The Floating Leaf Disk Assay for Investigating Photosynthesis
(A resource page) Brad Williamson
Introduction:
Trying to find a good, quantitative procedure that students can use for exploring photosynthesis
is a challenge. The standard procedures such as counting oxygen bubbles generated by an
elodea stem tend to not be “student” proof or reliable. This is a particular problem 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 photosynthesis.
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.
Materials: Optional:
Sodium bicarbonate (Baking soda) Buffer Solutions
Liquid Soap Colored Cellophane or filters
Plastic syringe (10 cc or larger) Leaf material of different ages
Leaf material Variegated leaf material
Hole punch Clear Nail polish
Plastic cups
Timer
Light source
1
Procedure:
1. Prepare 300 ml of bicarbonate solution for each trial.
o The bicarbonate serves as an alternate dissolved source of carbon dioxide for
photosynthesis. Prepare a 0.2% solution. (This is not very much it is only about 1/8 of a
teaspoon of baking soda in 300 ml of water.)
o 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 [Link] your solution generates suds then dilute
it with more bicarbonate solution.
2. Cut 10 or more uniform leaf disks for each trial.
o Single hole punches work well for this but stout plastic straws will work as well.
o Choice of the leaf material is perhaps the most critical aspect of this [Link] leaf
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 [Link] different plant leaves work for this [Link] classes have found
that in the spring, Pokeweed may be the best choice.
o Avoid major veins.
3. Infiltrate the leaf disks with sodium bicarbonate solution.
o Remove 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%).
o Pull a small volume of sodium bicarbonate solution into the syringe. Tap the syringe to
suspend the leaf disks in the solution.
o Holding a finger over the syringe-opening, draw back on the plunger to create a vacuum.
Hold this vacuum for about 10 seconds. While 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 of soap.
4. Pour the disks and solution into a clear plastic cup. Add bicarbonate solution to a depth of
about 3 centimeters. Use the same depth for each trial. Shallower depths work just as well.
5. For a control infiltrate leaf disks with a solution of only water with a drop of soap--no
bicarbonate.
6. Place under the 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 the disks are floating.
2
Data Collection and Analysis
These data are from an demonstration The point at which 50% of the leaf disks are
investigation using grape ivy leaf disks. floating (the median) is the point of
Minutes Disks reference for this procedure. By
extrapolating from the graph, the 50%
1 0
floating point is about 11.5 minutes. Using
2 0 the 50% point provides a greater degree of
3 0 reliability and repeatability for this
4 0 procedure. As Steucek, et. al. (1985)
described this term is referred to as the
5 0
ET50.
6 0
7 1
8 1
9 1
10 1
11 4
12 7
13 8
14 10
The problem with ET50 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
3
inverse or 1/ET50. This creates a graph like
this(data from Steucek, et al. 1985.):
