hypothesis for photosynthesis lab

Laboratory Manual For SCI103 Biology I at Roxbury Community College

9 photosynthesis.

In this lab, we will study the effect of light intensity and quality (wave length - color) on photosynthesis . As a measure of the rate of photosynthesis, we will monitor the rate of oxygen production. When plants that spend their life submerged in water release oxygen it forms bubbles, which we can count over a period of time to determine photosynthesis rate.

Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that can later be released to fuel the organisms’ activities (energy transformation). This chemical energy is stored in carbohydrate molecules, such as sugars, which are synthesized from carbon dioxide and water - hence the name photosynthesis, from the Greek phōs, “light”, and synthesis, “putting together”. In most cases, oxygen is also released as a waste product. Most plants, most algae, and cyanobacteria perform photosynthesis; such organisms are called photoautotrophs. Photosynthesis is largely responsible for producing and maintaining the oxygen content of the Earth’s atmosphere, and supplies all of the organic compounds and most of the energy necessary for life on Earth.

Although photosynthesis is performed differently by different species, the process always begins when energy from light is absorbed by proteins called reaction centers that contain green chlorophyll pigments. In plants, these proteins are held inside organelles called chloroplasts, which are most abundant in leaf cells, while in bacteria they are embedded in the plasma membrane. In these light-dependent reactions, some energy is used to strip electrons from suitable substances, such as water, producing oxygen gas. The hydrogen freed by the splitting of water is used in the creation of two further compounds that act as an immediate energy storage means: reduced nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP), the “energy currency” of cells.

In plants, algae and cyanobacteria, long-term energy storage in the form of sugars is produced by a subsequent sequence of light-independent reactions called the Calvin cycle; some bacteria use different mechanisms, such as the reverse Krebs cycle, to achieve the same end. In the Calvin cycle, atmospheric carbon dioxide is incorporated into already existing organic carbon compounds, such as ribulose bisphosphate (RuBP). Using the ATP and NADPH produced by the light-dependent reactions, the resulting compounds are then reduced and removed to form further carbohydrates, such as glucose.

The first photosynthetic organisms probably evolved early in the evolutionary history of life and most likely used reducing agents such as hydrogen or hydrogen sulfide, rather than water, as sources of electrons. Cyanobacteria appeared later; the excess oxygen they produced contributed directly to the oxygenation of the Earth, which rendered the evolution of complex life possible. Today, the average rate of energy capture by photosynthesis globally is approximately 130 terawatts which is about three times the current power consumption of human civilization. Photosynthetic organisms also convert around 100-115 thousand million metric tons of carbon into biomass per year.

The main source of light on Earth is the Sun. Sunlight provides the energy that green plants use to create sugars mostly in the form of starches, which release energy into the living things that digest them. This process of photosynthesis provides virtually all the energy used by living things. The primary properties of visible light are intensity, propagation direction, frequency or wavelength spectrum, and polarization, while its speed in a vacuum, 299,792,458 meters per second, is one of the fundamental constants of nature. Visible light, as with all types of electromagnetic radiation (EMR), is experimentally found to always move at this speed in a vacuum.

9.1 Intensity of light

Light is electromagnetic radiation within a certain portion of the electromagnetic spectrum (Figure 9.1 ). The word usually refers to visible light, which is visible to the human eye and is responsible for the sense of sight. Visible light is usually defined as having wavelengths in the range of 400-700 nanometres (nm), or 400 × 10 -9 to 700 × 10 -9 m, between the infrared (with longer wavelengths) and the ultraviolet (with shorter wavelengths). This wavelength means a frequency range of roughly 430-750 terahertz (THz).

Figure 9.1: Spectrum of light. V, violet; B, blue; G, green Y, yellow; O, orange; R, red

In this experiment (Figure 9.2 ), we will study the effect of light intensity on the photosynthetic activity of Elodea canadensis . We will vary the light intensity by changing the distance between the light source and the plant. We will count the emerging oxygen bubbles as an indicator of the photosynthetic activity of the plant.

Figure 9.2: Setup for photosynthesis experiment.

9.1.1 Experimental procedures

Before you begin with the actual experiment, write down in your own words the hypothesis for this experiment:

• Obtain a cylindrical test tube.
• Fill test tube with 0.3% sodium bicarbonate.
• Select a fresh, crisp sprig of Elodea about 15 cm in length.
• While the plant is still submerged, cut 2-3 mm from its base.
• Place the sprig upside down into the test tube filled with sodium bicarbonate. The sodium bicarbonate will absorb anu toxic materials that are released by the plant during photosynthesis.
• Keeping the plant submerged, position a light source 10 cm away and adjust so the light shines directly on the plant.
• Place the test tube in a beaker of water as shown in Fig. 9.2 to prevent overheating the plant. 1. 1. Allow the system to stand 7-10 minutes, or until bubbles begin to appear regularly.
• Count the bubbles produced each minute for a 5-minute period and average them. Record your findings in the table.
• Move the light back 20 cm from the plant, wait 5 minutes, and repeat counting. Record your findings in Table 9.1 .
• Move the light back 40 cm from the plant and repeat counting the bubbles.
• When you have finished recording your data, calculate the average number of bubbles for each 5 minute period and enter the result into the table.

Do the data support or contradict your hypothesis?

Figure 9.3: Appearance of bubbles indicates active photosynthesis.

9.2 Color of light

In this experiment, we will study the effect of the color of light on the photosynthetic activity of Elodea canadensis . We will use filter to expose the plant to light of only a limited range of wavelengths. We will again count the emerging oxygen bubbles as an indicator of the photosynthetic activity of the plant.

9.2.1 Experimental procedures

• Empty the test tube that you used in the previous experiment.
• Fill the test tube with fress 0.3% sodium bicarbonate.
• Place the Elodea sprig into the test tube and submerge it completely in the bicarbonate.
• Place the red colored filter between the test tube and the heat shield beaker and allow it to sit for 5 minutes.
• Count bubbles for 5 minutes as in the previous experiment. Record your findings in Table 9.2 .
• Remove the color filter and expose the plant to white light. Count bubbles again for 5 minutes in 1 minute intervals. Record your findings in Table 9.2 .
• Place the green colored filter between the test tube and the heat shield beaker and allow it to sit for 5 minutes.
• Count bubbles for 5 minutes. Record your findings in Table 9.2 . Table: (#tab:color) Color of light.

9.3 Determination of the light absorption spectrum of dye solutions

In this experiment, we will use a spectrophotometer to measure the differential absorption of light of different wavelength by water stained with food dyes.

Figure 9.4: Spectrophotometer and cuvettes with dye solutions.

9.3.1 Experimental procedures

• Take six cuvettes.
• Fill one cuvette with water.
• Fill each of the remaining five cuvettes with one of the color solutions listed in Table 9.3 .
• Insert the cuvette with water into the slot marked “B”.
• Insert the other cuvettes into the slots marked 1 to 5 and write down which color is in which slot.
• Following the instructions posted on the spectrophotometer, program the machine to take absorption measurements at wavelengths between 380-740 nm in 20 nm steps.
• Once the measurements are completed, write down the absorption number for each dye and wavelength.
• Use a spreadsheet program to graph your results.
• Compare your curves with the data shown in Figure 9.6 .

Figure 9.5: Cuvettes placed in the spectrophotometer.

Figure 9.6: Normalized absorption of red, green and blue dye solutions. Compare these data with your own results.

9.4 Chromatography

Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound’s partition coefficient result in differential retention on the stationary phase and thus affect the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for later use and is thus a form of purification. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture.

In this experiment, we separate a mixture of food dyes (a dark brown liquid). The mobile phase (separation buffer) is 1% NaCl in water, the stationary phase is chromatography paper.

9.4.1 Experimental procedures

• Obtain a small beaker.
• Add NaCl running buffer to the beaker until it reaches a height of about 5 mm.
• Obtain a strip of chromatography paper and put it down on the bench.
• Obtain the bottle containing the dark green food dye mixture.
• Obtain a glass capillary and insert the tip of the capillary into the food dye mixture liquid. A little bit of dye will ascend into the capillary.
• Remove the capillary and apply.
• Touch the left side of the chromatography paper about 1 cm above its lower end with the tip of the capillary. A little bit of green liquid will spread out on the paper. Lift the capillary and touch the paper again just to the right of the dye you just applied. Repeat this until you have a horizontal line of dye from the left to the right side of the paper.
• Place the chromatography paper into the beaker as shown below.
• Observe how the running buffer moves up the paper and separates the dye mixture into three components (red, yellow and blue.

Figure 9.7: Result of the Chromatography experiment.

9.5 Review Questions

• What is light?
• In your own words, describe the endproducts of photosynthesis.
• In your own words, describe what happens in photosynthesis.
• What is chlorophyll and what does it do?
• Where inside of plant cells does photosynthesis happen?
• What is chromatography and what is it used for?

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Floating Leaf Disk Photosynthesis Lab

I use this experiment to reinforce what we’ve been learning in the photosynthesis lessons. I will be using principles of experimental design, scientific method, and CER as well. The floating leaf disk photosynthesis lab is pretty accessible. The only special equipment is really some syringes to sink the floating disks ( I got mine overnight from Amazon ). That means that if you are like me and sometimes lose track of time, this is still a lab you can do relatively last minute.

Objectives:

• Understand the inputs and outputs of photosynthesis
• Investigate how the manipulation of variables like amount of sunlight and baking soda affects the photosynthetic rate in spinach leaves. (For my folks with NGSS standards this is modeling).

• Fresh spinach leaves
• Baking soda
• Distilled water
• Plastic syringes (without needles)
• Transparent cups or beakers
• A light source (lamp or direct sunlight)
• Stirring rod or spoon

Hypothesis:

Students can hypothesize whether the presence of baking soda and light will increase, decrease, or have no effect on the rate of photosynthesis in spinach leaf disks. Review the inputs and outputs of photosynthesis before asking students to make a hypothesis. Students may need support like setting up and if/then statement for them. If I/we __________, then _____ will occur. I like to have the I/we because that helps them identify the dependent and independent variable later.

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Procedure for the leaf disk experiment:

Preparation of solutions:.

• Prepare two solutions: one with baking soda and one without.
• For the baking soda solution, I usually dissolve about 1 grams of baking soda in 100ml of water. You could also use different concentrations of baking soda solutions.
• Use plain distilled water for the control solution.

Preparing the Leaf Disks:

• Use the hole punch to cut out uniform disks from spinach leaves. Aim for 10 disks for each trial. Try to avoid the veins in the leaf because the vein tissue doesn’t have chloroplasts.

Infiltrating the Leaf Disks:

• This is the hardest part.
• Label syringes according to instructions.
• Pull apart syringe and place leaf disks inside.
• Shake them down toward the tip.  Put two pieces back together and gently push air out of syringe.  Be careful not to crush the leaf disks.
• Fill a syringe halfway with the provided solutions (one syringe for each solution), by pulling back and pulling the liquid into the syringe.
• The leaf disks will be floating.  Make sure they are all in the water.
• Close the tip and pull back on the syringe plunger to create a vacuum. Hold for 10 seconds.
• This process helps remove air from the spongy mesophyll layer of the leaf disks, causing them to sink in the solution.
• Continue until all of the disks have sunk in the syringe.
• Remove plunger over container with the solution where you will conduct the experiment.
• Check out this video to see how it is done.

Setting Up the Experiment:

• This is where you can have a lot of fun.
• I make sure each group has a control (which depends on what they are testing). You can easily test amount of light or color of light if you have grow lamps. You can also test different concentrations of baking soda solution (I do a combination of both)
• Place an equal number of infiltrated leaf disks (about 10) at the bottom of two separate clear containers (I usually use beakers because I have them)—make sure that students have them labelled.
• Ensure that all disks are fully submerged and lying flat at the bottom.

Exposure to Light:

• Position the cups under a light source. Make sure both cups receive equal light intensity and duration (if light isn’t one of your variables).
• Start a timer when you expose them to light.

Observation and Data Collection:

• Observe the time it takes for leaf disks to rise to the surface. As photosynthesis occurs, oxygen accumulates in the disks, causing them to float.
• Record the time it takes for each disk to float to the top.

Repeat the Trials:

• Conduct at least two trials for each setup to ensure reliability of the data. Do more if time allows. I usually do have to do the data analysis the next class period.

Analysis and Conclusion:

• Compare the average time it takes for disks in each solution to float.
• Graph the data. This is a skill that students continuously need to work on. Have them identify what will go on the x and y axis.
• Discuss the results in relation to the hypothesis. Did baking soda enhance the rate of photosynthesis?

Discussion Points:

• Discuss the role of carbon dioxide in photosynthesis and how baking soda (sodium bicarbonate) may act as a source of carbon dioxide.
• Explore the importance of controlled experiments and variables. How do we eliminate other variables as possible causes for the results?
• Have students write up their conclusions using a claim-evidence-reasoning format . Their claim should either support or not support their hypothesis.

This lab provides a hands-on way for students to visualize and measure photosynthesis, and also use scientific methods and data analysis.

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9 Photosynthesis Lab Experiments for Your Class

Photosynthesis is one of the fundamental processes that occur in living organisms, particularly in plants. Understanding photosynthesis helps students appreciate the role of plants in the biosphere and their significance in sustaining life on earth. 

Teaching this topic in the science classroom helps students develop a deep understanding of the natural world, its complexity, and its interconnectedness. It also helps prepare them for future studies in biology, ecology, and other related fields. We’ve outlined both in-person experiments and virtual labs you can use to teach photosynthesis!

1. In-person lab: Measure rate of photosynthesis in spinach leaves

Prepare a solution of water and sodium bicarbonate by dissolving 1 g of baking soda in 1 liter of water. Collect several spinach leaves and place them in a beaker of water for a few minutes to hydrate them. Fill several test tubes or small beakers with the sodium bicarbonate solution. Place spinach leaves in each test tube or beaker, ensuring they are fully submerged. After 5 minutes, measure the amount of oxygen produced by the spinach leaves using a Vernier LabQuest or other data-logging equipment.

2. Virtual lab: Pigment Extraction

In Labster’s simulation, Pigment Extraction: Use photosynthesis to produce biofuel and reduce pollution , Roxy, the leader of a team of engineers, will take students on a journey over the sea and show them the most problematic facilities are the coal power plant and the fish farm. To mitigate the problem, students will help create a sustainable plan for energy production using sunlight, heat from a coal power plant, and nutrients from a fish farm.

3. In-person lab: Chromatography experiment

This experiment provides a hands-on opportunity for students to develop important scientific skills, such as making observations, collecting and analyzing data, and drawing conclusions based on evidence. Extract pigments from different leaves using rubbing alcohol and filter paper. The resulting chromatogram will show the different pigments present in each leaf, such as chlorophyll, carotenoids, and anthocyanins. By separating these pigments, students can investigate their individual roles in the process of photosynthesis.

4. Virtual lab: Algae Pigment Analysis

In Labster’s online lab, Photosynthesis: Algae pigment analysis , students will help Roxy, the lead engineer in an environmental project, determine if dark-colored algae can do photosynthesis using green light. They’ll use the Hill reaction and spectrophotometry to measure electron flow and find out if the pigments in the algae can use green light for photosynthesis.

5. In-person lab: Oxygen production experiment

This is an activity used to investigate the production of oxygen during photosynthesis in aquatic plants. It involves placing an aquatic plant, such as elodea or Cabomba, in a beaker of water and exposing it to light while measuring oxygen levels in the water over time.

6. Virtual lab: Electron Transport Chain 1

In Labster’s digital lab, Photosynthesis: Electron transport chain , students will observe the inner workings of the electron transport chain inside a plant cell and learn about the process of photosynthesis. Watch electrons flow, and molecules move during each step of the electron transport chain. How can a photon of light be converted into chemical energy?

7. In-person lab: Starch production experiment

This is an activity used to investigate the role of light in the production of starch in plants. The experiment involves placing a leaf in the dark for a period of time to deplete its starch reserves and then exposing it to light to observe the production of starch during photosynthesis.

8. Virtual lab: Electron Transport Chain 2

In Labster’s simulation, Electron Transport Chain: A rollercoaster ride that produces energy , students will help a group of engineers figure out if a mysterious dark alga can do photosynthesis using green light and measure this process with the Hill reaction. If it is, your work will help create a sustainable plan using sunlight and pollution sources for biofuel production.

9. In-person lab: Light intensity experiment

This experiment helps to understand the interplay between light and photosynthesis and how plants adapt to different light conditions in their environment. Use a light source of varying intensities and measure the rate of photosynthesis in a water plant, such as elodea, using a probe or sensor. Observe how the rate of photosynthesis increases with higher light intensity up to a certain point.

Questions for reflection

• Were any of these in-person or virtual labs new ideas for you?
• Which one can you implement in your classroom?

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Make a hypothesis about which color in the visible spectrum causes the most plant growth and which color in the visible spectrum causes the least plant growth.

How did you test your hypothesis? Which variables did you control in your experiment and which variable did you change in order to compare your growth results?

Analyze the results of your experiment. Did your data support your hypothesis? Explain. If you conducted tests with more than one type of seed, explain any differences or similarities you found among the types of seeds.

What conclusions can you draw about which color in the visible spectrum causes the most plant growth?

Given that white light contains all colors of the spectrum, what growth results would you expect under white light?

• Carry out an experiment to determine which colors of the light spectrum are used in photosynthesis as evidenced by plant growth.
• Measure plant growth under lights of different colors of the spectrum.

Carbon Cycle Lab- Photosynthesis and Respiration

In this lab, students will be testing whether or not aquatic plants do photosynthesis in the dark or light, and also testing if they do cellular respiration during the dark or light. The plant I usually use for this experiment is called elodea, which is available at any local pet store in the fish area. One nugget of information you will need to know- pet stores call it anacharis, not elodea. It is usually sold in bunches of 4-5 stems for a few bucks. Two big bunches should get you through the day. If they don’t have elodea, any other aquatic fish tank plant will work fine, but make sure it is a tall skinny plant that will fit down into your test tubes.

One reason this lab is great is because it can be used in multiple places in your curriculum: ~ Cells unit : When you are teaching cells, chances are you will be talking about chloroplasts and mitochondria. Along with these organelles you will be discussing photosynthesis and cellular respiration. This lab fits in great because it shows that plants not only do photosynthesis, but cellular respiration as well. ~ Ecology unit : During my ecology unit, we cover the 3 major biogeochemical cycles (water, carbon, and nitrogen). What better way to talk about the carbon cycle than to demonstrate the relationship between plants, animals, and gas exchange?

A great extension activity is to add aquatic animals to this experiment and see how the added respiration affects the color change. If you can get your hands on some small snails, they will fit great into the test tubes. I had trouble finding snails in Arizona, so I went to my local pet store and picked up two feeder goldfish. I filled up two large Erlenmeyer flasks with water and bromothymol blue, and turned one yellow. I added elodea and a goldfish to each flask. Next, I asked my students what will happen when we leave these in the light for 24 hours. The next day we came in and saw both flasks were a shade of bluish green (somewhere in the middle of where the two flasks began). If you don’t add a ton of bromothymol blue, and only leave the fish in for 24 hours the fish will not be harmed. Hopefully you are ready to start this experiment! If you have any questions, drop them in the comments below!

• Read more about: Cells , Experiments , Photosynthesis & Respiration

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Photosynthesis Virtual Lab - Plant Growth

"Which colors of the light spectrum are most important for plant growth?"

Site: https://nt7-mhe-complex-assets.mheducation.com/nt7-mhe-complex-assets/Upload-20190715/InspireScience6-8CA/LS12/index.html

(you can type "Plant Growth McGraw Hill" into search bar)

- Read the summary in the side bar which explains how colors of light affect plant growth.

- Read the procedure. Many of your tasks will be recorded in the journal which you will print out to turn in - there are 5 questions to answer in the journal, use complete, thoughtful sentences for each of these questions.

- You will also record your measurements in the table to be printed and turned in.

* Print your questions and tables to turn in.

Journal Questions

1. Make a hypothesis about which color in the visible spectrum causes the most plant growth and which color in the visible spectrum causes the least plant growth?

2. How did you test your hypothesis? Which variables did you control in your experiment and which variable did you change in order to compare your growth results?

3. Analyze the results of your experiment. Did your data support your hypothesis? Explain. If you conducted tests with more than one type of seed, explain any differences or similarities you found among types of seeds.

4. What conclusions can you draw about which color in the visible spectrum causes the most plant growth?

5. Given that white light contains all colors of the spectrum, what growth results would you expect under white light?

Photosynthetic Floatation

Photosynthetic organisms capture energy from the sun and matter from the air to make the food we eat, while also producing the oxygen we breathe. In this Snack, oxygen produced during photosynthesis makes leaf bits float like bubbles in water.

• Baking soda (sodium bicarbonate)
• Liquid dish soap
• Spoon or other implement (for mixing solution)
• Soda straw or hole punch
• Spinach leaves or ivy leaves
• 10-mL syringe (without a needle)
• Clear plastic cup (1-cup size) or 250-mL beaker
• Incandescent or 100-watt equivalent lightbulb in fixture (preferably with a clamp)
• Notepaper and pencil (or similar) to record results
• Optional: ring stand, foil, thermometer, ice, hot water, colored gel filters

• Make a 0.1% bicarbonate solution by mixing 0.5 grams baking soda with 2 cups (500 mL) water. Add a few drops of liquid dish soap to this solution and mix gently, trying to avoid making suds in the solution.

• Pour 150 mL of bicarbonate solution into the cup. Try to avoid making suds.

• Hold the syringe with the tip up, and expel the air by gently pushing on the plunger.

• Set up your light fixture so that it is suspended about 12 inches (30 cm) above the table. You may want to use a ring stand for this.

Turn on the light, start a timer, and watch the leaf disks at the bottom of the cup. Notice any tiny bubbles forming around the edges and bottoms of the disks. After several minutes, the disks should begin floating to the top of the solution. Record the number of floating disks every minute, until all the disks are floating.

How long does it take for the first disk to float? How long does it take for half the disks to float? All the disks?

When all the disks have floated, try putting the cup in a dark cabinet or room, or cover the cup with aluminum foil. Check the cup after about fifteen minutes. What happens to the disks?

Plants occupy a fundamental part of the food chain and the carbon cycle due to their ability to carry out photosynthesis, the biochemical process of capturing and storing energy from the sun and matter from the air. At any given point in this experiment, the number of floating leaf disks is an indirect measurement of the net rate of photosynthesis.

In photosynthesis, plants use energy from the sun, water, and carbon dioxide (CO 2 ) from the air to store carbon and energy in the form of glucose molecules. Oxygen gas (O 2 ) is a byproduct of this reaction. Oxygen production by photosynthetic organisms explains why earth has an oxygen-rich atmosphere.

The equation for photosynthesis can be written as follows:

$$\ce{6CO2 + 6H2O + \text{light energy} -> C6H12O6 + 6O2}$$

In the leaf-disk assay, all of the components necessary for photosynthesis are present. The light source provides light energy, the solution provides water, and sodium bicarbonate provides dissolved CO 2 .

Plant material will generally float in water. This is because leaves have air in the spaces between cells, which helps them collect CO 2 gas from their environment to use in photosynthesis. When you apply a gentle vacuum to the leaf disks in solution, this air is forced out and replaced with solution, causing the leaves to sink.

When you see tiny bubbles forming on the leaf disks during this experiment, you’re actually observing the net production of O 2 gas as a byproduct of photosynthesis. Accumulation of O 2 on the disks causes them to float. The rate of production of O 2 can be affected by the intensity of the light source, but there is a maximum rate after which more light energy will not increase photosynthesis.

To use the energy stored by photosynthesis, plants (like all other organisms with mitochondria) use the process of respiration, which is basically the reverse of photosynthesis. In respiration, glucose is broken down to produce energy that can be used by the cell, a reaction that uses O 2 and produces CO 2 as a byproduct. Because the leaf disks are living plant material that still require energy, they are simultaneously using O 2 gas during respiration and producing O 2 gas during photosynthesis. Therefore, the bubbles of O 2 that you see represent the net products of photosynthesis, minus the O 2 used by respiration.

When you put floating leaf disks in the dark, they will eventually sink. Without light energy, no photosynthesis will occur, so no more O 2 gas will be produced. However, respiration continues in the dark, so the disks will use the accumulated O 2 gas. They will also produce CO 2 gas during respiration, but CO 2 dissolves into the surrounding water much more easily than O 2 gas does and isn’t trapped in the interstitial spaces.

Try changing other factors that might affect photosynthesis and see what happens. How long does it take for the disks to float under different conditions? For example, you can compare the effects of different types of light sources—lower- or higher-wattage incandescent, fluorescent, or LED bulbs. You can change the temperature of the solution by placing the beaker in an ice bath or a larger container of hot water. You can increase or decrease the concentration of sodium bicarbonate in the solution, or eliminate it entirely. You can try to identify the range of wavelengths of light used in photosynthesis by wrapping and covering the beaker with colored gel filters that remove certain wavelengths.

This experiment is extremely amenable to manipulations, making it possible for students to design investigations that will quantify the effects of different variables on the rate of photosynthesis. It is helpful to have students familiar with the basic protocol prior to changing the experimental conditions.

Ask your students to think carefully about how to isolate one variable at a time. It is important to hold certain parts of the experimental setup constant—for example, the distance from the light source to the beaker, the type of light bulb used, the temperature of the solution, the height of the solution, and so on. Certain treatments may eliminate photosynthesis altogether—water with no bicarbonate, very low temperature, and total darkness.

A typical way to collect data in this assay is to record the number of disks floating at regular one-minute time intervals. This is easily graphed, with time on the x-axis and number of floaters on the y-axis.

To make comparisons between treatments, the number traditionally used is the time point at which half of the disks in the sample were floating, also known as the E50.

This experiment was originally described in Steucek, Guy L., Robert J. Hill, and Class/Summer 1982. 1985. “Photosynthesis I: An Assay Utilizing Leaf Disks.” The American Biology Teacher , 47(2): 96–99.

Photosynthesis

Instructor prep, student protocol.

• Plant Pigment Chromatography
• Floating Leaf Discs in a Vacuum
• NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments. HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
• Use a pencil to make a line two centimeters from one end of the chromatography paper.
• Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
• Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
• Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
• When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
• Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
• Next, place 8 mL of chromatography solvent in the beaker.
• Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
• Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
• When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
• Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
• Next, record the number of visible bands and describe their color and relative size.
• Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1. Click Here to download Table 1
• Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
• NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO 2 to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
• To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe “bicarbonate”, and label the other syringe “water”.
• Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
• Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
• Push as much air out as possible without expelling the solution or damaging the leaf discs.
• Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
• To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
• Then, release the vacuum. NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don't sink, you can repeat the vacuum creation up to three times.
• Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
• For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
• Place both cups under a light source.
• Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed. Click Here to download Table 3
• Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
• Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
• To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
• NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
• Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
• Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
• Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. Click Here to download Table 2
• Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
• Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
• Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
• Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50. NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
• Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
• Finally, compare your ET50 values and graphs with the rest of the class.