Measuring Photosynthesis: Leaf Disk Assay

0:00 [Intro Music] Have you thanked a primary producer today? You really should because we are 100% dependent on photosynthesis, the process by which plants and other primary producers use energy from the sun to convert water and carbon dioxide gas into sugars. In the process, they release oxygen, so our photosynthetic friends are really the reason we're alive and breathing. Look at these plants! Oh my gosh, this is gonna be fun! Okay. And the sugars they create get incorporated into more complex molecules that make up their sturdiest trunks, frilliest leaves, and juiciest fruits. So we rely on the products of photosynthesis every day for food, fuel, and shelter.

0:46 As you might imagine, the chemical reaction that literally enables all life on Earth has gotten a lot of attention from scientists, especially as climate change wreaks havoc on things that affect photosynthesis, like atmospheric carbon dioxide levels, rising temperatures, and droughts. And that also means we're gonna give it a lot of attention in biology class. Today, we're going to learn how to measure photosynthesis in the lab. Then, I'll leave you to design your own experiments to test what might change its rate. Scientists can measure this process in several ways. There are really fancy pieces of lab equipment that can tell you the rate of photosynthesis by directly measuring a leaf's consumption of carbon dioxide. Today we're gonna go with a slightly more budget friendly option that utilizes the other half of the photosynthesis equation, the production of oxygen gas.

1:31 Plant leaves are usually super gassy, not 'cause they've eaten too many beans, but due to their constant exchange of carbon dioxide and oxygen. This means that they'll float in water, but for this experiment we need them to sink. We'll put our leaves under a vacuum so that the water infiltrates them and pushes the gas out, causing them to sink. By adding baking soda to the water, we provide a source of carbon dioxide for the leaves to photosynthesize, and as they produce oxygen, they'll start to float again. We'll time how fast the flotation takes and use that as an estimate of photosynthetic rate. I'll go through that in much more detail as I walk you through the experimental setup. Let's get started! We'll need baking soda, aka sodium bicarbonate,

2:17 diluted dish soap—about 5 mL of soap in 250 mL of water, a pipette, 10 mL syringes without needles, living leaves—today I'm using baby spinach from the grocery store, a hole punch, clear cups or beakers and a way to label them, aluminum foil, a timer, and a light source. I'm using a grow light, but you can also use a desk lamp. Just make sure the bulb is super bright, something like 100 watts. And if you want to compare results across multiple experimental setups, the light source must be the same wattage across all of them. First thing I need to do is prepare a 0.2% solution of sodium bicarbonate in water, so when it dissolves,

3:03 there'll be plenty of carbon dioxide in there for our leaves to photosynthesize. I'm adding 0.6 grams of baking soda to this bottle of water and bringing it up to 300 mL. That solution goes into one of my cups, which I'll label "With CO₂.” I'll add plain water to another cup and label that one "Without CO₂.” This cup will serve as our control, and from now on, I'll duplicate every step of the experiment across these cups. Next, I'll use a pipette to add a drop of the soap solution to each cup. Leaves are covered in a waxy cuticle that repels water. The soap makes the surfaces of the leaves less hydrophobic, so it's easier for the solution to get drawn in by the vacuum. We don't want it so soapy that it causes suds, though.

3:49 If I see suds, I need to dilute what's in the cup with more bicarbonate solution or water. Now, it's time to prepare our leaves! Whole leaves are too big and irregularly shaped, so I'm actually going to make little uniform leaf disks with a hole puncher, 10 per cup. This is pretty straightforward, but just be sure to avoid big veins because they're harder to infiltrate with the vacuum. I'll be creating the vacuum using the syringes. This is the trickiest part of the experimental setup. I've labeled the syringes the same as my cups. First, I'll remove the plungers and load the leaf disks into the barrels, then gently replace the plunger so that only a tiny volume of air remains. Next, I'll pull around 5 mL of soapy bicarbonate solution

4:37 into the corresponding syringe and 5 mL of soapy water into the other. I'll invert the syringe, give it a few taps to make sure the disks are suspended in the solution, and then push the plunger until no air remains in the barrel. To create the vacuum, I'll hold my finger over the syringe opening and pull back on the plunger, holding the vacuum for 10 seconds, and occasionally swirling the syringe during that time to make sure the disks are suspended. The syringe will leave a tiny imprint on your finger for a little while, but we must all make sacrifices for our science. After 10 seconds, I'll release the vacuum. The plunger should spring back. If it doesn't, that means the vacuum wasn't very good, and you may need to try again or use a different syringe.

5:23 If it works, the solution will replace the airspaces in the disks, and the leaves should sink. Just be sure to release the vacuum gently so that the plunger doesn't spring back too aggressively. Sometimes even with a good vacuum, you'll need to repeat the procedure for the disks to sink. But if it doesn't work after 3 tries, the leaf disks will be pretty damaged. Best to start fresh with new disks and add more soap to your solution to help them sink better. And even after all that, you might only have 9 out of 10 disks sunk. But don't stress, just use that as your starting point for the experiment. Now that my disks have sunk, I'll pour them out into their corresponding cup. Before I repeat this process with my other syringe, I'm going to cover the first cup with aluminum foil so that the disks stay dark and don't start photosynthesizing.

6:11 Once all my disks are in their cups, it's time to put on my safety glasses and my gloves because I'm about to work with my bright light source, and that can be a shatter hazard. I'll put my cups under the light, take the aluminum foil off, turn the light on, and start my timer. The leaves in the "With CO₂" cup will start to photosynthesize and produce oxygen gas, allowing them to float to the surface. After a minute goes by, I'll record the total number of floating disks, then swirl the cup in case any disks have gotten stuck to the sides. Swirl... I'll keep recording every minute until all of the disks are floating in my "With CO₂" cup. The disks in my "Without CO₂" cup won't float

6:59 because they can't photosynthesize without a source of carbon dioxide. So, I actually waited about 15 minutes and 1 of my disks just didn't float. So, I think it probably got damaged during the vacuum, or something like that, and wasn't able to photosynthesize enough to get up there. And that's the end of the experiment! But, doing this with only 1 type of leaf in 1 environmental condition doesn't tell me much about what variables affect the rate of photosynthesis. Now that you know how to set up a leaf disk assay, it's time for you to investigate these questions. Think about the factors that could affect photosynthetic rate and have fun designing an experiment to test your hypothesis. Here's a little hint about data analysis. Since you'll be conducting multiple assays, it's important to have a consistent point of reference for comparing your data.

7:45 I'd suggest using the time point at which at least 50% of your leaf disks have risen. For my assay, that would be 7 minutes. Happy experimenting! And don't forget to thank a plant while you're at it! High five! [Outro music]