Exercise 4 – The effect of carbon dioxide and pH on the rate of photosynthesis
1. Use a cork borer to cut 100 or more leaf disks from a spinach leaf (spinach is rich in chloroplasts). Cut to either side of the largest leaf veins, not through them. Discard any disks that are bruised or torn. 2. The next step depends on which experiment you are doing: carbon dioxide or pH.
(a) If you are doing the carbon dioxide experiment, set up four 250 ml “side-arm” flasks, each containing 80 ml of one of the four premixed sodium bicarbonate solutions being tested. Use a separate flask for each concentration being tested. Label the flasks with a wax pencil. (b) If you are doing the pH experiment, set up four 250 ml “side-arm” flasks, each containing 80 ml of one of the four premixed pH/sodium bicarbonate solutions being tested. Use a separate flask for each pH value being tested. Label the flasks with a wax pencil.
3. Use forceps to carefully place 25 disks into each flask. Hold the disks by the edge so you don’t crush them! If the disks have not been crushed in the cutting and transferprocess, at this point they should all be floating. 4. Carry the flask(s) over to one of the sink faucets fitted with an “aspirator”. An aspirator generates suction that pulls air out of the flask and out of the leaf disk’s intercellular spaces. Connect the aspirator’s hose to the side arm of the flask. Put a one-hole stopper in the flask and hold your thumb over the hole. 5. Turn on the cold water full force. Watch carefully for 5-10 seconds as air bubbles escape from the edges of the leaf disks, and then release your thumb. DO NOT “BOIL” If you apply the vacuum for too long, the fluid will become turbulent and look like it is “boiling.” This boiling is not caused by a high fluid temperatures, but by low air pressures inside the flask. “Boiling” (too much suction) will permanently damage the intercellular spaces; the discs will never be able to reinflate the spaces and refloat. So take your thumb off the stopper hole as soon as any fluid turbulence starts. 6. After lifting your thumb off the stopper to release the vacuum, gently swirl the liguid around the flask and all the disks should sink. (Note that none of the disks will sink until you take your thumb off the hole!) Wait 10 seconds. You may need to repeat steps 5 & 6 again until fewer than 5-10 disks are left floating. 7. Pour the contents of the flask into a culture dish (a 4” glass bowl). Discard any disks that are still floating. Cover the the sunken disks or keep them away from bright light until you are ready to start timing the experiment! 8. Obtain two (2) partitioned plastic boxes with clear tops. Label the four chambers with the bicarbonate concentration or pH value using a wax pencil. Write on the sides (not the tops) of the boxes, so the labels don’t block the light. Pour fluid from the four labeled flasks into the four corresponding chambers. Use forceps to transfer 20 sunken leaf disks from each of the 4 labeled flasks into the 4 corresponding chambers. Again, hold the disks by the edge so you don’t crush them! Set the
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boxes under the lights. Place a large, glass bowl of room temperature water on top of the boxes to act as a heat shield. (See diagram below.) Turn on the light and keep track of the time.
9. Quantify differences between the four experimental treatments by recording the number of disks floating in each petri dish at 5 minute intervals for 45 minutes. To make a count: (a) turn off the light, and slip the petri dish out from under the beaker of water, (b) uncover the dish, gently swirl or jiggle the dish to free any stuck disks, (c) give the disks time to stop moving, then count the number of floating disks, (A disk standing on edge can be counted as 1/2.) (d) wipe off any water that has condensed on the underside of the cover. (e) replace cover, put the dish back under beaker, and turn on the light. 10. Use Table 2 (on the top of the next page) to record the number of re-floating disks. Label the columns with the sodium bicarbonate concentration or pH values you used.
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Table 2: The number of leaf disks floating after successive 5 minute intervals under four different conditions of ______________________ . (carbon dioxide or pH)
Time (min.)
0-
5-
10-
15-
20-
25-
30-
35-
40-
45-
Analysis and Discussion 1. What effect does pH — or bicarbonate (CO2 ) concentration — have on the rate of photosynthesis? Are the results what you expected? Reconsider the questions asked at the beginning of Part III: (a) pH. Does pH affect photosynthetic rate the same way it affected the browning reaction studied in Minicourse 2? Is there an optimal pH for photosynthesis? (b) Carbon dioxide (sodium bicarbonate) concentration. Does photosynthetic rate increase when carbon dioxide concentrations are increased? Does the rate of reaction keep increasing, or does it level off ?