BIOL 153L General Biology II Lab
Black Hills State University
Lab 4: Photosynthesis & Respiration II
As introduced in previous labs, photosynthesis is the process by which plants utilize light energy to
fix carbon into chemical energy in the form of sugar. The basic formula of photosynthesis is:
carbon dioxide + water + light energy –» carbohydrate + oxygen + water
The light reactions of photosynthesis occur in the chloroplast of photosynthetic eukaryotes. In the
chloroplast, photosynthetic pigments, which include green chlorophyll as well as pigments of other
colors, absorb light. The energy from the light is used to strip electrons from water, which releases
oxygen (O2) as a waste product. (Photosynthetic organisms are in fact responsible for the relatively
high levels of oxygen in the Earth's atmosphere!) NADPH and ATP are also generated during the
light reactions; these molecules are important later in the process of photosynthesis. True to their
name, the light reactions only occur when a photosynthetic organism is exposed to visible light.
In the dark reactions of photosynthesis, short-lived but high-energy compounds produced by the
light reactions (i.e., NADPH and ATP) are used to fix CO2 into sugar using a complex biochemical
pathway. Carbon dioxide enters cells via stomata (on the leaves of true plants) or via diffusion (for
algae and protists). In a multi-stepped process, the carbon dioxide is reduced—the carbon is fixed
and becomes part of a sugar molecule, and the oxygen combines with hydrogen ions to become
water. Note that the dark reactions do not require light, and may occur in a photosynthetic organism
in dark or light environments. However, if the photosynthetic organism is deprived of light for too
long, it will run out of the energy (ATP and NADPH) needed to run the dark reactions.
Like animals and other heterotrophs, plants break down food molecules for energy via respiration.
The difference is they are breaking down food (i.e., sugars, complex carbohydrates, fats, proteins)
that they made directly or indirectly via photosynthesis. Respiration occurs in the mitochondria,
and food molecules (sugars) are oxidized to release energy in the form of ATP – and oxygen is
consumed by this reaction, while carbon dioxide is released. The basic formula of respiration is:
carbohydrate + oxygen + water –» ATP (energy) + water + carbon dioxide
Note that for photosynthetic organisms, respiration and photosynthesis are opposing biochemical
processes—plants release carbon dioxide and use oxygen in respiration, but release oxygen and use
carbon dioxide in photosynthesis. Ultimately, for plants to grow and successfully reproduce, the
carbohydrate gains from photosynthesis must exceed the carbohydrate losses from respiration.
As described in last week's lab, photosynthesis in land plants may be studied using expensive
instrumentation, like the leaf porometer, that estimate gas exchange across leaf stomata. In many
ways, aquatic environments are better suited for this kind of research. Like their counterparts on
land, photosynthetic organisms in aqueous solutions will consume and release carbon dioxide and
oxygen based on the balance of respiration and photosynthesis – but amounts of these gasses may be
easily measured because they remain trapped in the water rather than escaping to the atmosphere.
Thus, photosynthetic rates may be measured by pH (solutions with abundant carbon dioxide are
acidic, owing to the formation of carbonic acid) or formation of air bubbles (which are oxygen
molecules produced by the light reactions of photosynthesis. Today we will use air bubbles – and
their buoyant properties—to look at factors influencing light and dark reactions of photosynthesis.
1
Introduction to "Leaf Disks"
Although terrestrial plants normally undergo gas exchange with the atmosphere, their leaves will
carry out dark and light reactions of photosynthesis for a day or two following their submersion in
water. Thus, we can estimate photosynthetic rates of higher plants in the absence of water limitation
by placing leaf pieces in aqueous solutions and measuring concentrations of carbon dioxide (e.g., by
determining pH of the water) or oxygen (e.g., by evaluating oxygen bubble formation). The latter is
particularly easy to assay, because oxygen bubbles make leaves buoyant – the rate of photosynthesis
will be linked directly to the time it takes leaf pieces to float in water!
Today you will examine factors that affect photosynthesis – such as availability of carbon dioxide,
amount and wavelength of light energy, and ambient temperature but not water related factors like
air humidity and soil moisture—by observing leaf disks of ivy (Hedera helix and H. hibernica) in an
aqueous bicarbonate solution. You will first use vacuum infiltration to remove oxygen from the leaf
disks and replace it with water; this causes the disks to sink. Following this treatment, oxygen that
accumulates in intercellular spaces of the leaf spongy mesophyll via photosynthesis will cause disks
to rise from the bottom of their container and approach the water surface. The time that it takes for
leaves to rise in the aqueous solution is a measure of photosynthetic rate achieved under conditions
prescribed to the container (e.g., availability of carbon dioxide, light exposure, temperature, etc.).
Anatomic features of plant leaves will be covered in depth later in the BIOL 153. Nonetheless, it is
worth reviewing basic leaf structures before proceeding with the exercise today. Recall that stomata
are located primarily on the bottom of leaf surfaces in ivy. Inside these stomata are loosely packed
cells (spongy mesphyll) with abundant air spaces; this is the primary site of gas exchange and, of
significance to today's lab, accumulation of oxygen for leaf disks in an aqueous solution. Above the
spongy mesophyll is the palisade mesophyll – one or more rows of densely packed cells in which
the light and dark reactions of photosynthesis occur – and then epidermis. Vascular tissue (xylem
and phloem) is contained in leaf veins that transect the spongy and palisade mesophyll. The images
below show cross sections of a hypothetical plant leaf with aforementioned structures and layers.
2
Getting Organized:
Watch the ksbioteacher video, Sinking Leaf Disks, before proceeding:
https://www.youtube.com/watch?v=vw8baZO89oc
1. You will be working together in groups of 3-4. Please move to a new table if you are alone or
have a single partner at your table.
2. Check that the following items are available on your lab table.
•
•
•
•
•
•
•
•
•
•
Filled beaker marked “Sodium Bicarbonate Solution”
Filled beaker marked “Distilled Water”
4 syringes (without needles)
6 small plastic cups with lids
1 paper punch
1 lamp
1 large Petri dish
1-2 probes
Ivy leaves
Three paper labels indicating treatment
3. Each group member should practice using the syringe to create a vacuum. Use beaker
marked “Distilled Water” for this exercise. Use syringe to draw up 10 mL of distilled water – then
point syringe toward ceiling and carefully push plunger to expel air bubbles. Once air bubbles are
expelled, place a thumb or index finger from one hand firmly over the syringe tip. Use the other
hand to pull the plunger. You should feel resistance! Hold for a few seconds and then release.
4. Each group member should practice emptying the syringe contents into the plastic cup
without spilling. Pull plunger almost—but not all the way—out of the syringe, then hold thumb
over syringe tip. Hold the syringe over the cup and carefully remove the plunger fully. Dump the
water into the plastic cup, and then dispose of this water in the lab sink.
Experiment 1: Testing effects of carbon dioxide concentration and light intensity on
oxygen production in ivy leaf disks. Let's start today by looking at two basic factors that
we'd expect to affect photosynthetic rates – the availability of carbon dioxide and light!
Motivation and Design of Experiment #1, Part A
Observation: Water naturally contains small amounts of CO2 that photosynthetic organisms use to
fix carbon during the dark reaction of photosynthesis.
Question: Would photosynthetic rates change if additional carbon is added to the water?
Method: Addition of baking soda (sodium bicarbonate, NaHCO3) to water will increase the
concentration of carbon dioxide in the solution. Thus, a solution of sodium bicarbonate will be used
to infiltrate a set of leaf disks used in the photosynthesis assay.
Control: A second set of leaf disks will be infiltrated with distilled water (lacks sodium
bicarbonate and other impurities) to compare with the above-described NaHCO3 infiltrated leaf
disks
3
PLEASE ANSWER THE QUESTIONS BELOW BEFORE STARTING EXPERIMENT
Why is baking soda added to the water in this experiment?
Why will the leaf disks float when photosynthesis occurs? Describe what a fast photosynthetic
rate will look like relative to a low photosynthetic rate (i.e., how can you tell a difference)?
Do you predict that photosynthetic rates will be higher or lower when baking soda is added?
Motivation and Design of Experiment #1, Part B
Observation: Plants require light to grow.
Question: Do the light reactions of photosynthesis occur in leaves placed in the dark?
Method: Prevent light exposure from contacting one container of leaf disks that is infiltrated with
sodium bicarbonate, and then assay photosynthetic rate on these leaf disks over time.
Control: A second set of leaf disks will be exposed with light to compare with the above-described
dark-treated container of leaf disks.
Why are both leaf disk containers (light + dark treated) infiltrated with sodium bicarbonate?
Would it be okay to use distilled water for one set of these leaves? Explain your logic.
Do you predict that photosynthetic rates will be higher or lower for dark-treated leaves?
Do you think it is possible to test effects of light and carbon dioxide with one multifaceted
experiment? Explain how this could work.
4
SET-UP EXPERIMENT #1:
1. Use paper punch to make 30 leaf disks. Make the punches relatively close together (so you
don’t run out of leaf) and avoid large leaf veins. Put the leaf disks into the large Petri dish.
2. Prepare 3 syringes and add 10 leaf disks to each. Tap the syringe so all leaf disks are near the
tip, and then replace plunger. Push plunger to the 5 mL mark—don’t squish leaf disks!
BE SURE TO READ STEP #3 BELOW CAREFULLY!!!
3a. For 2 syringes (with leaf disks), suck up 10 mL of sodium bicarbonate solution. The
sodium bicarbonate solution should be at the 15 mL mark on the syringe (leaf disks and air are
between 0-5 mL). Do not mix up this syringe with the syringe containing distilled water below!
3b. For 1 syringe (with leaf disks), suck up 10 mL of distilled water. The sodium bicarbonate
solution should be at the 15 mL mark on the syringe (leaf disks and air are between 0-5 mL). Do
not mix up this syringe with the syringes containing sodium bicarbonate solution above!
4. Hold syringes upright and expel air bubbles, as practiced earlier (see page 3).
5. Press thumb or index finger over syringe tips and pull plunger to infiltrate. Note the
bubbles erupting from leaf disks! (How cool is that?!) Maintain your hold on the plunger until this
bubbling stops, or at least greatly slows (~10-30 seconds).
6. Release plunger and tap syringe to dislodge leaf disks. Set syringe pointing up on table
(resting on the plunger)—do not mix up distilled water and sodium bicarbonate syringes! Wait a
few seconds to see if leaf disks sink. If any leaf disks are still floating, repeat steps #4-6 above. (If
leaf disks are still floating after three infiltration attempts, please consult instructor.)
7. When all leaf disks have sunk, remove plunger and add contents to plastic cup and put on
clear covers. Be sure to note which cup contains the distilled water! If necessary, add additional
sodium bicarbonate solution or distilled water to bring liquid to desired level for evaluating leaf disk
flotation. (Please see note on table about the proper solution level.)
8. Proceed quickly with next steps to minimize light exposure. Please note that if you aren’t
able to proceed quickly with the assay, you should put cups in dark place until you're ready.
9. Put distilled water cup and 1 sodium bicarbonate cup under light. Bend neck of lamp so that
it is the proper distance from cups. (Please see note on table about proper height of light above the
cups). Confirm that both cups are for sure getting comparable amounts of light.
10. Put 1 sodium bicarbonate cup in dark cupboard at lab table.
11. Start timer. Every minute count the number of disks that are floating and record data in
lab handout (page 6). Just before making your count, gently swirl cup to dislodge any disks that
are stuck together. Be sure to minimize the time the dark treatment is exposed to light.
12. Continue your assay until all disks float—or for 20 minutes, whichever comes first.
13. Remove dark treatment and dispose of liquid and leaf disks. Please use strainer at sinks to
capture leaf disks so that we do not clog the drains!
5
14. Put distilled water treatment under light on side bench—you will check this treatment
again at the end of the class. Record data in Table 3.
15. Put sodium bicarbonate light treatment in dark cupboard and set timer for 30 minutes.
At 30 minutes, count number of floating leaf disks. Record data in Table 2.
16. Record your data in the table below and then graph on the template (see page 7).
TABLE 1. Effects of CO2 and light treatments on photosynthesis. Please record the number
of leaf disks floating at each measurement time (1-20 minutes or until all disks floating).
No.
Minutes
Sodium bicarbonate
+ light treatment
Distilled water
+ light treatment
Sodium bicarbonate
+ dark treatment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
TABLE 2: Results of sodium bicarbonate + light treatment container placed in dark
cupboard following experiment. Please record the number of leaf disks floating after 30 min.
Sodium bicarbonate + light treated
container placed in dark cupboard
Minutes
30
TABLE 3: Results of distilled water + light treatment container left under lights on side table
following experiment. Please record the number of leaf disks floating at end of lab.
Distilled water + light treated
container left under lights
Time
End of lab (~2 hours under light)
6
Questions about carbon dioxide levels and light: After completing the graphing above,
please proceed to the next page and answer the questions presented.
7
1. Through the process of photosynthesis, ivy releases oxygen bubbles that become trapped in
the spongy layer of the leaf. Where did the plant obtain the oxygen?
The time required for 50% of the leaf disks to float—the median—is called ET50 (effective time).
Because some leaf disks may behave weirdly and float earlier or later than others, it is often more
meaningful to focus on median time to float rather than the total time required for all disks to float.
Note that smaller ET50 values indicate higher rates of photosynthesis (i.e., the more rapidly leaf
disks float the more photosynthesis is taking place in the leaf disks).
2. Inspect your graph and indicate below the ET50 for the three experiment treatments.
3. How did CO2 addition affect photosynthesis? (Use ET50 values to answer.)
4. How did light affect photosynthetic rates? (Use ET50 values to answer.)
5. This experiment was conducted with terrestrial leaves in water. How might effects of light
differ in real-world conditions where plants are exchanging gases in the atmosphere?
6. When a cup with floating leaf disks is placed in the dark, what happens? (Observe the
sodium bicarbonate + light cup with floating disks that was placed in the dark; you will need
to wait at least 30 minutes to answer this question.) Interpret this result.
7. Does photosynthesis occur over time for leaf disks in distilled water? (Observe the distilled
water cups placed on the side bench; you need to wait until the end of class to get this result.)
8
Experiment 2: Work with your lab partners to design your own experiment!
Now you will design an experiment to test other factor(s) that affect photosynthesis. The following
items are available for your use.
•
•
•
•
•
•
•
•
•
Ivy leaves of different ploidy levels (diploid H. helix and tetraploid H. hibernica)
Ivy leaves of different ages
Ivy leaves that have been exposed to light or that have been kept in the dark
Solutions with different levels of sodium bicarbonate
Solutions with different pH levels
Solutions with different temperatures
Red, green, and blue transparent cello filters
Fluorescent and LED bulbs
Blocks to position leaf disks at variables distances from light source
As a group, discuss the factor(s) you would like to test and how you will do this using available
supplies and the lab set-up. (Remember that there is space for only three leaf disk containers under
the lamps, though you could accommodate additional containers if they don't use a lamp treatment.)
If you want something that isn’t on the list, ask – we may be able to find it for you. Fill out the table
below with your observation, question, prediction, method, and control, following the format used
above on pages 3 and 4. When you have fully thought through your experiment and completed the
table, touch base with your instructor to confirm their approval for your approach.
Motivation and Design of Student Experiment
Observation:
Question:
Method:
Control:
9
Proceed with your experiment and record your data in the table below. Be sure to
identify the factor(s) being tested in the table and column headings!
TABLE 4: Student designed experiment evaluating effect of ____________________________
__________________________________________________ on photosynthesis in ivy leaf disks.
No.
Minutes
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
After completing the above data table for your experiment(s), please return to page 7
of the handout to graph your findings in the template provided! Then proceed to
questions posed on the next page of this exercise.
10
Questions about the 'design your own experiment' exercise. Answer the questions below to
summarize your findings. Then, as a group, present this information to your instructor and to
another student group. Everyone in the group should be active in the communication!
1. What factor(s) related to photosynthesis did you choose to study?
2. What was the motivation to study this factor? How does the factor you tested apply to the
real world (i.e., what might cause this factor to vary in nature and affect ivy)?
3. What was your prediction?
4. What was the method you used to test the prediction (e.g. variables and control)?
5. Using the graph of your data on page 7, summarize your results. How did your treatments
affect photosynthetic rates? How did this compare to results of the control?
6. Did you get the predicted result in the experiment? If so, what does this confirm about
your understanding of photosynthesis? If not, what may explain the unexpected results?
7. What potential sources of error were present in this experiment? How would you change
the experimental design if you were to run this experiment again?
11