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CELL RESPIRATION: A COMPUTER BASED LABORATORY

By Robert Goodman



Target Audience:

H.S. or A.P. Biology

Background:

Notes for teacher:

The creation of relatively inexpensive sensors that can be interfaced to computers or graphing calculators has provided numerous opportunities to improve the quality of the laboratory experience. The investigation described below is a simple technique for monitoring cell respiration in yeast. The results are graphed as they are collected giving students a virtually instant picture of what is occurring in their cuvettes.

Absorption is measured using a Vernier Colorimeter (set at 565nm) which is interfaced to a Macintosh computer (Vernier and several other companies make colorimeters which interface with Macintosh and IBM computers. These sensors could also be interfaced with a Texas Instrument Graphing Calculator). Data Logger Software is used when recording the results.

Yeast, brom thymol blue (BTB) and a sugar solution are placed in a cuvette. As the yeast give off carbon dioxide, the pH of the solution decreases and the BTB turns to brom thymol yellow (BTY). The graph shows a decrease in absorption as respiration proceeds. The slope of the curve is a measure of the rate of respiration. The Data Logger software will provide the slope of the best fit line for each graph. Three sugars, galactose, glucose and lactose are measured separately and distilled water serves as a control. (See graphs, bottom of page 3.)

There are a number of experimental twists which can be investigated:

  1. For example, the yeast I worked with (purchased in a local supermarket) could metabolize glucose, but not lactose or galactose. Students could measure the effects of exposing the lactose to lactase (purchased in drug store). Lactase is the enzyme which digests lactose. Lactose is a disaccharide composed of glucose and galactose. Since the yeast could metabolize glucose, but not galactose one would expect the respiration rate of a lactose/lactase solution to be intermediate, between galactose and glucose.

  2. Students could also measure respiration rates at different temperatures. One would expect the rate to increase until the temperature optimum of a rate limiting step is encountered.

  3. Students could also measure the respiration rate at various sugar concentrations. The rate should increase and then level off. In the 2% solutions tested, respiration proceeded at a maximum rate (i.e. higher concentrations of sugar did not result in steeper downward slopes).

Required of students:

Prior to lab students should review the overall equations for cell respiration (aerobic and fermentation). The structures of the mono and disaccharides to be tested should also be reviewed. If this is the first lab using sensors interfaced to computers, then additional preparation time must be provided for the students.

Preparation:

1/2 Hour- Packages of yeast are variable in their condition when purchased from the store. Therefore, the teacher or technician should test out the yeast in order to determine the range of absorbance to be set on the Y-axis. This takes about 10 minutes. Two grams of dried yeast were dissolved in 150 mL of tap water. The yeast should sit in water for a short period (about 1/2 hour) before the lab, giving them adequate time to use any stored sugar in their cells. If the yeast solution is left around too long the bacterial population will build up, interfering with the results. For example, if the bacteria metabolizes lactose, the results will be affected.

Class time:

1 Hour- This will, of course, vary depending on what parameters you ask your students to evaluate. One hour is enough time for the students to check 3 sugars, water and the effect of adding lactase to a lactose solution.


Lesson:

Summary:

All cells carry out the process of cell respiration in order to meet their energy needs. It is advantageous for cells to have the ability to metabolize different substrates. In this experiment, we will investigate which sugars baker's yeast can use for cell respiration. When yeast respires (anaerobically or aerobically) they give off carbon dioxide. When this carbondioxide reacts with water it forms a weak carbonic acid solution. We will use the indicator brom thymol blue (BTB) to monitor this reaction. When the pH of a BTB solution is lowered, it turns yellow. We will monitor that change with a colorimeter interfaced to a computer.

Materials:
  • 5-50mL Beakers
  • 1-1mL Pipette
  • 2-5mL Pipettes
  • 1-10mL Pipettes
  • 5 Cuvettes
  • 2% Glucose
  • 2% Lactose
  • 2% Galactose
  • Distilled Water
  • Yeast Extract
  • Macintosh Computer
  • Printer
  • Vernier Colorimeter
  • Serial Box Interface

Procedure:

  1. Turn on your Macintosh Computer and Colorimeter. Set the colorimeter to 565nm. Open the file which says experiments and then open the document which says colorimetry. Set your axes in the following way: X-axis, 0-300 seconds (independent variable), Y-axis, 1.75-2.00 Absorbance (dependent variable) (These ranges may vary according to your teacher's instructions.) Title the graph (i.e. Absorbance vs. Time for 2% glucose).

  2. Using a 10mL pipette, place 8mL of 2% glucose into a 50mL beaker.

  3. Using a 5 mL pipette, place 2 mL of Brom Thymol Blue (BTB) into the beaker.

  4. Using a 5mL pipette, place 2.5 ml of the BTB/glucose solution into a cuvette. Add 0.1 mL of yeast to the cuvette, cover the cuvette, shake it and place in the colorimeter.

  5. Initiate the test by pressing the "enter" key. Stop the test after 5 minutes (300 seconds) by again pressing the "enter" key.

  6. Print the graph and keep the hard copy for further analysis and include it in your laboratory report.

  7. Repeat procedures steps 2-6, except use 2% galactose instead of 2% glucose.

  8. Repeat procedures steps 2-6, except use 2% lactose instead of 2% glucose.

  9. Repeat procedures steps 2-6, except use distilled water instead of 2% glucose. Compare the slope of the 4 graphs obtained from this activity.

    • Rank order of sugars based on which ones the yeast can use most effectively for for respiration (from best to worst).
    • Why was it necessary to measure the respiration rate of distilled water?
    • What are some possible sources of experimental error?
    • Design an experiment to test out the effect of temperature or sugar concentration on the rate of respiration.

Method of Evaluation:

The instrument used to evaluate this activity is a laboratory report which includes title, aim, materials, outline of procedure, data, analysis of results (answers to questions) and a conclusion.

Extension//Reinforcement:

Have the students design and perform an experiment which shows the effect of adding lactase to the lactose solution prior to feeding it to the yeast. This should be related to the concept of lactose intolerance, common to many adults.


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