Materials Included In Kit

Additional Materials Required

Prelab Preparation

Germinate Pea Seeds—Begin two or three days prior to the laboratory

1. Place about 30 pea seeds per group into a shallow pan and cover them in warm water overnight. The dry seeds will absorb water, swell, and begin to germinate after about 12–16 hours.
2. Remove seeds from the water and place them in a moist paper towel.
3. Place the seed-containing paper towel into a paper bag, close the paper bag and place in a dark, warm place overnight or longer.

Water Bath—Prepare the night before

1. Fill the water baths in the locations where they will be used.
2. If the lab benches are cold, insulate the pans by placing them on newspaper.
3. Just prior to the laboratory, place some ice into each of the ice–water baths. This will save time during the laboratory period.

Safety Precautions

Potassium hydroxide solution is strongly corrosive and a severe skin and eye irritant. Avoid contace of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult 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. Excess potassium hydroxide solution and the saturated cotton balls may be disposed of according to Flinn Suggested Disposal Method #10. The pea seeds can be considered Type VI biological waste and disposed of in the normal garbage. The glass beads and the respirometer may be cleaned and reused. 

Lab Hints

• Enough materials are provided in this kit for three groups of students. This laboratory can reasonably be completed in one 50-minute class period. The data compilation and calculations can be completed the day after the lab.
• Ensure students label all materials with their group number or chemical used in the container.
• The volume of germinating pea seeds can vary greatly depending upon the species used. Adjust the number of seeds for the experimental setup so they fit easily into the respirometer bottles.
• Keep the temperature of the ice bath as close to 10 °C as possible. If the temperature rises even 2 °C, the results become questionable.

Teacher Tips

• Extend the lesson by determining the rate of respiration at 50°C.
• Extend the lesson by using crickets or other small animals in place of pea seeds.
• The Flinn Scientific, Inc. Alcohol Fermentation Demonstration Kit (Catalog No. FB1626) uses color change to show the depletion of oxygen and the production of carbon dioxide by yeast.

Sample Data

Observations and Analysis

Table 1

Table 2

Answers to Questions

1. This activity uses a number of controls. Identify at least three of the controls, and describe the purpose of each control.

   Atmospheric pressure, size of materials used to make the apparatus, type of materials used to make the apparatus, water source, temperature of the system (within each testing group), volume of materials used to make the apparatus. The purpose of each control is to ensure that only one parameter is tested in the laboratory.

2. Why is it important to react the carbon dioxide with potassium hydroxide in the respirometers?

   Carbon dioxide as a gas would negate any change in gas volume due to the use of oxygen by the seeds. The CO₂ must be reacted into a solid or a liquid so that the change in gas volume can only be due to the consumption of oxygen by the seeds.

3. Graph the results from the corrected difference column for the germinating peas and dry peas at both room temperature and 10 °C. For this graph you will need to determine the following:
   1. What is the dependent variable?

      Oxygen consumed (mL)

   2. What is the independent variable?

      Time (minutes)

   {13808_Answers_Figure_5}
4. Determine the rate of oxygen consumption of germinating and nongerminating peas during the experiments at room temperature and 10 °C.

   The rate of oxygen consumption of germinating peas at room temperature.

   {13808_Answers_Equation_4}
   The rate of oxygen consumption of nongerminating peas at room temperature.
   {13808_Answers_Equation_5}
   The rate of oxygen consumption of germinating peas at 10 °C.
   {13808_Answers_Equation_6}
   The rate of oxygen consumption of nongerminating peas at 10 °C.
   {13808_Answers_Equation_7}
5. Describe and explain the relationship between the amount of oxygen consumed and time.

   The amount of oxygen consumed by germinating seeds is relatively constant over time, indicating that the peas are continually using oxygen during cellular respiration.

6. Why is it necessary to correct the readings from the peas with the readings from the beads?

   Fluctuations in the temperature of the apparatus could potentially cause false changes in the slope of the line and create a false conclusion that oxygen usage increases or decreases with time.

7. Explain the effect of germination on the cellular respiration of pea seeds.

   Germinating seeds have a greater rate of cellular respiration than nongerminating (dormant) seeds because actively growing seeds have more cells performing cellular respiration at high rates due to increased water and oxygen requirements.

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

   The 25 g mammal will have a greater respiration rate than the 25 g reptile because the reptile is cold-blooded and at 10 °C cold temperature its metabolism would slow.

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

We know that animals use oxygen for respiration but what about plants? Plants create oxygen as a waste product of photosynthesis but do they require oxygen to live? Does respiration occur in both germinating and nongerminating or dormant seeds?

Objectives
After completing this laboratory, students 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 ungerminated versus germinated seeds in a controlled experiment.

Concepts

• Cell respiration
• Ideal gas law
• Germination
• Metabolism

Background

All cells need energy to function. Most cells require a very specific form of “cell fuel” called adenosine triphosphate or ATP. ATP is produced within the mitochondria of a cell in a process involving the metabolism or breakdown of glucose. This process is called cellular respiration. The most efficient form of cellular respiration uses oxygen and is called aerobic respiration. Aerobic respiration can produce up to 36 ATP molecules for every glucose molecule oxidized. Aerobic respiration is used by most species to produce ATP. Aerobic respiration can be divided into three stages: glycolysis, the Krebs cycle, and electron transport. It is the electron transport stage of aerobic respiration that requires oxygen (O₂) and produces ATP and the waste products carbon dioxide (CO₂), water and heat.

Anaerobic organisms do not use oxygen to release energy from glucose. Anaerobic respiration, also called fermentation, involves glycolysis or a modified electron transport system and only produces two ATP molecules for each glucose molecule, far fewer than the 36 produced by aerobic respiration. Consequently, relatively few organisms use anaerobic respiration exclusively.

The rate of aerobic cellular respiration can be determined by quantifying the change in concentration of one of the molecules required for the reaction or one of the products of the reaction. The amount of CO₂ or heat produced by the test organism can be measured or the amount of O₂ used by the test organism can be measured. The two gases, O₂ and CO₂, are easy to quantify because they follow the ideal gas equation: where

P is the pressure of the gas,
V is the volume of the gas,
n is the number of moles of gas

T is the temperature of the gas in Kelvin (°C + 273).

This law implies the following important principles about the properties of gases:

1. If the temperature and pressure are kept constant, then the volume of the gas is directly proportional to the number of moles of the gas.
2. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of moles of gas present.
3. If the number of moles if gas and the temperature remain constant, then the pressure is inversely proportional to the volume.
4. If the temperature changes and the number of moles of gas is kept constant, then either the pressure or volume (or both) will change in direct proportion to the temperature.
5. It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure.

In designing an experiment, scientific method specifies that all parameters must be kept constant except the one being tested. In the case of an experiment involving gases, the pressure and temperature of the experiment must be controlled to quantify change in volume and the number of moles of gas present in the experiment. In order to do this, the laboratory will be conducted in one lab session ensuring a constant pressure. A water bath provides a constant temperature. No new gas will be allowed to enter the test apparatus, called a respirometer, by sealing the air into the test apparatus, at the beginning of the experiment. The number of moles of each of the gases will change because of cellular respiration. Since cellular respiration involves two gases, the consumption of O₂ and the production of CO₂, CO₂ will be removed from the respirometer as it is produced. Potassium hydroxide (KOH) will react with the CO₂ produced during cellular respiration to form potassium carbonate (K₂CO₃) and water. Potassium carbonate is a solid and water is a liquid, therefore they do not factor into the ideal gas law. The number of moles of oxygen can then be seen as a change in the volume within the respirometer. A control sample containing glass beads will detect any changes in volume due to atmospheric pressure or temperature during the experiment which is then adjusted during the analysis of the experimental data.

Experiment Overview

In this laboratory, the amount of oxygen consumed by germinating versus dormant pea seeds will be measured over a period of time at two different temperatures.

Materials

Safety Precautions

Potassium hydroxide solution is strongly corrosive and a severe skin and eye irritant. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Prepare a room-temperature bath (≈25 °C) by filling a rectangular pan with 6 L of room temperature tap water. Across the short end of the rectangular pan, evenly space three large rubber stoppers (see Figure 2). Rest a thermometer on the opposite end of the pan from the rubber stoppers.
   {13808_Procedure_Figure_2}
2. Prepare a cold-water bath (≈10 °C) by filling a second rectangular pan with 4 L of cold tap water and 2 L of ice. Across the short end of the rectangular pan, evenly space three large rubber stoppers (see Figure 2). Rest a thermometer on the opposite end of the pan from the rubber stoppers. Add ice during the experiment as needed to keep the temperature at ≈10 °C.
3. Label the six French square glass bottles with the numbers 1 through 6 and with your group name or number.
4. Place a cotton ball in the bottom of each glass bottle and, using the graduated pipet, saturate the cotton with 1-mL of 15% potassium hydroxide. Note: It is important that exactly the same amount of KOH be used for each respirometer. Be careful not to get potassium hydroxide on the sides of the respirometer. Caution: Potassium hydroxide is strongly corrosive and a severe skin and eye irritant.
5. Gently tease a dry cotton ball so that it is the size of the inside area of the bottle and place on top of the saturated cotton. The dry cotton ball should cover the top of the wet cotton ball and protect the pea seeds and beads from the potassium hydroxide solution.
6. Place 25.0 mL of tap water in a 50-mL graduated cylinder. Add 10 germinating pea seeds to the graduated cylinder. The difference between the final volume and the original 25.0-mL reading is a direct measure of the amount of space (volume) required by the 10 germinating pea seeds. Record the volume of germinating pea seeds estimating to a precision of ±0.05 mL, in Table 1 on the Cell Respiration Worksheet. Remove the seeds from the graduated cylinder and place on a paper towel. Carefully blot the seeds dry and pour the seeds into bottle 1.
7. Fill the 50-mL graduated cylinder with 25 mL of water again and add 10 nongerminating (dry) peas to the water. The volume will be less than that of the germinating seeds. To compensate, add enough glass beads to attain an equal volume to the germinating seeds. Record the volume of nongerminating seeds and beads estimating to a precision of ±0.05 mL, in Table 1 on the Cell Respiration Worksheet. Remove the seeds and beads from the graduated cylinder and place on a labeled paper towel. Carefully blot the seeds and beads dry and pour the seeds and beads into bottle 2.
8. Fill the 50-mL graduated cylinder with 25.0 mL water again and add enough glass beads estimating to a precision of ±0.05 mL, to attain an equal volume to the germinating seeds. Record the volume of glass beads in Table 1 on the Cell Respiration Worksheet. Remove the beads from the graduated cylinder and place on a labeled paper towel. Carefully blot the beads dry and pour the beads into bottle 3.
9. Repeat steps 6–8 for bottles 4, 5 and 6. Bottles 1–3 will go into the room-temperature bath while bottles 4–6 will go into the cold-water bath.
10. Use a ruler and marker to mark the tapered end of each 1-mL serological pipet every 2 mm. These markings will be used to quantify the change in gas volume during the lab.
11. Coat the outside of the blunt end of the 1-mL serological pipets with petroleum jelly and insert each one into the hole of a #00 one-hole rubber stopper (see Figure 3).
    {13808_Procedure_Figure_3}
12. Coat the sides of the #00 one-hole rubber stoppers with petroleum jelly and insert each one into the mouths of the glass bottles.
13. Once the respirometers have been assembled, place them into the appropriate water bath. Make sure that the tapered end of the serological pipet is resting on the side of the rectangular pan (see Figure 4). Ensure that the volume markings on the pipet are facing up. Note: Bottles 1, 2 and 3 should be in the room-temperature bath, and bottles 4, 5 and 6 should be in the cold-water bath.
    {13808_Procedure_Figure_4}
14. After five minutes, carefully immerse the respirometers in the water, resting the pipet on top of the large rubber stoppers. Ensure that the volume markings on the pipets are facing up. They should not be moved after the data collection has begun. Note: A little water should enter the pipets and then stop. If the water continues to enter the pipet, remove the respirometer from the water bath and check for leaks.
15. Allow the respirometers to equilibrate for an additional five minutes.
16. Record the initial position of the water in each pipet to the nearest 0.001 mL (time 0) in Table 2 on the Cell Respiration Worksheet. Note: Readings are made by noting the movement of the water-air interface in the pipet.
17. Check the temperature in both baths and record in Table 2 on the Cell Respiration Worksheet.
18. Observe and record the water level in the six pipets every five minutes for 20 minutes.
19. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

13808_Student1.pdf