The Floating Leaf Disk
Assay for Investigating
(A resource page)
Trying to find a good,
quantitative procedure that
students can use for exploring photosynthesis is a challenge. The
such as counting
oxygen bubbles generated by an elodea stem tend to not be
“student” proof or
reliable. This is a
if your laboratory instruction emphasizes student-generated questions.
Over the years, I have found the floating leaf disk assay technique
to be reliable and understandable to students. Once
the students are familiar with the technique they can readily design
experiments to answer their own questions about
The biology behind the prodedure:
Leaf disks float,
normally. When the air spaces are infiltrated with solution
the overall density of the leaf disk increases and the disk sinks.
The infiltration solution includes a small amount of Sodium
bicarbonate. Bicarbonate ion serves as the carbon source for
photosynthesis. 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.
bicarbonate (Baking soda)
SoapPlastic syringe (10 cc or
larger)—remove any needle!
- Leaf material
- Hole punch
- Plastic cups
- Light source
Cellophane or filters
material of different ages
- Clear Nail
300 ml of bicarbonate solution for each
bicarbonate serves as an alternate
of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is
much it is only about 1/8 of a teaspoon of baking soda in 300 ml of
- Add 1 drop of dilute
liquid soap to this
solution. The soap wets the
hydrophobic surface of the
leaf allowing the solution to be drawn into the leaf. It’s
difficult to quantify this since liquid
soaps vary in concentration. Avoid
solution generates suds then dilute
it with more bicarbonate solution.
- Cut 10 or more
uniform leaf disks for each trial.
- Single hole
punches work well for this but stout plastic
straws will work as well.
- Choice of the
leaf material is perhaps the most critical
aspect of this procedure.The
surface should be smooth and not too thick. Avoid plants with hairy
leaves. Ivy, fresh spinach, Wisconsin Fast Plant
cotyledons--all work well. Ivy seems to provide very consistent
results.Many different plant leaves work for this lab.My classes have
found that in the spring, Pokeweed may be the best choice.
- Avoid major
- Infiltrate the
leaf disks with sodium bicarbonate
the piston or plunger and place the leaf disks
into the syringe barrel. Replace the plunger being careful not to crush
the leaf disks. Push on the plunger until only a small volume
of air and leaf disk remain in the barrel (< 10%).
a small volume of sodium bicarbonate solution into
the syringe. Tap
the syringe to suspend
the leaf disks in the solution.
a finger over the syringe-opening, draw back on
the plunger to create a vacuum. Hold
this vacuum for about 10 seconds.
holding the vacuum, swirl the leaf disks to suspend them in the
solution. Let off
the vacuum. The
bicarbonate solution will infiltrate the
air spaces in the leaf causing the disks to sink.
You will probably have to repeat this
procedure 2-3 times in order to get the disks to sink. If you
have difficulty getting your disks to sink after about 3 evacuations,
it is usually because there
is not enough soap in the solution. Add a few more drops
- Pour the disks
and solution into a clear plastic
bicarbonate solution to a depth
of about 3 centimeters. Use
depth for each trial. Shallower
work just as well.
- For a control
infiltrate leaf disks with a solution of only
water with a drop of
light source and start the timer.
At the end of each minute,
record the number of floating disks. Then swirl the disks to dislodge
any that are stuck against the sides of the cups. Continue until all of
disks are floating.
Data Collection and Analysis
These data are from
an demonstration investigation using grape ivy leaf disks.
The point at which 50% of the leaf
disks are floating (the median) is
the point of reference for this procedure.
By extrapolating from the graph, the 50% floating point is
minutes. Using the
50% point provides a
greater degree of reliability and repeatability for this procedure.
As Steucek, et. al. (1985) described this term is referred to
is that it goes down as the rate of photosynthesis goes up--it is an
inverse relationship and creates the following type of graph (data from
Steucek, et al. 1985.):
To correct for this representation of the data and present a graph that
shows increasing rates of photosynthesis with a positive slope the ET50
term can be modified by taking the inverse or 1/ET50.
This creates a graph like this(data
from Steucek, et al. 1985.):
this graph, the light was turned off at 14 minutes and the cups with
their floating disks (grape ivy) were placed in the dark.
Every minute, I removed the dark cover and counted how many were still
floating. Then I stirred the disks. Note that after
the disks begin to sink. Why? Cellular respiration
the oxygen from the cell spaces. The rate that the disks sink
an indirect measure of the rate of cellular respiration. Can
think of a way to how you might measure the gross rate of
photosynthesis with this technique?
Print and Web
Wickliff, J.L. and
Chasson, R.M. 1964. Measurement of
photosynthesis in plant tissues using bicarbonate solutions.
Bioscience, 14: 32-33.
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.
Tatina, Robert E. 1986.
to the Steucek and Hill Assay of Photosynthesis. The American Biology
Teacher, 48(6): 364-366.
Juliao, Fernando and
Henry C. Butcher
IV. 1989. Further Improvements to the Steucek and Hill Assay of
Photosynthesis. The American Biology Teacher, 51(3): 174-176.
Armstrong, Joeseph E.
1995. Investigation of Photosynthesis using the Floating Leaf Disk
Rukes, Kari L. and
Timothy J.Mulkey. 1994. Measurement on the Effects of Light Quality and
Other Factors on the Rate of
Photosynthesis. Bioscene, 20(3): 7-11. http://www.acube.org/volume_20/v20-3p7-11.pdf
Greenler, John. 1990.
Exploring Photosynthesis with Fast Plants. WisconsinFast Plant Notes,
4(1): 4-5. http://www.fastplants.org/pdf/activities/exploring_photosynthesis.pdf
BioPi listserv archives.
"Leaf Disks" for
a search to review a thread on the technique.
Dan Mott attached a copy of his lab using this technique to one of his
Richard, David S.
Measure of Photosynthetic Rate In Spinach Leaf Disks http://www.susqu.edu/FacStaff/r/richard/photosynthlab.html