigated using these same materials. Differences in the two procedures are noted:

1. Bubble a 10% solution of glucose and a 7% yeast/water mixture with an aquarium pump for 3–5 minutes then mix 8 mL of the “aerobic” yeast and glucose together in a fermentation tube. Mark this tube “aerobic.”
2. Prepare a second fermentation tube using the original protocol but substitute the 10% glucose solution for the 1% solution. Mark this tube “anaerobic.”
3. Invert both tubes in a smaller beaker and then put the beaker into a 40 °C water bath.
4. At either one to two minute intervals, mark the meniscus of the CO₂ bubble and allow the setups to run for 15 minutes. (Due to the rapid time between intervals, testing for glucose would not be necessary.)
5. At the end of 15 minutes, analyze the amount of CO₂ in each tube as before.

Answers to Prelab Questions

Write the statement and questions below on a separate sheet of paper, answer them, and then make the data table.

1. If living things release carbon dioxide in the process of using glucose for energy to grow (Equations 1 and 2) and a suspension of yeast and glucose are mixed together,

1. What do you predict will happen to the carbon dioxide level and the glucose concentration over time? (Make a prediction for both compounds.)
   

   The glucose level will drop as it is used by the yeast for energy and the CO₂ level will increase as it is released by the living yeast cells.

2. Do you think there will be a change in pH over time? (Briefly describe what you think will happen to the pH.)
   

   The pH of the solution may drop as a result of the formation of carbonic acid.

Sample Data

Table 1 results at room temperature 23.2 °C

Table 2 results when placed in a water bath between 28–30 °C

Answers to Questions

1. Plot the CO₂ level and glucose concentration over time on the same graph. Use different pencil colors or different shapes to mark each data point on the graph. Include a legend to show which line belongs to which data set.

See sample data and graphs for what student data and graphs should approximate.

2. Was the group’s prediction (Prelab Activity 1a) supported by the results? Explain!

Student answers will vary.

3. 1. What happened to the pH of the mixture from the beginning to the end?

      It decreased.
      

   2. Explain the reason for the change in pH. (What do you think was being produced to affect the pH and how does that compound affect pH?)

      As CO₂ is produced, it dissolves in the water to produce a weak acid called carbonic acid. As more carbonic acid is produced, the solution becomes more acidic and the pH drops.

4. Based on the graph of the data, during which time period did the yeast appear to be growing/consuming glucose the fastest? Use specific data to support your answer.

At room temperature during the middle 10–20 minutes, the CO₂ level dramatically increased meaning a lot of work (cell division) was being done. During that time the amount of CO₂ level more than tripled from 1.5 to 5.25 mL.

5. Why did the lid of the fermentation tube have holes in it?

To allow the yeast–glucose mixture to escape as it is displaced by the CO₂ gas being produced.

6. Was a control used in this experiment? No

1. If yes, what was it?
2. If no, what would a control setup contain?

   A tube with only yeast and no glucose or a tube containing only glucose and no yeast.

3. Why would a control tube be necessary?

   To compare CO₂ production with and without an energy source or CO₂ production with and without the presence of living organisms.

7. 1. Although not visible or tested for, what other compound was present inside the tube (refer to the Background section).

      Ethyl alcohol

   2. What evidence from the data supports your answer! (Hint: What happened to the color of the solution in the small beaker?)

      When CO₂ is produced from the breakdown of glucose, ethyl alcohol is also produced when no oxygen is present. The resazurin changed color as the level of oxygen in the fermentation tube decreased.

8. Was the process occurring inside the tube mostly aerobic or anaerobic? Explain your choice.

Anaerobic, because the resazurin, as an indicator of oxygen depletion, lost its color over time.

9. 1. How could this experiment be modified to increase the amount of CO₂ produced? (Base the answer on your results.)

      Increase the concentration of glucose and/or slightly increase the temperature.

   2. If the change suggested in Question 9a was done, what do you predict would happen to the amount of the “invisible” compound present in the mixture? (Question 7)

      The amount of ethyl alcohol should increase because the environment inside the fermentation tube would become anaerobic more quickly.

   3. Why would this occur?

      Increasing the temperature and/or concentration should increase yeast cell reproduction. The increased number of cells would then metabolize the glucose more quickly, producing more CO₂ and causing the mixture to become anaerobic quicker. This, in turn, would result in an increased production of alcohol.

10. List three things that could be done, as far as procedures, to improve the results?

Student answers will vary.

• • Better marking of the tube at the correct time interval
  • More precise interpretation of the glucose test strip color.
  • Ensuring that any foam inside the tube is also included when marking the location of the CO₂ gas bubble.
  • Making sure that the temperature of any water bath that is used is more precisely maintained throughout the experiment.
  • Using a pH meter instead of pH paper would provide more precise pH values than guessing at colors.

Teacher Handouts

10658_Teacher1.pdf

References

Black, S., Moore, R., Haugen, H., ed. Biology Labs That Work: The Best of How-To-Do-Its, National Association of Biology Teachers: Reston, VA. 2000, Vol. II. pp. 47–50.

Introduction

Are breathing and respiration two words for the same process? Not really! Breathing may be thought of as something all aerobic (air-breathing) organisms do—inhaling or taking in oxygen. On the other hand, respiration is the process that converts food into usable energy and is carried out by individual cells. There are two types of respiration, one requires oxygen, the other—called anaerobic (without air) respiration—does not. Fermentation is the word commonly used for anaerobic respiration. Which type of respiration is better? It depends on the type of organism and where it lives, but the focus of this activity is fermentation.

Concepts

• Aerobic respiration

• Fermentation
• Anaerobic respiration
• Glucose utilization

Background

The process of fermentation has been around since ancient times. This anaerobic breakdown of sugar may be used to produce alcoholic beverages or Kimchee (fermented cabbage popular in Korea) depending on the type of plants involved. Prior to 1860, this process was thought to involve only natural chemical reactions, like rust forming on iron when air and moisture are present. Experiments performed by Louis Pasteur (1822–1895) demonstrated that living microorganisms played an essential role in this process. As these microbes consume glucose (C₆H₁₂O₆) and produce energy, additional compounds are made. When no oxygen is present, the fermentation reaction may produce carbon dioxide (CO₂) and alcohol (C₂H₅OH), as seen in Equation 1. When oxygen is present (aerobic respiration), the products are as indicated in Equation 2.

Since aerobic and anaerobic respiration (fermentation) are only carried out by living organisms, studying how these processes occur in other organisms can help us understand many types of human metabolic disorders or physical problems, such as muscle cramps, lactose intolerance, diabetes and hypoglycemia.

Experiment Overview

Using yeast cells, students will investigate the relationship between glucose concentration and carbon dioxide production over time. Resazurin, a color indicator, will be used to help detect changes in the yeast’s environment, namely the presence of oxygen and changes in pH. These changes will be “visible” via distinctive color changes. Note: Color changes may range from dark violet (pH 6.5) to pink, peach, then orange (pH 3.8).

Materials

Prelab Questions

Write the statement and questions below on a separate sheet of paper, answer them and then make the data table.

1. If living things release carbon dioxide in the process of using glucose for energy to grow (Equations 1 and 2) and a suspension of yeast and glucose are mixed together

1. What do you predict will happen to the carbon dioxide level and the glucose concentration over time? (Make a prediction for both compounds.)
2. Do you think there will be a change in pH over time? (Briefly describe what you think will happen to the pH.)

2. Follow the layout of Figure 1 to make a data table with seven rows for recording data for 30 minutes:

Safety Precautions

Although materials in this activity are considered non-hazardous, wear safety glasses or goggles whenever working with chemicals, heat or glassware in the laboratory. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Put 20 mL of tap water into a 50-mL beaker and measure the temperature of the water. Record in the data table. Add one drop of resazurin solution to the water in the beaker. Record the initial color and the pH of the water. Note: Use pH paper to get more precise readings. The color changes of the resazurin can only provide approximations of pH. Record resazurin color changes in the data table.
2. Add 8 mL of the glucose solution to the fermentation tube.
3. After stirring/swirling the yeast suspension, add sufficient yeast to fill the tube almost to the top (within 5 mm). Screw on the cap, hold fingers over the holes and quickly invert the tube one time.
4. Unscrew the cap and immediately dip a glucose test strip into the mixture. Wait 30 seconds then, compare the color of the test strip with the color chart provided by the instructor. Based on the color, record the glucose concentration in the data table.
5. Immediately after dipping the test strip, continue filling the tube with the yeast suspension until there is a liquid bubble above the lip of the tube. Doing this will help eliminate large air bubbles inside the tube.
6. Screw on the cap (a few drops of liquid will come through the holes), turn the tube upside down and place it in the small beaker of colored water. Record the time in the data table and begin timing. (If an air bubble is already present in the tip of the tube, mark the bottom of the bubble with a marker. This will be the starting point of the experiment.)
7. After 5 minutes, use a dark colored permanent marker to mark the location of the growing gas bubble (Any foam present in the tube must also be included in each measurement.) Note: Keep the tube upside down while marking.
8. After marking the tube, lift it out of the water, wipe the cap with a paper towel, and hold a glucose test strip under the cap. Let a few drops of the solution drip on the end of the strip and immediately put the tube back in the beaker of water.
9. Wait 30 seconds and use the color chart again to approximate the glucose concentration. Record the level of glucose in the data table and any observable color change in the data table.
10. Repeat steps 7–9 at least five additional times for a total of seven measurements.
11. When all the measurements have been completed, dispose of the tube’s contents as directed by the instructor and carefully dry the outside of the tube.
12. Hold a laminated “reader card” (see Figure 2) next to the tube and line up the markings. Use the marks on the tube to determine the amount of CO₂ produced at each time interval.

13. Record the CO₂ levels in the data table.

Student Worksheet PDF

10658_Student1.pdf