The Floating Leaf Disk Assay for Investigating Photosynthesis The biology behind the procedure: 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 ions serve as the carbon source for photosynthesis as well as provide dissolved CO 2 (Also a necessary ingredient). 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 rises is an indirect measurement of the net rate of photosynthesis. Materials: 1. Sodium bicarbonate (Baking soda) 2. Liquid Soap 3. Plastic syringe (10 cc or larger 4. Leaf material 5. Hole punch 6. Plastic cups 7. Timer 8. Light source Procedure: 1. In a clear plastic cup, prepare approximately 200 ml of bicarbonate solution (a depth of about 3 cm). Label this cup With CO 2. a. The bicarbonate serves as an alternate dissolved source of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is not very much it s about 1/8 of a teaspoon of baking soda in 300 ml of water!) Too much bicarbonate will cause small bubbles (CO 2 )to form on the surface of the leaf which will make it difficult to sink the leaf disk. 2. Fill a second cup with only water to be used as a control group. Label this cup Without CO 2. 3. Throughout the rest of the procedure you will be preparing material for both cups, so do everything for both cups simultaneously.
4. Add 1 small drop of dilute liquid soap to the cups. 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 suds. If your solution generates suds then dilute it with more bicarbonate solution. 5. Prepare leaf discs a. Use a hole punch to punch out 20 uniform leaf disks (10 for each cup) Avoid major leaf veins. b. Pull the plunger out of the syringe and put 10 leaf disks in the syringe barrel. Carefully replace the plunger being careful not to crush the leaf disks. 4. Infiltrate the leaf disks with solution. a. Push on the plunger until only a small volume of air and leaf disk remain in the barrel (< 10%). b. Put the syringe tip in the cup with the solution. Pull a small volume of solution into the syringe. Tap the syringe to suspend the leaf disks in the solution. c. 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 might have to repeat this procedure several times in order to get the disks to sink. You may have difficulty getting the disks to sink even after applying a vacuum three or four times. Generally, this is usually an indication that you need a little more soap in the bicarbonate solution. (Some leaf surfaces are more water repellent than others are.) Adding a bit more soap usually solves the problem. 6. Once your leaf discs are ready, empty them out of the syringe and into the cup of solution. 7. Place the cups under a light source and start timing. a. At the end of each minute, record how many leaf discs are floating you might want to gently swirl the cup to be sure they are not
sticking to the plastic. (Don t be surprised if it takes 5-7 minutes to see results) b. Continue timing until all the discs are floating. c. Record your results in the data table. Sample results: Time (minutes) Disk Floating 1 0 Disks floating 2 0 12 3 0 4 0 10 5 0 8 6 0 6 Disks floating 7 1 8 1 4 9 1 2 10 1 0 11 4 0 5 10 15 12 7 13 8 Time in minutes 14 10 The point at which 50% of the leaf disks are floating is the point of reference for this procedure. By interpolating from the graph, the 50% floating point is about 11.5 minutes. Using the 50% point provides a greater degree of reliability and repeatability for this procedure.
Floating Leaf Disk Assay Student data sheet Name: Control: water Time (min) Disks floating Time (min) Disks floating 0 8 1 9 2 10 3 11 4 12 5 13 6 14 7 15 Sodium Bicarbonate solution Time (min) Disks floating Time (min) Disks floating 0 8 1 9 2 10 3 11 4 12 5 13 6 14 7 15 Graph the results use two different colors/symbols and graph both water and sodium bicarbonate solution on the same graph.
Analyze/Conclude: Could the way you perform the procedure affect the outcome? If the outcome changes, does it mean the net rate of photosynthesis has changed? Explain your thinking. Extend your Thinking: List several environmental variables that might affect the net rate of photosynthesis. How do you predict each of these would affect it? List at least 2 features or variables of the plant leaves might affect the net rate of photosynthesis? Explain how and why.