Leaf Disc Assay
Introduction
Today you will be using an assay, which will allow you to measure the relative rates of
photosynthesis. It is called a Floating Leaf Disk Assay. In this activity you will investigate some of the
factors (light intensity) that influence photosynthesis. To do this you will use small round disks of spinach
leaves (or other fresh, chlorophyll rich, leaves). If you force the air out of the intercellular spaces of
these disks they will sink in water. If you allow these disks to go through photosynthesis the air spaces
will eventually fill with oxygen and the disks will float again. The faster the rate of photosynthesis, the
sooner the disks will float.
Materials
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Meter stick
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Light with 100 watt bulbs and reflectors
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Four 100mL beakers
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Four small culture dishes (should fir on top of beakers) or
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Petri dishes
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One large culture dish (large Petri dish)
One 50mL graduated cylinder
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Paper punch or straw
*NaHCO3 is called sodium bicarbonate, or more
Fresh spinach (or other dark green, fresh) leaves
One 12mL plastic syringe
Timer or stop watch
320 mL 0.4% NaHCO3 solution at 35˚C
Bottle of 10% dish washing solution (on main lab
bench)
Tape and markers for labels
commonly, baking soda
Pre-Lab HELP! (Same as usual... just a few hints/reminders)
 Write out your purpose, hypotheses and procedure
 Purpose Write the purpose for doing this lab. (1-2 sentences)
 Hypothesis Make one hypothesis to predict the outcome of the assay using
different intensities of light. Make sure to justify your hypotheses (you may use
"If/Then/Because (IV/DV/Mechanism)" statements). Identify the control group
that will be used for comparison.
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Procedure
Procedure Summarize all of the steps below in your journal (or type if
preferred), don’t forget to include your data table!
1. Adjust the height of the lamps according to the chart below. Light Intensities at each lamp height have
been calculated for you.
Beaker
Light Intensity
Distance from table top to rim of light (cm)
1
100%
13cm
2
50%
18cm
3
25%
26cm
4
12.5%
35cm
*This step will probably be done ahead of time for you
2. Label the 100mL beakers 1-4.
3. Fill the four small culture dishes half full with tap water.
IF YOUR CLASS IS LESS THAN 60MIN LONG, SKIP TO STEP 19! RETURN TO STEPS 4-18 AT
END!
* If your class period is less than 60min, steps 4-18 will be done for you ahead of time,
skip from step 3 to step 19, you will THEN RETURN to steps 4-18 after you have placed your
experiment under the light.
4. Fill the large culture dish three quarters full with tap water.
5. Punch out 50 disks from the spinach leaves; do not punch through the large veins. Place the leaf disks
into the water in one of the small culture dishes as you punch them out.
a. NOTE: Punch out more leaves if you plan to do extra credit too!
6. Measure 20mL of tap water and add one drop of 10% dish soap solution.
7. Remove the plunger from the syringe. Put your finger over the tip.
8. Place the 10mL of soap solution and the 50 leaf disks in the syringe. Be sure all the disks are in contact
with the solution.
9. Insert the plunger and invert the syringe (the plunger is now at the bottom).
10. Remove your finger from the tip and slowly push up on the plunger to remove the air from the syringe.
11. Cover the tip of the syringe with your finger.
12. Pull back on the plunger with your other hand (DON’T PULL THE PLUNGER OUT! JUST PULL IT
BACK!) to create a vacuum in the syringe. Hold the plunger back for 10 seconds. Gently shake the
syringe during this time.
a. This will pull all the air out of the leaf disks so that they will sink.
13. Keeping your finger over the tip of the syringe, let go of the plunger and allow it to return to its
original position.
14. After the plunger has returned to its original position force out any air bubble that may have arisen by
gently pushing up on the plunger.
15. Repeat two to three times until all the disks sink.
16. Place the tip of the syringe in the large culture dish of water, pull back on the plunger and fill the
syringe with water.
17. Keep the tip of the syringe in the water but remove the plunger from the syringe.
18. Lower the syringe into the water and tip it up so the disks are released into the culture dish. The disks
should sink. Do not use any that float.
19. Add 80mL of 35˚C NaHCO3 (located in the heated water bath) solution to each 100mL beaker.
20. Use forceps to transfer 10 leaf disks into each beaker. Place them in the solution edge-on so air is not
trapped under them.
21. Place each beaker under the correct lamp (use chart above).
22. Cover each beaker with a culture dish with water to absorb the heat from the lamp.
23. Turn on the lamp and start timers.
** IF YOU PREVIOUSLY SKIPPED STEPS 4-18- While 2 of your group members monitor your
experiment (steps 24-25), the other 2 to 3 partners need to go back and complete steps 4-18 to prepare
the lab for the next class (as was done for you!)
24. Note the amount of time its takes for 5 of the 10 disks to rise to the surface in each beaker.
25. Record your results in the table below.
Beaker
Distance (cm) Light Intensity
Time for 5 disks to rise (min:sec)
1
13cm
100%
2
18cm
50%
3
26cm
25%
4
35cm
12.5%
Observations
1. What is the dependent variable in this experiment?
2. What is the independent variable in this experiment?
3. What are 3 control variables in this experiment? (NOTE: There were more than 3!)
4. Graph time vs. light intensity. Make sure to use proper graphing techniques and label your graph. MUST
USE EXCEL! SEE EXCEL HELP LINK ONLINE IF YOU NEED ASSISTANCE!
5. What is the relationship between light intensity and rates of photosynthesis?
Conclusion
1. What can you conclude about light intensity and photosynthesis?
2. What other variables may influence rates of photosynthesis?
3. Write 1 paragraph (3-6 sentences) summarizing any other conclusions you may have about
photosynthesis, any source of error in your experiment, and any improvement you would make to
better test your hypothesis if you were to do the experiment over again. (Required for all formal lab
reports, you DO NOT have to do this twice, this is just a reminder!)