Set up 6 fermentation tubes with the ingredients listed in ...



Laboratory Exercise on Cellular Respiration

Procedure 1: Production of CO2 During Anaerobic Respiration

*NaF is poisonous so be extremely careful with it.*

1. Set up 6 nested tubes with the contents listed in the table below. The listed substances should be placed in the smaller tube of the pair. The yeast should be added last and you should add enough to completely fill the smaller tube. Since no yeast is added to test tube 2, completely fill the tube with water.

2. When you have completed filling the tubes, place the larger tube over the smaller tube and carefully invert them. If you accidentally end up with a small air bubble at the top measure, it before proceeding to the next step. You will need to subtract the size of this initial bubble from the size of the bubble present at the conclusion of the experiment. Record the resulting number in Table 2.

3. Number the tubes and place them in a 400 water bath for at least 40 minutes.

4. Remove the tubes from the bath and measure the size of the air bubble (CO2) in each tube. Measurements should be taken in millimeters using the supplied ruler. Record your measurements in Table 1. Remember to subtract the initial bubble size from the final bubble size if necessary.

|Table 1: Production of CO2 by Yeast in the Presence of Different Compounds |

|Tube |Yeast* |3M |.1M |5% |Water |CO2 |

|# | |Na Pyruvate |NaF (inhibitor) |Glucose | |Size of |

| | | | | | |Bubble |

|1 |X |- |- |- |6 mL | |

|2 |- |- |- |2.0 mL |Fill tube | |

|3 |X |- |- |2.0 mL |4.0 mL | |

|4 |X |- |.5 mL |2.0 mL |3.5 mL | |

|5 |X |- |2.0 mL |2.0 mL |2.0 mL | |

|6 |X |2.0 mL |2.0 mL |2.0 mL |- | |

|* An X in this column means that the appropriate tube should be topped off with the yeast suspension. Tube 2 should be topped off |

|with water. |

Important information to remember when analyzing your results:

1. Yeast produce ATP by breaking sugar (glucose) down to ethanol. This process is called fermentation.

2. The initial steps of fermentation are the same as those in glycolysis and yield pyruvate as an end product.

3. Pyruvate is then converted to ethanol. CO2 is produced as a waste product during this reaction.

4. NaF inhibits some of the enzymes needed for the steps leading to the production of pyruvate. It does not inhibit the conversion of pyruvate to ethanol.

Before answering the following questions set up the next experiment as explained in Procedure 2

Questions

1. Why must yeast convert the pyruvate to ethanol?

2. How large do you predict the gas bubble in test tube 1 will be? Why?

3. How large do you predict the gas bubble in test tube 2 will be? Why?

4. Which of the tubes to you think best represents the normal situation? Why?

5. NaF is an enzyme inhibitor added to Tubes 4 and 5. How would you expect it to affect the size of the gas bubble produced by the yeast.

6. What test tube should you compare Tube 4 with in order to determine if your prediction concerning the affect of NaF is correct?

7. What information can you obtain by comparing the size of the gas bubbles in Tubes 4 and 5?

8. Tube 6 has the same contents as Tube 5 except that Na pyruvate has been added to Tube 6. What affect do you predict the addition of Na pyruvate will have on the size of the gas bubble? Why?

9. How would you expect the size of the gas bubble in Tube 6 and Tube 3 to compare.

10. What variables are present in this experiment? Think carefully about this one.

11. Did your results match the predictions made in the above questions? If not think about why.

The answers to these questions should assist you in preparing your laboratory report. If you are unable to fully answer the questions you may be missing important information.

Procedure 2: Production of CO2 During Aerobic Respiration

|Materials |

|3-100 mL beakers |2.5 mM NaOH with dropper |

|1-150 mL beaker |phenolphthalein |

|1-100 mL graduated cylinder |Elodea |

|culture solution |Small fish |

Procedure

1. Place 75 mL of culture solution in each of the three beakers.

2. Determine the volume of the test organisms as follows:

a. Fill the graduated cylinder to the 100 mL mark with water.

b. Pour approximately 50 mL of the water from the graduated cylinder into the 150 mL beaker. Place several sprigs of Elodea into the beaker.

c. Carefully pour water from the graduated cylinder into the beaker until the water level in the beaker reaches the 100 mL mark. Make certain that none of the leaves break the surface of the water.

d. Note how much water remains in the graduated cylinder. This amount represents the volume of Elodea.* Record the number in mL in Table 2.

Please note that measurement markings on beakers are not accurate and are therefore not usually used for measuring.

3. Carefully remove the Elodea from the beaker and place it in one of the 100 mL beakers containing culture solution.

4. Repeat steps a-d in step 2 above for the fish.

5. The third 100 mL beaker serves as the experimental control.

6. Carefully remove the fish from the beaker and place it in the second beaker containing culture solution.

7. Cover all 3 100 mL beakers with plastic film or a petri dish.

8. Place the Elodea in one of the drawers and close it.

9. Wait 15 minutes.

While you are waiting, work on the questions for both experiments.

10. Return the fish and Elodea to the stock containers.

11. Add one drop of phenolphthalein to each of the three 100 mL beakers. Remember that phenolphthalein is an indicator that turns pink in a basic solution.

In the next part of the experiment, you will determine how much NaOH must be added to each of the 100 mL beakers for the solution in each one to become basic (indicated by the phenolphthalein turning pink).

12. Using a dropper add NaOH drop by drop to the control beaker. In Table 2 record the number of drops required to turn the solution pink.*

13. Repeat Step 12 with the beaker that had contained the Elodea. Match the color to the control beaker.

14. Repeat Step 12 with the beaker that had contained the fish. Match the color to the control beaker.

*You must decide on the level of pink you wish to use as an indicator. This color will be the control that you should match in the other two beakers.

|Table 2: CO2 Production in Aerobic Organisms |

|Organisms |Total |mL of NaOH |Relative |Respiration rate |

| |volume of |added to reach |respiration |per mL of |

| |organism (mL) |end point1 |rate of organism2 |organism3 |

|Beaker 1 | | | | |

|(control) | | | | |

|Beaker 2 | | | | |

|(Elodea) | | | | |

|Beaker 3 | | | | |

|(fish) | | | | |

|1 Number of drops of NaOH added to turn the solution pink. |

|2 Calculate this value by subtracting the number of drops of NaOH required to turn the |

|solution pink in the Control Beaker from the number of drops required to turn the |

|solution in the specified beaker pink. |

|3 Calculate this by dividing the relative respiration rate for the organism by the volume |

|of the organism (in mL). |

Important Information

1. CO2 combines with water to form carbonic acid (H2CO3).

2. Phenolphthalein is clear in an acid solution and pink in a basic solution.

Questions

1. The culture solution for this experiment was dechlorinated and slightly acidic. Why was it important to start with a solution that was slightly acidic?

2. Would you expect the culture solution to become more or less acidic as the organisms respire? Why? Using this information what hypothesis might you formulate for this experiment?

3. Why did you place the beaker containing the Elodea in a dark place?

4. Do you expect the solution that contained the Elodea to be more acidic than the solution that contained the fish? Why?

5. Why did you need to calculate the relative rate of respiration per mL of organism?

6. Did your results support you hypothesis? Think about why.

These questions should assist you in preparing your written lab report. If you do not fully understand the answers, you may be missing important information.

Author: Dr. Bette Jackson, FGCU, Ft. Myers, FL 33965

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