1. Lab 5 – Cell Respiration Don’t step in pea. Side note: This is a stand-in for Lab 6 from your investigations manual. The next lab we do will correspond to Lab 5.

2. The Background Everyone respires. Everyone. – Plants, animals, fungi, bacteria, archaea…everyone. – That includes peas. We’re going to measure respiration in peas using a respirometer. The important question for you is…how? – What would be a good measurable proxy substance for quantifying respiration?

3. The Experiment C 6 H 12 O 6 + 6O 2  6CO 2 + 6H 2 O + ATP We’re going to measure O 2 consumed. However, there’s an issue here… – In a reaction, O 2 levels are going to drop and CO 2 is going to be produced. – How are we going to know if gas level changes are due to CO 2 or O 2 ? Get rid of the CO 2 !

4. The Experiment We’re going to use KOH (potassium hydroxide) to capture the CO 2 : CO 2 + 2KOH  K 2 CO 3 + H 2 O With all the CO 2 being captured by KOH, any change in gas level is due to O 2 being consumed. The more O 2 consumed in a certain amount of time, the greater the respiration rate.

5. The Experiment: Comparisons Test subject: Alaska pea seeds. Variables: – Temperature – Germination Dry versus germinating seeds Control group: – Beads. Just plain ol’ (pea-sized) beads.

6. The Experiment: Comparisons So you’ll have six total respirometers: 1.Room temperature, germinating seeds 2.Room temperature, dry seeds and beads 3.Room temperature, beads [control] 4.10 °C (chilled), germinating seeds 5.10 °C (chilled), dry seeds and beads 6.10 °C (chilled), beads [control]

7. The Experiment: Procedure At your lab tables are plastic water bath bins. One is filled with water already – this is the room temperature (25 °C or 77 °F). Create the chilled water bath by getting some ice from the Styrofoam cooler. Make sure you have a thermometer in the bath to get the temperature. – You want it to be around 10 °C. Now you prepare the respirometers in two sets of three.

8. The Experiment: Procedure 1.Fill a 100 mL graduated cylinder to 50 mL with tap water and add 25 germinating peas. – They’re the ones already in a bowl of water. 2.Determine the volume of germinating peas, then take the peas out and put them on a paper towel. 3.Repeat steps 1-2 for dry peas, except you’ll need to add beads until the dry peas + beads take up the same volume as germinating peas. 4.Repeat steps 1-2 for beads only. – Heads-up – we’re using just beads in one respirometer because ambient temperature or air pressure in the room may change. – This allows us to correct our results as yet another control. 5.Repeat steps 1-4 for the other water bath.

9. The Experiment: Procedure Assemble each respirometer as follows: Get a vial (make sure it’s dry). Put in an absorbent cotton ball. Moisten with 15% KOH. – Keep the vial dry and don’t get the sides wet. Put in a “non-absorbent cotton” (polyester) ball. Add: – Germinating Peas OR – Dry Peas and Beads OR – Beads Put in a stopper and pipette (tapered side out). Add a washer for weight. 15% KOH

10. The Experiment: Procedure Follow the diagrams in the lab to create a sling out of tape (your bath may already have one). – The pipettes should be out of the water. Let the vials equilibrate for seven minutes, then call me over. – I will add a tiny drop of red food coloring to the inside of your pipette. – This is not in the printed lab but will help with finding the water line. Next, put the whole respirometer in the water. – The pipettes should be in the water. – Quickly check for leaks (only some water should come in) and arrange them so you can read the pipettes through the water. – Get your hands out of the water as quickly as possible.

11. The Experiment: Procedure Let them equilibrate for three more minutes and then record the starting position of water in the pipette (nearest 0.01 mL). Record the temperature too. – By the way, this is a 1 mL pipette. Just…FYI. Record the water level in the pipette every five minutes for twenty minutes. – Should this be going up or down? Complete the analysis questions.

12. Tips and Tricks Insert the pipette as far as you can without stabbing any peas (AKA don’t clog the pipette). When you put the respirometer completely under water, look immediately for the water line. – If you don’t see water coming into the pipette, quickly try to get some of the air bubble out. – Flick it or tap it till some water goes in to a measurable point. – If that fails, carefully pull the pipette out of the stopper just slightly. The water should be drawn in further. According to the ideal gas law, even a small increase in temperature will cause a dramatic increase in pressure and therefore a detrimental increase in volume. – In other words, if the water temperature rises, particularly in the cold water bath, the water will exit the respirometer. – Add ice continuously to keep the temperature as constant as possible.

13. Tips and Tricks: Gas Laws PV=nRT – remember that from chemistry? Suppose we have 0.01 mol gas in our vials at a temperature of 8 °C (281 K) and pressure of 101.3 kPa. Therefore: – (101.3)(V) = (0.01)(8.314)(281) – V = 23.06 mL What happens if the temperature rises to 13 °C? – (101.3)(V) = (0.01)(8.314)(286) – V = 23.47 mL These are 1 mL pipettes, remember? – Our volume has expanded and our dye may be ejected.

14. Clean-Up Dump the ice water into the sink at your station. – If there is another AP Bio class after yours, do not dump the ambient water. – If you’re the last class of the day, do dump the ambient water. – DO SO CAREFULLY! It’s very easy to spill everywhere. Disassemble the respirometers and dry them. – They should be as dry as possible. – Leave them upside down when you’re done. Throw out the cotton. Return the peas to their respective containers.