Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Phosphate buffer solution. Prepare at least one day prior to making the chloroplast suspension.
   1. Dilute 100 mL of the 10X concentrated phosphate buffer to 1 L with deionized water.
   2. Cover and store on ice or in a refrigerator until ready to use.
   3. The solution is stable for four weeks if kept cold.
2. Chloroplast suspension. Prepare the chloroplast suspension as close to lab time as possible (within the same day). All materials and solution used in the preparation of the chloroplast suspension should be refrigerated overnight and kept cold prior to making the suspension. This includes the pitcher of the blender, the beakers, the funnel and the phosphate solution.
   1. Remove the ribs from the fresh baby spinach leaves.
   2. Place approximately 10 g of the fresh baby spinach leaves on a piece of paper towel behind a heat sink (large beaker filled with tap water) in front of a 100W light source for 1–3 hours prior to preparing the chloroplast suspension.
   3. Add the 10 g of baby spinach leaves to 1 L of cold phosphate buffer solution in a blender.
   4. Blend for 30 to 40 quick pulses followed by a 30-second blend.
   5. Place a 1-L beaker in an ice bath. Place four layers of cheesecloth into a large funnel and place the funnel into the beaker.
   6. Filter the suspension through the cheesecloth in the funnel and into the beaker. Squeeze the cheesecloth to retrieve as much buffer as possible. Note: You should be able to retain 90% of the original volume (≈900 mL).
   7. Pour the chloroplast suspension into an amber bottle or completely cover the beaker with aluminum foil. Label the container “unheated chloroplast suspension.”
   8. Store on ice or in a refrigerator until ready to use. Note: During the experiment, the unheated chloroplast suspension must be kept on an ice bath and in the dark prior to being incubated by the students.
   9. The success of this laboratory hinges upon the preparation of the chloroplast suspension.
      1. Too concentrated or too weak of a suspension will adversely affect the results. Test the suspension prior to the lab. If the DCPIP is reduced in the unheated sample within 5 minutes of light exposure, dilute the chloroplast suspension with additional buffer or add fewer milliliters of suspension to each test tube. Note: Even adding as few as 5 drops is possible if the chloroplast solution is very concentrated.
      2. If the DCPIP is not significantly reduced within 15 minutes, increase the amount of chloroplast suspension added or decrease the concentration of the DCPIP solution.
      3. (Optional)Test the activity of the chloroplast suspension. Set a warmed up spectrophotometer at 600 nm. Zero the spectrophotometer with 5 mL of the phosphate buffer. Insert a cuvet containing 5 mL of the new chloroplast suspension. An ideal absorbance reading is 0.300–0.400.
3. Heated chloroplast suspension.
   1. Each student group will need enough heated chloroplast suspension for eight test tubes. Based upon the testing above multiply the calculated amount by 15 to determine the minimum amount of chloroplast suspension that must be heated to complete the lab. Note: The total amount heated must be no more than 800 mL (⅘ of prepared amount). If it is, then prepare more chloroplast suspension.
   2. Place the calculated amount of unheated chloroplast suspension into a 1-L borosilicate glass beaker.
   3. Place the beaker in a 50 °C hot water bath for 5 minutes. Note: Do not boil the suspension as it will clump. If it clumps, use a glass stirring rod to break the clumps apart.
   4. Pour the heated chloroplast suspension into an amber bottle or completely cover the beaker with aluminum foil. Label the container “heated chloroplast suspension.”
   5. Store on ice or in a refrigerator until ready to use. Note: During the experiment, the heated chloroplast suspension should be treated to the same conditions as the unheated suspension, therefore, keep it on an ice bath and in the dark prior to being utilized by the students.
4. 2,6-Dichlorophenolindophenol (DCPIP) solution, 0.1% (also known as 2,6-Dichloroindophenol, DPIP and DCIP).
   1. Using a magnetic stirrer and stir bar, dissolve 0.1 g of DCPIP in 100 mL of deionized water. Note: Slightly lowering the pH of the solution enables the DCPIP to more easily dissolve.
   2. Transfer the solution to an amber bottle with cap or completely cover the beaker in aluminum foil.
   3. Store on ice or in a refrigerator until ready to use. Note: During the experiment the DCPIP solution must be kept on an ice bath and in the dark prior to being incubated.
   4. The solution is stable for two weeks if kept cold.
5. Incubation area. Use one of the three methods described (see Figure 2 in the Procedure section).
   1. Place a 1-L beaker filled with tap water between a 150W flood lamp and the area in which students will incubate their samples. The water serves as a heat sink for the light so that the chloroplast suspensions do not become heated.
   2. Turn on a bright overhead projector and point the light directly at the area in which students will incubate their samples. The overhead projector should be located close to the incubation area to ensure a very bright light source.
   3. Turn on a bright overhead projector and place the test tubes in a beaker filled with water on the transparency area of the overhead projector.

Safety Precautions

Although DCPIP is considered nonhazardous, it may stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. 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. 2,6-Dichlorophenolindophenol may be disposed of according to Flinn Suggested Disposal Method #5. Phosphate buffer may be disposed of according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this Super Value Kit for 150 students working in pairs, or for 75 student groups. Both parts of this laboratory activity may be completed in one class period. If necessary, have students clean and label all glassware the day prior. Also, the prelaboratory assignment should be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.

Teacher Tips

• This lab may also be completed using a spectrophotometer. The optimal absorbance will occur at 600 nm.
• Extend the activity by using colored filters to block specific wavelengths of light from reaching the unheated chloroplasts or by testing the rate of photosynthesis at different temperatures or different light intensities.

Answers to Prelab Questions

1. As the chloroplasts capture light energy to form ATP and NADPH what will happen to the blue DCPIP?

   The blue oxidized form DCPIP will be reduced to the colorless form DCPIPH.

2. Refer to a textbook or other reliable source for a diagram of the reactions that occur in photosynthesis and the electron transfer chain. Draw the reaction, inserting DCPIP where NADP occurs in the reaction chain.
   {10973_PreLabAnswers_Figure_3}

Sample Data

Note: Results obtained using a chloroplast suspension in which the absorbance measured 0.313 at 600 nm in a 13 x 100 mm test tube.

Answers to Questions

1. Describe any color change(s) observed in test tubes A–C. Explain any observed color change in terms of the rate of photosynthesis.

   No photosynthesis is occurring in the dark or heated samples. For colors see the chart above.

2. Based on the evidence observed in this experiment, did the suspensions containing heated chloroplasts undergo photosynthesis? How does this result affect the use of the standard test tubes 1–7 as controls for interpreting the rate of photosynthesis?

   No photosynthesis occurred in the heated samples.

3. What factors might account for heated chloroplasts being inactive?

   Heat causes the proteins to deform halting photosynthesis.

4. What reasons account for the difference in the rate of photosynthesis between the unheated chloroplasts that were incubated in the light and those that were kept sealed in aluminum foil?

   Without light photosynthesis was inhibited; therefore, the electron transport chain was not functioning and the DCPIP was not reduced.

5. What other factors besides lack of light and temperature might affect photosynthesis?

   The wavelength of the light, the amount of water available, and the amount of carbon dioxide gas all affect the rate of photosynthesis.

Introduction

In plants, algae, and some types of bacteria, photosynthesis is the process that traps the energy from sunlight, called photons, to convert carbon dioxide and water to glucose and oxygen, and also to make adenosine triphosphate (ATP). ATP is the “fuel” used by all living things. Pigments within these autotrophs (auto = self, troph = nourish) help to capture the energy from the Sun.

Concepts

• Photosynthesis
• Light reaction vs. light-independent reaction
• ATP
• Autotrophs
• Electron transfer

Background

Photosynthesis is a complex process in which light energy, carbon dioxide, and water are converted to chemical energy in the form of glucose and other carbohydrates. Of all of the pigments found in plants, only chlorophyll a directly captures light energy and converts it to chemical energy. When light energy is absorbed by chlorophyll a, it boosts an electron within the chlorophyll molecule to a higher energy level. High-energy electrons are transferred via a series of accessory pigments to NADP+, which is reduced to NADPH, and the excess energy leads to the production of ATP. In a higher order plant, the reduction occurs within the thylakoid of the plant’s chloroplasts (see Figure 1). Synthesis of carbohydrate occurs in a separate series of light-independent reactions, which use NADPH and ATP to convert CO₂ to a series of sugars. These light-independent reactions are sometimes called dark reactions.

In order to study photosynthesis, scientists have developed methods to observe the transfer of electrons that occurs in the light reactions. One compound that allows scientists to monitor the absorbance of light energy by chloroplasts is the indicator 2,6-dichlorophenolindophenol (DCPIP). DCPIP is useful because it changes from its blue oxidized form (DCPIP) to a colorless reduced form (DCPIPH) when it accepts electrons. DCPIP competes with and thus intercepts the electrons meant for NADP+ in the electron transport chain. Simply adding DCPIP to a solution containing chloroplasts and exposing the mixture to a light source will cause the DCPIP to become reduced.

Experiment Overview

The rate of photosynthesis in chloroplasts will be studied by adding the indicator DCPIP and following the color changes produced as it is reduced. The rate will be compared in samples of unheated chloroplasts exposed to light with those that are not exposed. Photosynthesis rates of heated chloroplasts will also be compared to the unheated chloroplasts.

Materials

Prelab Questions

1. What will happen to the blue DCPIP as the chloroplasts capture light energy to form ATP and NADPH?
2. Refer to a textbook or other reliable source for a diagram of the reactions that occur in photosynthesis and the electron transfer chain. Draw the reaction, inserting DCPIP where NADP occurs in the reaction chain.

Safety Precautions

Although DCPIP is considered nonhazardous, it may stain skin and clothing. When working with chemicals wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part A. Constructing a Standard Curve

Constructing a standard curve is a very important technique used in biology and chemistry laboratories around the world. A standard curve is used to determine the amount of a substance contained in an “unknown sample” by comparing its color versus a series of solutions containing known amounts of the substance. In this lab the amount of blue DCPIP within each sample will be used to determine the amount of DCPIP reduced during the incubation of the chloroplast suspensions with light. Precise and accurate volumetric measurements are necessary when constructing a standard curve.

1. Label the top area of seven test tubes with the numbers 1–7. Place the test tubes in the test tube rack.
2. Using separate serological pipets for each solution or mixture add the specified amounts of DCPIP and heated chloroplast suspension listed in the following table into each test tube.
   {10973_Procedure_Table_1}
3. Cover each test tube with a 1-inch square piece of Parafilm and invert to mix. Note: Ensure the Parafilm will not shield any of the liquid in the tube from exposure to the incubation light.

Part B. Electron Capture and Photosynthesis

1. Prepare an incubation area using one of the setups pictured in Figure 2.
   {10973_Procedure_Figure_2_Exposure of chloroplast suspension to light}
2. Work in a darkened room while completing this laboratory experiment.
3. Obtain three additional clean test tubes. Label them A, B and C.
4. Cut a 3" x 5" piece of aluminum foil and use it to cover the outside walls and bottom of test tube B. Use a second piece of aluminum foil to make a loose lid for the same test tube. Note: This is the dark test tube. It will contain the same components as the unheated test tube but the aluminum foil will shield the chloroplasts from light. Place the dark test tube into the test tube rack.
5. Place the remaining test tubes A and C into the test tube rack.
6. Using serological pipets add the amounts listed in the table below into each test tube.
   {10973_Procedure_Table_2}
7. Add the unheated chloroplast suspension to the unheated and dark test tubes, A and B, respectively. Add the heated chloroplast suspension to the heated test tube C.
8. Cover each test tube A–C with Parafilm and invert to mix. Note: Ensure the Parafilm will not shield any of the liquid in the tube from exposure to the incubation light.
9. Compare the color of each test tube A–C with the Standard Curve tubes constructed in Part A. Determine the Equivalent DCPIP Standard in each test tube and record the values under time zero in the data table on the worksheet. Note: Remember to recover the dark test tube with aluminum foil after checking its color.
10. Place the test tube rack in the incubation area prepared in step 1. Turn on the light and incubate only test tubes A–C for 5 minutes.
11. Turn off the light and invert each test tube. Compare the color of each of test tubes A–C with the Standard Curve (test tubes 1–7). Determine the Equivalent DCPIP Standard and record the values under time 5-min in the data table on the worksheet.
12. Repeat steps 10 and 11 twice more for a total exposure time of 10 minutes.

Student Worksheet PDF

10973_Student1.pdf