A Measure of Photosynthetic Rate In Spinach Leaf Disks
In this technique, the intercellular spaces of spinach leaf disks are infiltrated with a sodium bicarbonate solution, which causes them to sink in the solution. As photosynthesis occurs, oxygen collects in the intercellular spaces and refloats the leaf disks.
Procedure
1. Using a punch made from a small diameter soda straw, cut 10 leaf disks from young actively growing spinach leaves by supporting the leaf with your index finger while pressing and using a twisting motion of the straw. A.
2. Remove the plunger from a clean 10-ml syringe. Blow the 10 disks into the body of the syringe. Be sure the leaf disks are near the tip of the syringe as you re-insert the plunger so as not to damage the disks. B
3. Insert the tip of the syringe into a beaker of 0.1% sodium bicarbonate solution and draw about 8 ml into the syringe. The leaf disks should be floating at this time. C
4. Hold the syringe tip upward and expel the air by depressing the plunger carefully.
5. Seal the tip of the syringe using the index finger of your left hand. Pull back on the plunger, creating a partial vacuum within the syringe. If you have a good seal, it should be hard to pull on the plunger and you should see bubbles coming from the edge of the leaf disks. D
6. Simultaneously, release your index finger and the plunger. Some of the leaf disks should start to sink. Tap the side of the tube to dislodge bubbles on the edges of the disks. E
7. Repeat steps 5 and 6 until all disks sink. Do not overdo these steps!! You have been successful if the disks sink to the bottom. Don't repeat "just to be sure" as it is possible to damage the cells of the leaves.
8. Once you have removed the air from the disks, proceed with your experimental treatment, using colored filters, etc. Make sure all syringes used are filled to the same level by adding or expelling bicarbonate solution. If colored filters are used, they are wrapped around the body of the syringe.
9. Set the syringes, tips up, in front of the flood lights or other light source selected, making sure to place a jar of water between the syringes and the light as a heat absorber. The lights are best held in a clamp on a ring stand. F
10. As photosynthesis proceeds, accumulating oxygen will cause the disks to float. At 2 minute intervals, invert the syringes to agitate the leaf disks and immediately return them to their positions in front of the lights. Count the number of disks floating after each interval until all disks are floating.
The basic kit for this module includes:
(2) 10-ml syringes
wax pencil
soda straw punch
young spinach leaves
50 mL beaker for sodium bicarbonate solution.
100 mL of 0.1 % sodium bicarbonate solution.
(1) 150 watt flood light in fixture
ring stand and clamps
large jar for heat absorber
Some possible experiments: Design protocols to investigate the following
Adapted from Steucek,G.L. and Hill,R.J. (1982) Am. Biol. Teacher, 471:96-99, as modified by Linda Holler, Susquehanna University.