Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Bake about half of each of the seeds at 150 °C for 15 minutes, or boil the seeds for 5 minutes. These are the control seeds.
2. Germinate Seeds—Begin two or three days prior to the laboratory (see Sample Data for germination times).
   1. Place the remaining half of the seeds into shallow pans and cover them in warm water overnight. The dry seeds will absorb water, swell, and begin to germinate.
   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. Seeds with a radicle present work well for this investigation.
3. Fill several large containers with tap water and allow the temperature to equilibrate overnight.

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. Hot glue guns and liquified hot glue will burn. 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 seeds can be considered Type VI Biological Waste and disposed of in the normal garbage.

Lab Hints

• Enough materials are provided in this kit for 8 groups of students. Three seed types are included—one dicot and two monocots. This laboratory activity can reasonably be completed in three 50-minute class periods. The construction of the respirometers will take part of one lab period, the baseline activity will take a second class period, and the inquiry activity will take a third full lab period or more depending upon the students’ procedures. The inquiry assignments may be completed before coming to lab, and the data compilation, calculations and the summative assessment should be completed after the lab.
• Students should adjust the size of the chamber portion of the respirometer by deciding how much space their experiment will need. See the Sample Data section for guidance. Larger syringes can be used for larger specimens but longer capillary tubes are also required.
• Seeds with the radicle visible but prior to the emergence of the cotyledon(s) seem to work best for this experiment.
• Adjust baking and boiling times for very small or very large seeds.
• Attempting to germinate the baked or boiled seeds leads to smelly, rotting seeds. These rotting seeds may respire due to bacterial or fungal growth.
• 500-mL (GP1060) or 1000-mL (GP1061) tall form beakers work very well for this investigation, especially if the inquiry investigation involves warming water on a hot plate where the plastic cups are not an appropriate vessel.
• Keep the hot glue gun available at all times during this investigation. Students will need to replace broken capillary tubes immediately in order to continue with the experiment.

Teacher Tips

• Refresh the concept of respiration with the Flinn freebie “Do Animals Give off CO₂?” Contact Flinn Scientific and request digital publication 10793.
• Link cellular respiration to the human body with the Flinn Student Lab Kit “Respiration,” Flinn Catalog No. FB1975.
• The health and safety of any animals used in the inquiry portion of this lab must be considered prior to ordering or collecting the animals. Provision for long-term maintenance or euthanasia must also be considered. Many states require special collection permits before specimens can be captured from local ecosystems. No plants or animals used in a laboratory should be released into the wild, even if they originated there. Laboratory specimens may become invasive or have become infected during culturing and therefore may harm the local ecosystem.

Further Extensions

Alignment with the Concepts and Curriculum Framework for AP^® Biology 

Big Idea 1: The process of evolution explains the diversity and unity of life.

Enduring Understandings
1B1: Organisms share many conserved core processes and features that evolved and are widely distributed among organisms today.

Big Idea 2: Biological systems utilize free energy and molecular building blocks to grow, to reproduce and to maintain dynamic homeostasis.

Enduring Understandings
2A1: All living systems require constant input of free energy.
2A2: Organisms capture and store free energy for use in biological processes.
2B3: Eukaryotic cells maintain internal membranes that partition the cell into specialized regions (e.g., mitochondria).
4A2: The structure and function of subcellular components, and their interactions, provide essential cellular processes.
4A6: Interactions among living systems and with their environment result in the movement of matter and energy.

Big Idea 4: Biological systems interact, and these systems and their interactions possess complex properties.

Enduring Understandings
4A2: The structure and function of subcellular components, and their interactions, provide essential cellular processes.
4A6: Interactions among living systems and with their environment result in the movement of matter and energy.

Learning Objectives

• The student is able to describe specific examples of conserved core biological processes and features shared by all domains or within one domain of life, and how these shared, conserved core processes and features support the concept of common ancestry for all organisms (1B1 & SP 7.2).
• The student is able to justify the scientific claim that organisms share many conserved core processes and features that evolved and are widely distributed among organisms today (1B1 & SP 6.1).
• The student is able to justify a scientific claim that free energy is required for living systems to maintain organization, to grow, or to reproduce, but that multiple strategies exist in different living systems (2A1 & SP 6.1).
• The student is able to use representations to pose scientific questions about what mechanisms and structural features allow organisms to capture, store, and use free energy (2A2 & SP 1.4, SP 3.1).
• The student is able to use representations and models to describe differences in prokaryotic and eukaryotic cells (2B3 & SP 1.4).
• The student is able to construct explanations based on scientific evidence as to how interactions of subcellular structures provide essential functions (4A2 & SP 6.2).
• The student is able to apply mathematical routines to quantities that describe interactions among living systems and their environment, which result in the movement of matter and energy (4A6 & SP 2.2).

Science Practices
1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively.
2.2 The student can apply mathematical routines to quantities that describe natural phenomena.
3.1 The student can pose scientific questions.
6.1 The student can justify claims with evidence.
6.2 The student can construct explanations of phenomena based on evidence produced through scientific practices.
7.2 The student can connect concepts in and across domains to generalize or extrapolate in and/or across enduring understandings and/or big ideas.

Sample Data

Sample Results Graph 1. Respiration Rate Germinating Mung Beans at 22.7 °C

Table 1. Seed Types Tested

References

AP Biology Investigative Labs: An Inquiry-Based Approach. College Entrance Examination Board: New York, 2012.

Biology: Lab Manual. College Entrance Examination Board: New York, 2001.

Introduction

If you ask for someone to define respiration, most people will describe breathing, the inhalation and exhalation process. Respiration is also a cellular phenomenon; one that is actually about energy. In eukaryotes, this process occurs within mitochondria.

Concepts

• Cellular respiration
• Germination
• Aerobic vs. anaerobic respiration
• Ideal gas law
• Metabolism
• Respirometer

Background

All cells and therefore organisms and ecosystems need energy to function. The cycle of energy through the system is driven by a 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.

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. On the reactants side of the equation the amount of O₂ consumed by the test organism can be measured. On the products side of the equation the amount of CO₂, the increase in temperature, the amount of ATP or the amount of water produced 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 in atm
V is the volume of the gas in L
n is the number of moles of gas (moles)
R is the gas constant:

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

By controlling the pressure, temperature and the number of moles of gas allowed within a closed system, the only factor that can change is the volume of the gas. In this experiment the number of moles of oxygen consumed equals the number of moles of carbon dioxide produced. Carbon dioxide (gas) can be removed from the closed system because it can be reacted with potassium hydroxide to form solid potassium carbonate and liquid water. Thus the carbon dioxide gas is scrubbed from the system and the number of moles of oxygen consumed can be observed. The instrumentation used for this is called a respirometer. There are many types of respirometers, in the case of this experiment a closed-system, whole specimen respirometer will be constructed and used to test the respiration rate of whole seeds or insects.

Experiment Overview

In the Baseline Activity, the amount of oxygen consumed by germinating versus control seeds will be measured within a closed-system respirometer. The results of this baseline activity provide a procedure and model for open-inquiry and student-designed experiment—see the Opportunities for Inquiry section. Investigate factors that may affect the rate of cell respiration in various eukaryotic organisms.

Materials

Safety Precautions

Potassium hydroxide solution is strongly corrosive and a severe skin and eye irritant. Avoid contact with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Hot glue guns and liquified glue sticks will burn. Do not wear chemical-resistant gloves as they will melt. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Respirometer Construction

1. Ensure the plunger is pushed all the way into the syringe.
2. Use the hot glue gun to secure a nut to the base of the plunger. Allow the hot glue to cool. The nut acts as a weight to keep the syringe from floating during the experiment.
3. The next part of the procedure requires two people.
   1. Insert a capillary tube into the end of the syringe where a needle would typically sit. The capillary tube should fit into the end hole and rest on the plunger inside the syringe.
   2. Hold the capillary tube seated on the plunger and projecting straight out of the syringe.
   3. Add a bead of hot glue around the capillary tube creating an airtight seal.
   4. Quickly place one hex nut onto the capillary tube (see Figure 1). Add more hot glue only if necessary to secure the nut. Excess glue may cause problems if the capillary tube must be replaced later.
      {11146_Procedure_Figure_1}
   5. Do not allow the capillary tube to shift as the hot glue cools.
   6. After the glue has cooled, gently pull back on the plunger to ensure the glue has not plugged the capillary tube or syringe. If the capillary tube is plugged, carefully remove it and try again with a new capillary tube.
4. Repeat steps 1–3 using a second syringe, capillary tube and hex nuts.

Baseline Activity

1. Prepare a room-temperature water bath by filling a 16-oz cup with room temperature tap water.
2. Place a thermometer in the water, on the edge of the cup so the temperature scale is readable.
3. Prepare the respirometers.
   1. Draw a small amount of the red manometer fluid into the capillary tube and pull it down the length of the tube. Then eject the fluid back out. This coats the inside of the tube with a thin soapy film that helps prevent the manometer fluid from sticking.
   2. Place ½ of a cotton ball in each respirometer.
   3. Use a glass stirring rod to push the cotton ball into the respirometer so that it is next to the capillary tube.
   4. Use a graduated pipet to saturate the cotton ball with 0.5 mL of 15% potassium hydroxide. Note: 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. Place a small amount of the nonabsorbent fiberfill into each respirometer.
   6. Use the glass stirring rod to push the fiberfill into the respirometer next to the saturated cotton ball. This will protect the specimen from the alkaline solution. Note: Excess KOH may be ejected from the syringe. Make sure the capillary tube is pointed into a sink or waste container.
   7. Repeat steps a–f for the second respirometer.
4. Place the 10 germinating mung bean seeds into one respirometer and the same number of control mung bean seeds in the second respirometer.
5. Ensure the capillary tube is pointed down in a sink or container and replace the plunger and adjust it until the top of the plunger is at 4 mL. Ensure the two chambers have the same volume. Note: Excess KOH may be ejected from the syringe as the plunger is replaced and the volume is adjusted. Make sure the capillary tube is pointed into a sink or waste container.
6. Place the respirometers into the water bath with the capillary tubes pointing out of the water. Make certain the respirometer chamber is completely submerged in the water. It may be necessary to place another hex nut around the capillary tube to make the respirometer sink.
7. Top off the cup with room temperature water (see Figure 2).
   {11146_Procedure_Figure_2}
8. Wait 3–5 minutes for the system to equalize.
9. Use a pipet to add red manometer fluid to the tip of each capillary tube. The manometer fluid should be drawn into the tip of the capillary tube. If it is not, lift the respirometer and pull down slightly on the plunger to pull the fluid into the tube. The manometer fluid seals the respirometer creating a closed system.
10. If the manometer fluid is ejected from the respirometer wait a few more minutes for the respirometer to equilibrate. If the manometer fluid is still ejected from the respirometer, there is a leak in the respirometer. Reglue if necessary.
11. Use a permanent marker to mark the bottom edge of the red manometer fluid. Also record the temperature. This is the initial reading.
12. Mark the bottom edge of the manometer fluid and record the temperature every minute for 10 minutes or until the manometer fluid enters the chamber.
13. Remove the respirometers from the water bath and dry with a paper towel.
14. Use a ruler to measure the distance the manometer fluid traveled from the origin to the mark for each one minute interval. The change in height is proportional to the change in volume and so the distance traveled per minute equals the respiration rate.
15. Calculate the corrected volume for each reading by subtracting or adding any volume change for that same time period that was recorded on the control respirometer. This corrects for any change in volume that occurred in the control respirometer that occurred due to fluctuations in air pressure or temperature.
16. Construct a graph to show the rate of respiration.

Cleaning the Respirometer

1. Use forceps or a bent paper clip to remove the seeds, fiberfill, and cotton from the respirometer.
2. Dampen a cotton ball or paper towel with isopropyl alcohol to remove the respirometer readings from the capillary tube.
3. Rinse the inside of the respirometer with deionized water and use the plunger to eject any fluid in the capillary tube into the sink or other container.
4. Once dry, the respirometer can be reused.

Opportunities for Inquiry

1. Consider the following questions while reflecting upon your knowledge of using a respirometer to measure the respiration rate of a eukaryotic organism.
   1. Do nongerminating, old, cooked or frozen seeds respire?
   2. Does the type of organism affect respiration rate?
   3. Does the amount of time from the start of germination or the age of the animal affect the respiration rate?
   4. Do monocot and dicot seeds differ in their respiration rates?
   5. What environmental factors may affect respiration rate?
   6. Are there ways to control an increase or decrease in the rate of respiration?
   7. Of the factors identified in the above questions, which can be replicated as an experiment in the laboratory?
2. Plan, discuss, execute, evaluate and justify an experiment to test a question regarding respiration.
   1. Decide upon one question that your group would like to explore.
   2. Develop a testable hypothesis.
   3. Discuss and design a controlled procedure to test the hypothesis.
   4. List any safety concerns and the precautions that will be implemented to keep yourself, your classmates, and your instructor safe during the experimental phase of this laboratory.
   5. Determine what and how you will collect and record the raw data.
   6. How will you analyze the raw data to test your hypothesis?
   7. Share your hypothesis, safety precautions, procedure, data tables and proposed analysis with your instructor prior to beginning the experiment.
   8. Once the experiment and analysis are complete, evaluate your hypothesis and justify why or why not the hypothesis was supported by your data.
   9. Present and defend your findings to the class.
   10. Make suggestions for a new or revised experiment to modify or retest your hypothesis.