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investigating photosynthesis: floating leaf disk assay(Adapted from: )Requirements for Lab Report: For this practical you will need to write a FULL lab report (refer to IA Lab Format for required sections/formatting). You may use the information below as the introduction/background information. You may also use the research question, materials list, and procedure as listed on this handout. Introduction & Background Information:Photosynthesis is undoubtedly the most important process occurring on Earth. It fuels ecosystems and replenishes our atmosphere with free oxygen. Like all enzyme-driven reactions, photosynthesis can be measured by either the disappearance of substrate or the accumulation of product (or by-products).The general, summary equation for photosynthesis is:6CO2(g) + 6H2O + light energy → C6H12O6 + 6O2(g) In the case of photosynthesis, we can, therefore, either measure the rate of reaction by the loss of carbon dioxide or the accumulation of sugars or oxygen. In this lab, we will use a system that measures the accumulation of oxygen. Because the spongy mesophyll layer of leaves is normally infused with gases (O2 and CO2), leaves — or disks cuts from leaves — normally float in water. If the gases are drawn from the spongy mesophyll layer by using a vacuum and replaced with an aqueous solution with a small amount of bicarbonate, then the density of the leaf disk will increase, and the leaf disk will sink in water.With the bicarbonate serving as an alternate source of carbon dioxide, photosynthesis can occur in a sunken leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy mesophyll and the leaf disk will once again become buoyant and rise in a column of water. Therefore, the rate of photosynthesis can be indirectly measured by the rate of rise of the leaf disks. However, there’s more going on in the leaf than that! You must also remember that cellular respiration is taking place at the same time as photosynthesis in plant leaves (remember plant cells have mitochondria, too!). Aerobic respiration will consume oxygen that has accumulated in spongy mesophyll. Consequently, the two processes counter each other with respect to the accumulation of oxygen in the air spaces of the spongy mesophyll. So now we have a more robust measurement tool — the buoyancy of the leaf disks is actually an indirect measurement of the net rate of photosynthesis occurring in the leaf tissue.Research Question: After exposing leaf discs to two different intensities of light, will there be a difference in the rate of photosynthesis in terms of generation of O2 gas? Variables:-Dependent: Amount of time it takes for leaf discs to float (Rate of Photosynthesis)-Independent: Light Intensity (Treatment 1: Full light, Treatment 2: No light)-Controls: VariableHow is it being controlled?Temperature of roomTemperature of room will remain at room temperature.Temperature of C02 solutionTemperature of CO2 solution will remain at room temperature. Material used for leaf discsLeaf discs will all be made from same batch of spinach leaves.Size of leaf discsLeaf discs will be made using a hole punch for consistent size and shape.Sodium carbonate concentrationSame concentration of CO2 substrate will be provided for all leaf discs.TimingTiming will be done using a stopwatch to avoid human error.Light IntensityLight intensity will be controlled by placing all cups containing leaf discs 15cm from light source. An isolated room with no infiltrating light will be used for the leaf discs undergoing the “No Light” treatment.Materials:-living spinach leaves-baking soda (sodium bicarbonate)-liquid soap -1 plastic syringe without needle (10cc or larger)-hole punch-plastic cups-timer-light source-1mL plastic pipette-100mL graduated cylinder-permanent marker-plastic spoon-ruler with cm units-medium sized beaker (200-300mL)Procedure:1. Prepare a beaker of dilute soap solution by adding a few drops of soap into about 200mL of water. 2. Prepare 1000 mL of 0.2% bicarbonate solution for. This can be done by dissolving 2.0g of baking soda in 1000 mL of water. The bicarbonate will serve as a source of carbon dioxide for the leaf disks while they are in the solution.3. Label 5 plastic cups “Light”. Pour 200 mL of bicarbonate solution into each plastic cup and label “Light”. 4. Using a plastic pipette, add 1 drop of a dilute liquid soap solution to the bicarbonate solution in each cup. It is critical to avoid suds. If either solution generates suds, then dilute it with more bicarbonate or water solution. The soap acts as a surfactant or “wetting agent” — it wets the hydrophobic surface of the leaf allowing the solution to be drawn into the leaf and enabling the leaf disks to sink in the fluid.427863053975005. Using a hole punch, cut 50 uniform leaf disks. Avoid major leaf veins.6. You must now draw the gases out of the spongy mesophyll tissue and infiltrate the leaves with the sodium bicarbonate solution. To do this:a) Remove the plunger from both syringes. Place all leaf disks into the syringe barrel.b) Replace the plunger but be careful not to crush the leaf disks. Push in the plunger as far in as you can to get rid of as much air as possible without crushing any leaf discs. c) Using the bicarbonate solution from one of your prepared cups, pull a small volume of sodium bicarbonate solution into each syringe (enough to submerge all leaf discs). Tap each syringe to float the leaf disks in the solution.d) You now want to create a vacuum in the plunger to draw the air out of the leaf tissue. Create the vacuum by holding a finger over the narrow syringe opening while drawing back the plunger. Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspend them in the solution. Now release the vacuum by letting the plunger spring back. The solution will infiltrate the air spaces in the leaf disk, causing the leaf disks to sink in the syringe. If the plunger does not spring back, you did not have a good vacuum, and you may need a different syringe. You will probably have to repeat this procedure 2-3 times in order to get the disks to sink. (If you have any difficulty getting your disks to sink after about 3 evacuations, it is usually because there is not enough soap in the solution. Try adding a few more drops of soap.) Placing the disks under vacuum more than three times can damage the disks. 7. Empty the discs and the solution from the syringe into the 5 cups labelled as “Light” by removing the plunger and inverting the syringe into each cup. Divide leaf discs so that each cup has 10 leaf discs. Use a plastic spoon or tweezers if necessary.8. Place all “Light” cups under the light source and start the timer, make sure each of your cups are at the same distance from your light source. At the end of each minute, record the number of floating discs (a floating disc is one that is no longer touching the bottom of the cup). Make sure to gently swirl the cup to dislodge any disc that might be stuck against the side of the cups. Continue until all of the disks are floating in the cup OR until you have reached 25 minutes. 9. Dispose of all materials as directed.10. Repeat Steps 2 – 7, but for the “No Light” treatment. 11. Once discs are divided evenly into each cup of bicarbonate solution again, place all “No Light” cups in the No Light condition room and start timer. 12. At the end of each minute, record the number of floating discs (a floating disc is one that is no longer touching the bottom of the cup). Make sure to gently swirl the cup to dislodge any disc that might be stuck against the side of the cups. Continue until all of the disks are floating in the cup OR until you have reached 30 minutes.13. Rinse all materials with tap water. Place any glassware on the drying rack. Dispose of all liquid wastes down the sink and all solid waste in the trash.-112143395260NOTE FOR DATA PROCESSING: Repeated testing of this procedure has shown that the point at which 50% of the leaf disks are floating (the median) should be used as the point of reference for this assay when comparing different experimental treatments. The 50% point, or ET50, provides a greater degree of reliability and repeatability for this procedure. Extrapolate this point from the graph you construct from your data. ................
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