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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 standardprocedures 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 readilydesign 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.  Asphotosynthesis 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.

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Materials:

• Sodium bicarbonate (Baking soda)
• Liquid SoapPlastic syringe (10 ccor larger)—remove any needle!
• Leaf material
• Hole punch
• Plastic cups
• Timer
• Light source

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Optional:

• Buffer Solutions
• Colored Cellophane or filters
• Leaf material of different ages
• Variegated leaf material
• Clear Nail polish

Procedure:

• Prepare 300 ml of bicarbonate solution for each trial.o
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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 intothe leaf.  It’s difficult to quantify this since liquid soaps vary in concentration. Avoid suds.If your solution generates suds then dilute it with more bicarbonate solution.
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• Cut 10 or more uniform leaf disks for each trial.
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o Single hole punches work well for this but stout plastic straws will work as well.
o Choice of the leaf material isperhaps the most critical aspect of this procedure.The 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 results.Many different plant leaves work for this lab.My classes have found that in the spring, Pokeweed may be the best choice.
o Avoid majorveins.
• 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%).
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o Pull a small volume of sodium bicarbonate solutioninto 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...