AP Biology Lab: Photosynthesis

(adapted from AP College Board Photosynthesis lab)

Background and Pre-lab:

Photosynthesis fuels ecosystems and replenishes the Earths atmosphere with oxygen. Like all
enzyme-driven reactions, the rate of photosynthesis can be measured by either the
disappearance of substrate, or the accumulation of product.

The equation for photosynthesis is:

6CO2 + 6H2O C6H12O6 + 6O2 (+ H2O technically)

To determine the rate of photosynthesis one could measure the production of oxygen, which is
released when photosynthesis occurs, or the consumption of carbon dioxide.

Leaf Structure and Function:

In this investigation, you will use a system

that measures the accumulation of oxygen in
the leaf. Consider the anatomy of the leaf.
The leaf is composed of layers of cells. The
spongy mesophyll layer is normally infused
with gases, such as oxygen and carbon
dioxide. Leaves (or disks cut from leaves) will
normally float in water because of these
gases. If you draw the gases out from the spaces, then the leaves will sink because they
become more dense than water. If this leaf disk is placed in a solution with an alternate source
of carbon dioxide in the form of bicarbonate ions, then photosynthesis can occur in a sunken
leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy
mesophyll, causing the leaf to become buoyant and float.

While this is going on, the leaf is also carrying out cellular respiration. This respiration will
consume the oxygen that has accumulated and possibly cause the plant disks to sink. In other
words, the buoyancy of the leaf disk is actually an indirect measurement of the NET rate of
photosynthesis occurring in the leaf tissue.

Pre-lab Questions:
1. What is the function of the stoma (or stomata)? The function of the stoma is to allow
carbon dioxide and release oxygen.

2. What will happen to the leaf disks if you remove the air from the spongy mesophyll? I f
you remove the leaf disks from the spongy mesophyll, then the leaf disks will sink.
 3. How will air return to these air spaces? Air can only return to this space if the leaf disk is
placed in a solution where carbon dioxide is immersed in the form of bicarbonate ions.
Photosynthesis will then occur to allow gases to accumulate.

4. Instead of carbon dioxide, what will be used as the reactant in this lab? S
odium
Bicarbonate

Materials:

Sodium Bicarbonate (Baking soda), 2g Leaves (spinach or ivy)

Water, 450 mL Hole punch
Liquid soap 2 Cups
2 Plastic syringes (10 mL or larger) 1 Beaker
Marker Timer
Tape Light source
1 Disposable Pipette 2 Stirring rods

Procedure:
1. Collect leaf disks by punching holes in the dark green portion of a leaf (try to get them
between the large veins). You will use a total of 20 disks.

2. Make a solution of sodium bicarbonate by mixing 200 mL of water to 2g of baking soda.

Pour this solution into a cup until it is full. Label this With CO2.

3. Add the same amount of water to another cup. Label this Without CO2.

4. Make a diluted solution of liquid detergent (3 drops of soap to 70 mL of water). This has
already done for you; it is located in the graduated cylinder on the cart.

5. Add one drop of this dilute soap solution to both the beaker of bicarbonate solution and
the beaker with water do not generate suds.

6. Draw the gases out of the spongy mesophyll tissue and infiltrate the leaves with the
solution by performing the following steps.

a. Remove the plunger from both syringes. Place 10 leaf disks into each syringe.

b. Replace the plunger, but be careful not to damage any of the leaf disks. Push in
the plunger until only a small volume of air and leaf disk remain in the barrel
(<10%).

c. Pull a small volume (5 cc) of sodium bicarbonate plus soap solution from your
beaker into one syringe and a small volume of water plus soap into the other
syringe. Tap each syringe to suspend the leaf disks in the solution. Make sure
 no air remains. Move the plunger to get rid of air from the plunger before you
attempt Step d.

d. You now want to create a vacuum in the plunger to draw the air out of the leaf
tissue. This is the most difficult step to master. Once you are able to do this you
will be able to complete the entire exercise successfully. Create the vacuum by
holding a finger over the narrow syringe opening while drawing back the plunger.
Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf
disks to suspend them in the solution. Now release the vacuum by letting the
plunger spring back. Tap the syringe a few times to allow the disks to sink.

i. If the plunger does not spring back, you did not have a good vacuum and
you may need a different syringe. You may have to repeat this procedure
two or three times in order to get the disks to sink (If you have any
difficulty getting your disks to sink after three tries, it is usually because
there is not enough soap in the solution. Try adding a few more drops of
soap to the beaker and replacing the liquid in the syringe).

ii. Placing the disks under vacuum more than three times can damage the
disks and they may need to be replaced.

7. Pour the disks and the solution from the syringes into the appropriate cups by holding
the syringe over the beaker and removing the plunger.

8. Place both cups under a light source and start the timer.

9. Record the number of disks floating at the end of each minute, swirl the disks to dislodge
any that may get stuck to the beaker or to each other. The experiment should continue
until all the disks are floating.

Data

Time (mins.) # of Light Discs # of Light Discs

Floating in Baking Floating in Water
Soda Solution

1 1 2

2 1 2

3 2 2

4 3 2

5 3 2

6 3 2
7 3 2

8 3 2

9 3 2

10 3 2

11 3 2

12 3 2

13 3 2

14 4 2

15 4 2

16 4 2

17 4 2

18 5 2

19 5 2

20 5 2

Analysis:

To make comparisons between experiments, a standard point of reference is needed. Repeated

testing of this procedure has shown that the point at which 50% of the disks are floating (ET50) is
a reliable and repeatable point of reference.

Insert a graph of your data for the experiment. X-axis is Time (min) and Y-axis is # floating
disks. Be sure to include a key for each sample set. There should be four lines on your graph.
Determine the ET50 for each set of leaf disks.

ET50 of Light Disks floating in water = (0,0)

ET50 of Light Disks floating in baking soda solution = 18 minutes

Write a summary of your data. Was there a difference in the rate of photosynthesis between the
light disks with CO2 and without CO2? Describe this difference (if any).

The rate of photosynthesis for the light disks with CO2 was higher than that of the light disks
without CO2. This is indicated by the fact that the light disks with CO2 steadily increased in
number while the number of light disks without CO2 remained at a constant value.

Conclusion:

Answer the following in your conclusion: What would account for your results? Summarize
photosynthesis and relate to the differences between the leaf samples. What environmental
factors might affect the rate photosynthesis? How and why do you think these environmental
factors would affect the rate of photosynthesis?

The rate of photosynthesis increases with the presence of CO2 in a given environment. This
coincides with the fact that photosynthesis is a process that is closely related to the
concentration of gases within a plants cells. Both carbon dioxide and water are used as
reactants in photosynthesis to generate the products of oxygen and glucose. When placed in a
solution that provides said reactants under a light source, this jump starts photosynthesis, hence
why the leaves were floating in such solution during the lab trials. While there is a strong
correlation between the presence of gases and the rate of photosynthesis, there are, however,
factors that could affect it. These factors are the concentration of CO2 and water in a solution as
well as the amount of ambient light. The concentration of both CO2 and water in a solution is
important to consider as there is a direct relationship between the number of molecules in the
reactants with the number of molecules as the products. As the amount of reactants increase,
so do the products, and vice versa. Additionally, the amount of ambient light can change with
certain weather conditions, and can either decrease or increase the amount of light available for
photosynthesis. This is, thus, a direct relationship, as the more light available the more likely
photosynthesis is to occur.