Cell Respiration Pre-Lab - quigly biology



Cell Respiration Pre-Lab

PREDICTIONS:

What do you predict will happen in this experiment regarding

A. The relative volume of oxygen consumed by germinating and nongerminating seeds

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B. The effect of different temperatures on seed germination

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HYPOTHESES:

You are therefore, testing two different hypotheses in this experiment. What are they?

Hypothesis 1:

Null__________________________________________________________________

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Alternative____________________________________________________________

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Hypothesis 2:

Null_________________________________________________________________

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Alternative____________________________________________________________

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1. What is the equation for the ideal gas law? ________________________________________

Define each variable in the equation.

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2. In this experiment, what are we going to measure?

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3. What parts of the ideal gas law should be held constant in order for us to measure the variable we intend to study in this experiment?

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4. What is the purpose of KOH in this experiment?

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5. How are you going to measure the variable you identified in question 2?

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6. What exactly is the vial containing glass beads used for?

(Yes- I know it is a control--explain what it is controlling for !)

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7. How are you going to calculate ml of oxygen consumed for each vial?

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8. This activity uses a number of controls. Identify at least 3 of the controls and describe the purpose of each control.

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Cell Respiration

OVERVIEW

In this lab you will work with seeds that are dormant and seeds that have been stimulated to germinate because they have been soaked in water. A seed contains an embryo plant and a food supply surrounded by a seed coat. When the necessary conditions (moisture etc…) are met, germination occurs and the rate of cellular respiration greatly increases. A dormant seed still has a plant embryo, but the embryo is not undergoing mitosis and is therefore not needing to produce large quantities of ATP. In this experiment you will measure oxygen consumption during germination. You will measure the change in gas volume in respirometers containing either germinating or nongerminating grass seeds. In addition, you will measure the rate of respiration of these seed at two different temperatures.

OBJECTIVES

Before doing this lab you should understand:

• how a respirometer works in terms of the gas laws

• the general processes of metabolism in living organisms

After doing this lab you should be able to:

• calculate the rate of cell respiration from experimental data relate gas production to respiration rate

• test the effect of temperature on the rate of cell respiration in nongerminated vs. germinate seeds in a controlled experiment.

INTRODUCTION

Cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria within each cell. Cellular respiration involves a series of enzyme mediated reactions. The equation below shows the complete oxidation of glucose. Oxygen is required for this energy releasing process to occur.

C6H12O6 + 6O2 → 6CO2 + 6 H2O + 686 kcal of energy/mole of glucose oxidized

By studying the equation above you will notice there are three ways cellular respiration could be measured. One could measure the:

1. Consumption of oxygen --how many moles of oxygen are consumed in cellular respiration?

2. Production of carbon dioxide--how many moles of carbon dioxide are produced by cellular respiration?

3. Release of energy during cellular respiration.

In this experiment, the relative volume of oxygen consumed by germinating and nongerminating (dry) peas at two different temperatures will be measured.

BACKGROUND INFORMATION

A number of physical laws relating to gases are important to the understanding of how the apparatus that you will use in this exercise works. The laws are summarized in the general gas law that states:

PV = nRT

P = pressure of gas

V = volume of gas

n = number of molecules of gas

R = gas constant (its value is fixed)

T = temperature of gas (Kelvin)

This law implies the following important concepts about gases:

A. If temperature and pressure are kept constant,

then the volume of gas is directly proportional to the number of molecules of gas.

B. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas present.

C. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume.

D. If the temperature changes and the number of gas molecules is kept constant, then either pressure or volume (or both) will change in direct proportion to the temperature.

It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure.

In this experiment, the CO2 produced during cellular respiration will be removed by KOH and will form solid K2CO3 according to the following reaction.

CO2 + 2KOH → K2CO3 + H2O

Since the carbon dioxide is being removed, the change in the volume of gas in the respirometer will be directly related to the amount of oxygen consumed. In the experimental apparatus if water temperature and volume remain constant, the water will move toward the region of lower pressure. During respiration, oxygen will be consumed. Its volume will be reduced because the carbon dioxide produced is being converted to a solid. The net result is a decease in gas volume within the tube and a related decrease in pressure in the tube. The vial with glass beads alone will permit detection of any changes in volume due to atmospheric pressure changes or temperature changes. The amount of oxygen consumed will be measured over a period of time.

Each group will set up 3 microrespirometers. Each group will have a DIFFERENT temperature of water (warm, room, cold).

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|RESPIROMETER |TEMPERATURE |CONTENTS |

| |… | |

|A | |Germinating Seeds |

| |… | |

|B | |Nongerminating (Dry) seeds |

| |… | |

|C | |Beads |

PROCEDURE:

1. Prepare a water bath at the assigned temperature: ________________

2. Assemble 3 respirometers:

a. Label 3 vials A, B and C

b. Place circle of absorbent cotton (approximately the size of a nickel) into bottom of each vial.

c. Carefully soak with 1-2 ml of 15% KOH solution

Do not allow any KOH to touch side walls of vials

d. Place circle of non absorbent cotton into vial directly on top of KOH/cotton circle. This will keep KOH solution from touching peas during experiment

3. Find the volume of 25 germinating peas.

• Fill a 100 ml graduated cylinder to 50 ml with water

• Place the peas in the cylinder and measure the increase in water volume_____________

• Place peas on paper towel

• These are germinating peas to be used in respirometer A

4. Determine volume of 25 non-germinating peas

• Fill 100 ml graduated cylinder with 50 ml water

• add 25 non-germinating peas

• Add glass beads to raise volume to equal that obtained with swollen germinating peas

• Remove peas and beads. Place them on paper towel

• Nongerminating peas and beads will be used in respirometer B

5. Determine the volume of glass beads

• fill the 100 ml graduated cylinder with 50 ml water

• add glass beads to raise volume so it equals volume of germinating peas as determined previously

• place glass beads on paper towel

• Glass beads to be used in respirometer C

6. Place non germinated seeds + glass beads into vial B. Insert Cork/pipet assembly into vial

7. Wrap parafilm tightly around seams (cork and tube) to seal any potential leak

8. Make sure ice water bath has equilibrated to the correct temperature

9. Place piece of masking tape over water bath to suspend pipet tips out of water during equilibration phase

[pic]

10. Place each respirometer into water bath with calibrated side of pipet facing up so that measurements can be taken.

11. Allow respirometers to equilibrate in water bath for 5 minutes.

12. After equilibration phase, immediately submerge each respirometer (put them all the way into the water). Pipettes will take on some water. Make sure vial does not fill up with water. If it does, there is a leak which must be corrected.

13. Arrange vials so you can read volume markings on each pipet. Weight vials to keep them submerged.

14. Record starting point (time 0) volume of each pipet.

15. Take readings of volume of water in each pipet every 5 min. for 20 min. Record these values in your data table.

16. Collect Vial A, B and C class data for the other temperature. Record in data table.

17. Correct volumes measured for changes in environmental variables:

• Vial C measures volume changes due to environmental variables. For vial C, the environmental effects are calculated by subtracting the volume at a given time (Tx) from staring volume at starting time (T0)

apparent ml of oxygen consumed = Vial C volume at Tx - Vial C volume at T0

• Environmental effects must be applied to data collected for vial A as shown below:

ml of O2 consumed vial A =

[vial A vol. at Tx - Vial A vol. at T0] - [vial C vol. at Tx - Vial C vol. at T0]

• Environmental effects must be applied to data collected for vial B as shown below:

ml of O2 consumed vial B =

[vial B vol. at Tx - Vial B vol. at T0] - [vial C vol. at Tx - Vial C vol. at T0]

Data Tables:

Room Temperature

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|Time (T) |Volume Readings |apparent ml of oxygen consumed in vial|ml oxygen consumed vial A |ml oxygen consumed vial B |

|min. | |C | | |

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| |vial A |Vial B |Vial C | | | |

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Ice Water Bath:

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|Time (T) min. |Volume Readings |apparent ml of oxygen consumed in vial |ml oxygen consumed vial A |ml oxygen consumed vial B |

| | |C | | |

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| |vial A |Vial B |Vial C | | | |

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Warm Water Bath:

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|Time (T) min. |Volume Readings |apparent ml of oxygen consumed in vial |ml oxygen consumed vial A |ml oxygen consumed vial B |

| | |C | | |

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| |vial A |Vial B |Vial C | | | |

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EVALUATION:

1. Graph your corrected data for vials A & B for all three temperatures (you will have more than one line!)

• What is the independent variable?__________________________________

• What is the dependent variable?___________________________________

• Data from all temperatures should be plotted on the same graph.

• Draw the best fit straight line through the data points.

[pic]

2. What accounts for the difference in oxygen consumption seen between germinating and

Non-germinating seeds?

3. Why do the glass beads seem to be using oxygen?

4. Why are the readings corrected using the glass bead values?

5. From the slope of the four lines on the graph, determine the rate of oxygen consumption of germinating and dry peas during the experiments at room temperature and 10ºC . Show your work!!!!!

Recall that rate = Δy/Δx

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|CONDITION |CALCULATIONS |RATE: ml O2/minute |

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|Germinating seeds at cold temp. | | |

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|Germinating seeds at room temp. | | |

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|Germinating seeds at warm | | |

|temperature | | |

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|Dry seeds at cold temperature | | |

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|Dry seeds at room temperature | | |

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|Dry seeds at cold temperature | | |

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6. Compare the rate of oxygen consumption at cold and room temperature. Why are they different?

7. Compare the rate of oxygen consumption at warm and room temperature. Why are they different?

8. Compare the rate of oxygen consumption at cold and warm temperatures. Why are they different?

9. What basic cellular process is responsible for the oxygen consumption?

10. Explain why water moved into the respirometer pipettes.

11. What is the effect of germination (vs. Nongermination) on seed respiration?

12. Discuss whether these results support your original hypotheses.

13. Summarize your conclusions from this lab.

14. If you used the same experimental design to compare the rates of respiration of a 25g reptile and a 25g mammal, at 10ºC what results would you expect? Explain your reasoning.

15. If respiration in a small mammal were studied at both room temperature and 10ºC, what results would you predict? Explain your reasoning.

Part II: Developing and Implementing Your Research Plan

Reviewing the Variables

Below is a table of variables that provides a starting point for developing research questions on cellular respiration. A number of variables that you may wish to investigate are listed in the table.

|Environmental Variables |Seed Variables |Organism Variables |

|1. Temperature |1. Stage of germination |1. Type of seed |

|2. Light (possible confusion with photosynthesis) |2. Age of seeds |2. Mixed seeds |

|3. Conditions for germination |3. Condition of seeds |3. other tissues |

|4. Pressure |4. Seed size |4. Other organisms |

|5. Oxygen availability |5. Surface area of seed | |

|6. Time of the day |6. Others | |

|7. Others | | |

Suggested Topics

In the previous section, you examined variables to investigate for independent research. However, you may still be having some difficulty trying to get started with your research. Listed below are a few ideas that you might want to investigate. Notice that the questions are limited and provide a suggestion for developing an answer.

1. Do seeds just starting to germinate consume oxygen at a greater rate than seeds that have been germinating for several days?

2. Does the temperature of germinating seeds affect the rate of cellular respiration?

3. Do insects respire faster or slower at higher temperatures?

4. Do monocot plants respire at different rates than dicot plants?

5. Do boiled peas respire? If so, how does the respiration rate compare with that of unboiled peas?

Decide on the question you want investigate for independent research and write it below:

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Developing and Implementing Your Plan

Complete the following chart in your lab notebook and get your teacher’s approval. Materials needed must be those already found in the lab or those that your group can bring with you on the day you run your experiment. You MUST request materials needed from the prep room at least 24 hours prior to running your experiment.

|Null Hypothesis: |

|Alternative Hypothesis: |

|Materials: |

|Independent Variable |

|Dependent Variable(s) |

|Controlled Variable(s)/Constants |

|How will data be recorded? |

|What kind of data analysis will you perform? How will it be done? |

Student Presentation

Research biologists keep a record of their work in their laboratory or field notebook. They communicate the final results of their work to others a number of ways. They may publish an article or make a presentation at a meeting. To keep others informed about work that is just getting started, biologists often use a form of presentation known as a poster. You will be producing and presenting a poster of your research.

Use the information from the enzyme catalysis lab to prepare a mini-poster to present your findings.

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These questions must be answered BEFORE you will be allowed to conduct this lab!

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