Introduction:

    The prevailing teaching-lab procedures such as counting bubbles or manometrically measuring increasing air pressure within the closed vessel due to the oxygen gas generated by an elodea stem tend not to be "student" proof or reliable, The following floating-leaf-disk assay technique is far more reliable and consistent. Furthermore, once the students are shown via diagrams that there is considerable air space within the spongy mesophyll of leaves, this method is understandable to students. After practicing the technique they can readily design experiments to answer their own questions about photosynthesis.

Materials:

1. A solution of sodium bicarbonate (Baking soda): use about ¼ teaspoon per liter of tap water.
2. Liquid Soap
3. 1 liter vacuum filter flask with a hand or motorized vacuum pump and stopper.
4. Leaf material such as spinach
   Spinach is good providing it is fresh, and dandelion leaves are even better.
5. Hole punch
6. Transparent plastic cups or beakers
7. Timer
8. Light source (the light box of an overhead projector)

Optional Materials:

9. Buffer Solutions
10. Colored Cellophane or filters
11. Leaf material of different ages
12. Variegated leaf material
13. Clear Nail polish

Procedure:

1. Main solution
   • Add 1 drop of dilute liquid soap to two liters of tap water. Collect about a drop of the liquid soap on the tip of a finger, and then mix it into the water. The soap wets the hydrophobic surface of the leaf allowing the solution to be drawn into the leaf. Itıs difficult to quantify this since liquid soaps vary in concentration. Avoid suds. If your solution generates suds then dilute it with more water. Set aside about 1 liter of this, which will be used for your control (the sample lacking CO₂).
   • Use the remainder to prepare a liter of bicarbonate solution for each trial. The bicarbonate serves as an alternate dissolved source of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is not very much‹itıs about ¼ of a teaspoon of baking soda in a liter of water.) Too much bicarbonate will cause small bubbles (CO₂) to form on the surface of the leaf which will make it difficult to sink the leaf disk.

2. Leaf preparation
   • For a class of 10 pairs of students, cut 100 or more uniform leaf disks for each trial
   • Single hole punchers 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 leaf surface should be smooth and not too thick. Avoid plants with hairy leaves. Ivy, fresh spinach, Wisconsin Fast Plant cotyledons‹all work well. Any number of plants work. My classes have found that in the spring, Pokeweed may be the best choice.
   • Avoid major veins.

3. Infiltration of leaf disks with sodium bicarbonate solution.
   • As shown in the figure next to the materials section, above, place all the freshly punched disks into a 1L vacuum-filter flask with about 800 mL of the bicarbonate water. Make things easy for yourself: fill your flask at least 90% full so that you do not have a large volume of air to remove.
   • If possible, put a Cartesian Diver (an inverted tube filled with trapped air - this is your internal barometer).
   • Firmly push the stopper in place and attach the vacuum pump. (Water-tap aspirators are not strong enough for what is needed.)
     
   • Do each of the following steps in dim light!
   • Remove air
     • As you begin withdrawing air you will note that bubbles are escaping from the bottom of the barometer (the inverted tube). This is because your making a vacuum expands the volume of the air in the tube. It is doing likewise on a microscopic scale in all of the leaves' mesophylls. Tiny bubbles begin appearing on the leaf surfaces.
     • Continue pumping until no more bubbles escape from the bottom of the barometer.
     • Swirl the flask to move all the disks down into the liquid. You may see bubbles adhering to the disks. Critical: swirl more vigorously to dislodge the bubbles. When all disks are below the surface and NOT floating at the top, let the air back into the flask. The sudden influx of air will cause the liquid to rise far up the barometer. You want the final trapped bubble to be only about 5% of the volume of the inverted tube. If more than 5%, you haven't attained a high enough vacuum. In similar fashion, the liquid will move into the spongy mesophylls through the leaves stomata (the plural of stoma).
     • Do this evacuation several times until ALL the disks will come to a rest on the BOTTOM of the flask after ambient air pressure is restored above the solution.

4. Distribute the disks to the various groups. (Though not amenable to groups, see speedier alternatives.)

5. Individual set-ups
   • Pour the disks and solution into a clear container such as a plastic cup or glass beaker. Add bicarbonate solution to a depth of about 3 centimeters. Use the same depth for each trial. Shallower depths work just as well.
   • This experimental setup includes a control. The leaf disks in the cup on the right were infiltrated with a water solution with a drop of soap‹no bicarbonate.  
   • Expose to the light source and start the timer. Every 30 seconds, record the number of floating disks. Then swirl the disks to dislodge any that are stuck against the sides of the cups. Continue until HALF of the disks are floating. Usually by 7 minutes, half of the disks will have risen.

Click THIS for a page for recording your data. If you are planning on using photos in your report, it is strongly recommended that your vessel have flat sides so as to eliminate distortion.

* http://home.earthlink.net/~bioteacher/LeafDisk.htm The reason for modifying Williamson's procedure is two-fold: (a) gaining more constancy - all disks have been treated the same way prior to light-exposure, and (b) it obviates the problem that a number of students have great difficulty in using a syringe to evacuate air from the disks.

Alternatives

More Speed: Rather than distributing the sunken disks to various groups, ease them undistributed in the flask, which can itself be set directly upon the light box. The process is made a little quicker by reattaching the vacuum pump and withdrawing just a little air such that the bubble in the manometer doubles in volume. Illumination causes the disks to rise much sooner than without doing this expansion.

Testing for light intensity quantitatively: If one sheet of stationery blocks a certain proportion of the light, two sheets block twice that much and three exactly three times that much and so on.

The Action Spectrum: The classic notion that leaves absorb in both the red and blue and are thus most photosynthetic with those colors is often not true. Often leaves that are exposed to long-term, bright sunlight predominantly use blue light, while those growing in the shade use both blue and red. The reason is that red light penetrates down to the understory. Indeed, it has been found that many forest-floor flowers monitor infrared light for their blooming. (Evidence is also accumulating that photosynthesis takes place around deep-sea vents using infrared light exclusively (remember that IR = heat rays).

1. The action spectrum: Method A: Using a spectrum which has been printed onto a transparency and setting the "speedy" flask, above, on the transparency will find some disks over the red end, others over other colors. Which ones rise sooner than others? (A printable spectrum for a transparency.) The photo (by LW) to the right shows several disks lifting off the red portion of the spectrum. Also notice the small size of the bubble in the "barometer." The vacuum in this instance was produced by a hand-held pump.
   

2. The action spectrum: Method B: Because transparency colors are not highly discriminating in which colors allowed to pass through, a better way to do this is by using 15 cm x 15 cm squares of colored glass which have been pre-checked for their spectral transmissions. Cobalt blue glass is renowned for letting no reds, yellows, greens or oranges through. By looking at the sun through a spectroscope interposing members from a collection of colored glasses will allow you to quickly ascertain the best discriminators. You should then use a light meter to correct for the relative opacities of the various glasses. Thus, looking down at the light box:

   

Temperature Effects: Because the method works so rapidly, temperaturres do not change appreciably over the course of light-exposure. If needed small amounts of hot water can be stired in.