BIOLOGY: Photosynthesis Lab (Leaf Disc Method)

PHS SCIENCE (2021-2022)

INTRODUCTION: Photosynthesis is arguably the most important biological process on earth. Simply put,
photosynthesis transforms solar energy into chemical energy. Organisms that perform photosynthesis
use the sun’s energy to rearrange the atoms in carbon dioxide and water to produce sugars and oxygen.
In this lab, we will be examining the effect of increased carbon dioxide on the rate of photosynthesis in
spinach leaf disks. Leaf disks normally float. When the air spaces are infiltrated with solution the overall
density of the leaf disk increases and the disk sinks. As photosynthesis proceeds, oxygen is released into
the interior of the leaf, which changes the buoyancy--causing the disks to rise. Since cellular respiration is
taking place at the same time, consuming oxygen, the rate that the disks rise is an indirect measurement
of the net rate of photosynthesis.

Purpose:

Examine the effect of increased carbon dioxide on the rate of photosynthesis in spinach leaf disks by
using a solution containing carbon dioxide and another containing none. We will learn the effects the
different amounts of carbon dioxide has on the rate of a spinach leaf rising, which is the indirect
measurement of the net rate of the leaf’s photosynthesis.

Materials:
- 0.2% Sodium Bicarbonate Solution
- dilute liquid soap
- 2 plastic syringes
- spinach leaves
- hole punch & trays
- plastic cups
- timer
- lamp with reflector
- fluorescent light bulbs of various
Watts
- water
- stirring rod
- label tape & marking pen
- transfer pipettes
- ring stand and clamp Fig 1 - Leaf Cross Section

Procedure → Part I (“Set-Up”):

1. Set up your two plastic cups: one labeled WITH CO2 and the other labeled WITHOUT CO2. Fill
each up ~⅔ of the way full with the appropriate solution.
2. Add 2-3 drops of the dilute soap solution to BOTH cups and lightly stir.
3. Using a hole-punch, cut 20 leaf disks from the spinach leaves. Avoid any major veins in the leaf.
4. Draw gases out of the spongy mesophyll tissue and infiltrate the leaves with the sodium
bicarbonate solution by the following:
a. Remove the plunger from a syringe. Place 10 leaf disks into the syringe barrel.
b. Replace the plunger. Push the plunger down until only a small amount of air is left in the
barrel. Be careful not to crush the leaf disks.
c. Pull up a small amount (5 mL) of the sodium bicarbonate plus soap solution into the
syringe.
 d. Invert the syringe and tap the syringe to suspend the leaf disks in the solution (at the
top).
e. Press the plunger to remove any air from the syringe
f. Create a vacuum in the syringe to draw the air out of the leaf disk tissue. Do this by
placing your finger / thumb over the opening in the syringe while pulling down on the
plunger.
g. Do this for about 10 seconds, swirling the syringe at the same time.
h. Release the syringe and let it spring back. The leaf disks should now sink.
i. You may have to repeat this procedure a few times until all 10 leaf disks have sunk. If it
still doesn’t work after 3 tries, you probably need to add more soap to your solution
then repeat.
5. Remove the plunger from the syringe and pour the solution and discs back into the “with CO2”
cup.
6. Repeat #2 and #3 for 10 more leaf disks, but this time use the “without CO2” (water) solution

Procedure → Part II (Experiment)

1. Place both cups directly under the light bulb that is about 2 inches above the cups.
2. Start the timer immediately after placing the cups below the light source.
3. After each minute, record the number of floating disks in the table below.
4. Gently swirl the cup (or stir the solution with the stirring rod) after each minute.
5. Continue until all leaf disks are floating in the cup with the bicarbonate solution (“with CO2”).
6. Once done, calculate the ET50 as a reference point. The ET50 is the “estimated time” it took
for HALF of the discs to float in the water. If 50% of the disks never floated, you may need to
extrapolate out (estimate) based on the results.

Quantitative Data:
Table 1 – Total Leaf Disks Floating after each Minute
40
Ligh
t 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16
Set-Up Bul mi mi mi mi mi mi mi mi mi
min min min min min min min
b n n n n n n n n n
(W)
10
with CO2 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2
0
without 10
CO2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0

Table 2 - ET50 (Estimated time for half to them to float)

Light
Set-Up Bulb ET50
(W)
30
with CO2 100
minutes
without CO2 100 Never
General Notes & Observations (Include images?):
● After 6 minute mark, leaves in the solution containing carbon dioxide started to sink slower and
was staying about halfway up the water level.
● The leaves in the solution without carbon dioxide did not get any higher in the water and
instead stayed at the bottom of the container throughout the whole experiment.
● Bubbles formed around the leaves in the solution with carbon dioxide.
● With CO2:

● Without CO2:

Post-Lab Questions:
1. What was the purpose of adding CO2 to the water?
For plants to use the CO2 to trigger photosynthesis for them to start absorbing oxygen
 2. What impact did adding CO2 have on your results?
Started the photosynthesis reaction of the leaves

3. Besides the CO2, what other outside variables/factors (at least two) in your experiment could
impact results? How so?
A slight difference in the amount of light the two groups received and slight errors in the amount of CO2
added to the CO2 solution. The groups might have received different amounts of light due to the light
source not being perfectly in the middle of the two. The amount of CO2 could have been different than
predicted due to leftover liquids in/on tools used for moving the leaves.

4. What caused the leaf disks to float?

The leaves photosynthesizing and releasing oxygen into themselves allowed them to float due to their
buoyancy becoming higher.

5. Look back at your data tables/collected evidence. What patterns & trends do you notice overall?
Refer to SPECIFIC data points & observations.
At the 12 minute mark, 2 leaves started floating in the test group with CO2, while no leaves rose to the
surface in the test group without CO2.

6. Hypothesize what you would observe if you conducted the experiment with a higher intensity
lightbulb (i.e. 150W or 200W). Be sure to explain your reasoning.
The photosynthesis reaction would have happened at twice the speed due to more sunlight being
available and absorbed by the leaves. This is because photosynthesis uses the energy from the light it
absorbs to fuel the reaction along with CO2.

7. Hypothesize what you would observe if you conducted the experiment with twice the amount of
CO2 added. Be sure to explain your reasoning.
The reaction would have allowed twice the number of leaves to float because CO2 is used up and the
amount is fixed unlike changing the intensity of the lightbulb, which would allow more leaves to
participate in the reaction due to the limiting reactant doubling.

8. What might be some other ways to measure the rate of photosynthesis? (Hint: Consider the
overall equation of photosynthesis)
Measuring the amount of water left in the container and the concentration of CO2 in the solutions.