7.2: Experimental Design
In this experiment you will examine how different light conditions effect the rate of the light dependent reaction of photosynthesis. The rate of this reaction can be estimated by measuring oxygen production in disks cut from spinach leaves.
Leaves are riddled with gas-filled intracellular spaces, evident by observing a leaf floating on water. Small circular disks are prepared from spinach leaves. The leaf disks are then placed in a solution of sodium bicarbonate and subjected to a vacuum. The trapped gases will be removed and replaced by the bicarbonate solution causing the disks to sink to the bottom of the flask. The disks will be divided into 3 petri dishes and placed under different light conditions – dark, room light, and under a sunlamp. As the light dependent reaction occurs oxygen will diffuse into the intracellular spaces replacing the sodium bicarbonate solution with this gas. Once enough oxygen has been generated, the leaf disks will regain buoyancy and turn on one edge or float to the surface. The percentage of leaf disks turned or floating on edge after 20 minutes will be used to measure the rate this phase of photosynthesis.
Effect of different light conditions on the rate of photosynthesis
Materials
- Spinach leaves, cork borer, wood or thick cardboard
- 0.2 % sodium bicarbonate solution
- 250 ml flask side armed flask with 1-holed rubber stopper
- Petri dishes – 2 clear, 1 black per group
- Sunlamp
- Culture dish
- Forceps
Procedure
- Pour the sodium bicarbonate solution into the 3 petri dishes, filling 2/3 of the way. Fill the 250 ml side armed flask with approximately 100 ml of sodium bicarbonate solution.
- Take spinach leaves and cut 60 disks using the core borer. Spinach leaves may be stacked together. Avoid areas that contain large veins.
- Add spinach disk to the 250 ml side armed flask.
- Attach the vacuum tubing to the side arm of the flask. Put the rubber stopper firmly over the mouth of the flask. Use tape or your finger to secure the hole in the stopper.
- Attach flask to vacuum nozzle. Leave the disks under the vacuum for 15 – 20 second increments. Turn off the vacuum, slowly release the tape/finger on the hole in the rubber stopper to check if the disks have sunk to the bottom of the flask. Repeat this process until all disks have sunk to the bottom of the flask. Do not over aspirate as this will damage the plant tissue.
- Pour the disks into the culture dish, discard any that may still be floating.
- Using forceps gently transfer 15 – 20 disks to each of the 3 petri dishes. The black dish represents “the dark” light condition. Place one of the clear dishes under the sunlamp. Place the second clear dish on the bench top away from the sunlamp.
- Wait 20 minutes, you can answer many of the questions in the analysis section of this lab while you wait.
- After 20 minutes count the number of disks that have floated or are standing on edge in each petri dish and calculate the percentage floating/standing on edge. Record the data in table 7.3 and generate a bar graph to represent the data.