Materials Included In Kit

Additional Materials Required

Prelab Preparation

Obtain enough leaves and types so that all groups can perform the experiment.

Safety Precautions

Hydrochloric acid is toxic by ingestion or inhalation and is severely corrosive to skin and eyes. Sodium bicarbonate is slightly toxic by ingestion. Keep water and all other solutions away from electrical cords and outlets. Please review 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. The leaf disks and excess leaves may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The sodium bicarbonate solution may be disposed of according to Flinn Suggested Disposal Method #26b. If acidic solutions are made during the open inquiry portion of this activity they may be neutralized and disposed of according to Flinn Suggested Disposal Method #24b.

Lab Hints

• Use a bulb that is at least 40 W. The sample data provided was obtained using a 40 W bulb that was 18 cm from the top of the lab bench.
• Ivy leaves should ideally be obtained from a plant so they are as fresh as possible.
• Use the freshest possible spinach leaves for optimal results. Precut spinach in the bags will work but it takes 5–10 minutes longer on average for the disks to float.
• Do not use wilted spinach. The best pieces are found on the stiff leaves and are not cut through veins.
• If students perform the experiment as done in the Baseline Activity until the disks float and then place the cup with the floating disks in a dark location the disks will resink. This is due to plant respiration consuming oxygen bubbles.
• Rinse plastic cups from the Baseline Activity for use in the Opportunities for Inquiry portion.
• One molar hydrochloric acid is included in the kit in order to test pH. Buffers of various pH values may also be used.

Teacher Tips

• Students often have the misconception that plants only undergo photosynthesis and animals undergo cellular respiration. Plants also have mitochondria and respire.
• Have students view prepared microscope slides of leaf cross-sections to review leaf anatomy prior to beginning this lab.
• Complete a stomata peel to study leaves as part of the Baseline Activity. Contact Flinn Scientific and request publication 10226, Lasting Impressions—Counting Stomata.

Further Extensions

• Extend the activity using Mitochondrea in Action, Flinn Catalog No. FB1823.

Alignment with the Curriculum Framework for AP^® Biology 

Big Idea 1: The process of evolution drives 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 organisms require constant input of 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 such as chloroplasts.

Big Idea 4: Biological Systems interact and these systems and their interactions process 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 & 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 & 6.1).
• Justify the 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 & 6.1).
• Use representations and models to describe differences in prokaryotic and eukaryotic cells (2B3 & 1.4).
• Construct explanations based on scientific evidence as to how interactions of subcellular structures provide essential functions (4A2 & 6.2).
• Apply mathematical routines to quantities that describe interactions among living systems and their environment, which result in the movement of matter and energy (4A6 & 2.2).

 

Answers to Prelab Questions

1. What is the purpose of creating a vacuum with the floating leaf disks?

   When immersed in the experimental solution oxygen bubbles are trapped in the air pockets of the mesophyll layer of the plant leaf. By creating a vacuum the air is drawn out of the leaf and replaced with the bicarbonate solution, allowing the disk to sink.

2. Why, in this experiment, are the floating leaf disks placed in a solution that contains sodium bicarbonate?

   The bicarbonate ions serve as the carbon source for photosynthesis.

3. What causes the disks in the bicarbonate solution to rise after they are placed under a light source?

   During the photosynthesis process oxygen is produced changing the buoyancy of the disk causing it to rise.

4. Why is the rate at which the disks float an indirect measurement of the net rate of photosynthesis?

   Cellular respiration is occurring simultaneously which consumes the oxygen as it is produced by photosynthesis.

Sample Data

Graphical Analysis

Calculate the ET₅₀, the time required for 50% of the leaf disks to float by making a graph measuring Number of Disks vs. Time (min) with the disks from the “w/CO₂” cup.

Opportunities for Inquiry

Students may choose a multitude of variables to test in this portion of the laboratory activity. Following is a table of variables that students might come up with to test. Those with an asterisk were tested by Flinn with sample results provided. Environmental Variable—pH of Solution

In order to test only one variable at a time the original 0.2% sodium bicarbonate solution was made with one drop of soap. To alter the pH, approximately 2-mL of 1 M hydrochloric acid was added to the solution. The results were: the disks began floating more quickly at a lower pH. The sample data is listed below and it is also visualized graphically. Plant or Leaf Variable—Type of Plant

This experiment was also tested using fresh spinach leaves. The ivy leaves are represented by the diamonds and the squares represent the fresh spinach. The ivy leaves floated significantly sooner than the spinach leaves. This may be due to the type of plant or the fact that although the spinach leaves were fresh and had stems, they were not directly cut from the plant and tested immediately. They were purchased from the grocery store.

Introduction

A variety of procedures and techniques may be used to study the rate of photosynthesis. However, the most practical method in a high school laboratory setting is the floating leaf disk technique. Examine the factors that affect photosynthesis in land plants.

Concepts

• Photosynthesis
• Cellular processes

Background

The sun plays an important role to life on Earth. Plants contain chloroplasts that capture light energy from the sun and convert it to chemical energy stored in sugar and other organic molecules. The name of this overall conversion process is photosynthesis.

To maintain order, grow and reproduce, living systems require free energy. Sufficient energy levels are required for individual organisms to grow and also to ensure the survival of populations and ecosystems. Different organisms use various strategies to capture, use, and store free energy. Autotrophic organisms capture free energy from the environment through photosynthesis and chemosynthesis. Heterotrophic organisms harvest free energy and carbon compounds produced by other organisms.

Photosynthesis occurs in the chloroplasts within cells. The photosynthesis process occurs in a series of enzyme-mediated steps that capture light energy to form energy-rich carbohydrates. The overall process is summarized by Equation 1.

The net rate of photosynthesis can be determined in two ways—by measuring the production of oxygen, O₂ or the consumption of carbon dioxide, CO₂. Traditionally the rate of photosynthesis is calculated by measuring the consumption of carbon dioxide. However, this is difficult to perform with the equipment present in a traditional educational setting. Accurately measuring the production of oxygen is difficult because aerobic respiration occurs at the same time as photosynthesis; thus consuming oxygen as it is produced. Therefore, measuring oxygen production is equivalent to measuring net photosynthesis. A measurement of respiration in the same system would allow the estimation of gross production.

The ratio of the rate of photosynthesis (Equation 1) to the rate of cellular respiration (Equation 2) can be indirectly determined using the floating disk assay. The floating disk assay uses the overall rate at which oxygen is produced as a measure of the balance between the two reactions. Disks of leaf tissue are vacuum-infiltrated to replace intercellular air with liquid. As photosynthesis takes place, if the rate of photosynthesis exceeds the rate of cellular respiration, the accumulating oxygen imparts buoyancy to the leaf disk, and it floats. Conversely, if the rate of the respiration exceeds the rate of photosynthesis, the decreased oxygen will eventually cause the leaf disk to sink.

Experiment Overview

In the Baseline Activity, the skills needed to perform a leaf disk assay will be used to compare one variable to a control. The analysis of the results of the baseline activity will provide guidance for open-inquiry, student-designed experiments—see the Opportunities for Inquiry section for further information. Explore environmental, plant-type and even methodology in the inquiry portion of this lab. The results of the baseline activity will be analyzed and graphed, then a procedure will be developed to study an environmental, method, or plant variable that affects the rate of photosynthesis.

Materials

Prelab Questions

1. What is the purpose of creating a vacuum with the floating leaf disks?
2. Why, in the Baseline Activity, are the floating leaf disks placed in a solution that contains sodium bicarbonate?
3. Why is this method performed using leaves and not roots?
4. Why is the rate at which the disks float an indirect measurement of the net rate of photosynthesis?

Safety Precautions

Sodium bicarbonate is slightly toxic by ingestion. Wear chemical splash goggles whenever chemicals, heat or glassware is used. Keep water or other solutions away from electrical cords and outlets. Follow all normal laboratory safety guidelines.

Procedure

Baseline Activity

1. Using a permanent marker, label one syringe “w/ CO₂” and the other “control.” Repeat labels on the plastic cups as well.
2. Prepare 200 mL of 0.2% sodium bicarbonate solution by dissolving 0.4 g of sodium bicarbonate in 200 mL of distilled or deionized water.
3. Add one drop of liquid dish soap to the solution. Note: Take care to add the smallest possible drop. Too much soap will cause unnecessary suds in the solution.
4. Prepare 200 mL of the control solution (no carbon dioxide) by adding one drop of soap to 200 mL of distilled or deionized water.
5. Pour enough bicarbonate solution into the cup labeled “w/ CO₂” so it is approximately 3 cm full.
6. Fill the cup labeled “control” 3 cm with the solution made in step 4.
7. Using a single-hole punch, cut out 20 leaf disks. Avoid cutting leaf disks over major veins in the leaf.
8. Remove the plunger from the syringe.
9. Place 10 leaf disks into the barrel of each syringe.
10. Carefully replace the plunger to avoid crushing the leaf disks. Push the plunger until only a small volume of air and leaf disks remains in the barrel, less than 10% of the volume. Note: Steps 10–18 will also need to be performed with the control solution. This may be done simultaneously by another group member or one after the other.
11. Using the syringe labeled “w/ CO₂,” pull a small volume of the sodium bicarbonate solution into the syringe, about 3 mL. Tap and swirl the syringe to suspend the leaf disks in the solution.
12. Place the syringe tip cap on the syringe.
13. Make sure the disks are suspended in solution by lightly shaking the syringe and then draw back the plunger to create a vacuum. Hold the vacuum for approximately 10 seconds. While holding, swirl the leaf disks to keep them suspended in solution.
14. Release the vacuum. The bicarbonate solution will infiltrate the air pockets in the leaf causing the leaf disks to sink. Repeat step 13 up to three times until all the disks sink. If the disks do not sink after three trials add a second very small drop of soap to the bicarbonate solution and repeat steps 10–14.
15. Add fresh sodium bicarbonate solution (step 3) into a clean 200-mL beaker to a depth of approximately 3 cm.
16. Transfer the leaf disks and the solution in the syringe to the beaker.
17. Place the leaf-disk solution under a light source. The top of the beaker should be approximately 8" from the light.
18. Start the timer. At the end of each minute, record the number of floating disks. Then swirl the disks to release any disks that are stuck against the side of the cups. Continue for 15 minutes or until all disks float.

Analysis

Graph the data from both trials and mark the point for each trial where 50% of leaf disks floated. If 50% did not float, do not mark anything. The time at which 50% float is called the ET₅₀. The change in ET₅₀ is a good relative measurement of photosynthesis. The shorter the ET₅₀, the greater the rate of photosynthesis.

Opportunities for Inquiry

1. Consider the following questions while reflecting upon your knowledge of photosynthesis.
   1. How might biotic and abiotic factors in the environment, such as light, pH, temperature, etc. affect the rate of photosynthesis?
   2. Do all leaf types photosynthesize at the same rate? Does the type, color or age of the leaf affect the rate of photosynthesis? Do all plant tissues have the same rate of photosynthesis?
   3. How do method variables such as depth of solution, data collection method, etc vary the rate of photosynthesis?
2. Plan, discuss, evaluate, execute and justify an experiment to determine how an environmental variable, plant/leaf variable, or method variable affects the rate of photosynthesis.
   1. Develop a testable hypothesis.
   2. Discuss and design a controlled experiment to test the hypothesis.
   3. List any safety concerns or precautions that will be taken to protect yourself, your classmates and your instructor during the experiment.
   4. Determine how you will collect and record raw data.
   5. How will you analyze raw data to test your hypothesis?
   6. Review your hypothesis, safety precautions, procedure, data tables and proposed analysis with your instructor prior to beginning your experiment.
   7. Once the experiment and analysis are complete, evaluate your hypothesis and justify why or why not the hypothesis was supported by your data.
   8. Present and defend your findings to the class.
   9. Make suggestions for a new or revised experiment to modify or retest your hypothesis.