Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.

Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
Lab 4A: Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis Cells are able to utilize carbohydrates through the process of glycolysis (“sugar- splitting”) in the cytoplasm. In the absence of oxygen , many organisms can utilize fermentation to produce a small amount of ATP, which can be readily used to power cellular processes. However, in the presence of oxygen , cells may utilize aerobic respiration , which is the process by which cells are able to convert chemical energy to greater amounts of ATP, largely through the production of reduced electron carriers as intermediates using the redox reactions of the citric acid cycle. In eukaryotic cells , the citric acid cycle occurs within the matrix of the mitochondrion , while the inner membrane of the mitochondrion is enriched with many of the enzymes necessary for the final reactions of cellular respiration, containing the electron transport chain and ATP synthase for oxidative phosph orylation. Through these processes, the chemical energy from reduced electron carriers is transformed into ATP. (See Figure 1 ). Figure 1 : Eukaryotic glucose metabolism. (Iwasa, J and Marshall, W (2016) Karp’s Cell and Molecular Biology, 8 th edition. Wiley. P. 173) We will be examining the rate of aerobic respiration in two organisms: yeast ( Sacchromyces cerevisiae ) and Euglena . The terminal electron acceptor of the electron transport chain of aerobic respiration is oxygen (see Figure 2 ). Therefore, by determining the rate at which oxygen is utilized by the cell under different conditions, we can monitor the effects of these variables on the overall rate of respiration. Figure 2: Mitochondrial components of the electron transport chain for oxidative phosphorylation and ATP production. (Urry, et al. (2019) Campbell: Biology in Focus, 3 rd edition, page 156) In the broadest sense, photosynthesis is the process whereby cells absorb the energy of visible light, and use this energy to synthesize organic compounds. The most important light absorbing pigment in this process is chlorophyll . In eukaryotes, chlorophyll is found embedded in the thylakoid membranes of chloroplasts . The overall process is shown in the familiar summary equation for photosynthesis: 6CO 2 + 6H 2 O – light → C 6 H 12 O 6 + 6O 2 Of course, this equation oversimplifies the actual chemistry of the process. The Hill reactions (often referred to as the “light reactions” ) provide the primary step in photosynthesis. It photolyzes water, producing oxygen, and traps energy in the form of ATP and reduced electron carriers (NADPH). This reaction occurs in the thylakoid membranes of chloroplasts. (See Figure 3 on the following page ). I n the second phase of photosynthesis, which occurs in the stroma of the chloroplast, the energy-rich products of the Hill reactions (NADPH and ATP) are used for the synthesis of organic molecules in the Calvin cycle . These reactions do not have a direct dependence on light, and are often referred to as the “ dark reactions ”. Do not confuse this as being equivalent to “dark conditions”! In these reactions, carbon dioxide is reduced (or “fixed”) to produce carbohydrates Because the products of the Hill reactions (ATP and NADPH) are the reactants in the Calvin cycle, and the products of the Calvin cycle (ADP and NADP + ) are reactants in the Hill reactions, these two processes are interdependent . This means that neither set of reactions can occur in the absence of the other for long. Therefore, despite being often referred to as the “dark reactions”, the Calvin cycle will cease to function in the absence of light. 2 Figure 3. An overview of the light-dependent (Hill) reactions. (By Somepics – Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=38088695) In this lab, we will be focusing on how alterations in temperature influence the rate of aerobic respiration in both yeast and Euglena . As aerobic respiration is largely dependent on enzymatic reactions, we would expect that the rate of oxygen use will be altered in a manner similar to that seen for most enzymatic activity. That is, as temperature increases to the optimal temperature for enzymatic function, the rate will increase, as more reduced electron carriers will be produced. However, once temperatures increase to those which result in the denaturation of the relevant enzymes, and hence a loss of function, the rate of oxygen usage should decrease as reduced electron carriers are no longer produced, and thus the ETC is no longer active. In this laboratory exercise, we will measure the rate of aerobic respiration as a function of temperature using an oxygen electrode . We will also measure the rate of photosynthesis in Euglena cells as a function of temperature using the oxygen electrode. You will measure the rate of oxygen evolution in “ light conditions ” to determine the observed rate of photosynthesis . However, as Euglena also contain mitochondria, they will utilize aerobic respiration for the production of ATP, regardless of whether light is present or not. Therefore, we will also measure the rate of oxygen consumption in “ dark conditions ” to determine the rate of aerobic respiration. By adding the absolute rates of oxygen consumption in the dark and oxygen evolution in the light we can determine the actual rate of photosynthesis . We will also add a proton ionophore, CCCP, to our assays in order to examine it’s effect on the rates of aerobic respiration and photosynthesis. CCCP functions to dissipate the proton gradient established by the electron transport chains of both processes, but it 3 has a differential effect in our cellular systems. These observations will be provided with you along with the data for these experiments, and an explanation of why this occurs will need to be included in your final lab report. PROCEDURE Basic procedure for calibrating and using the oxygen probe Your lab instructor will demonstrate the proper procedure for calibrating and using the oxygen electrode. The temperatures and times may differ somewhat depending on which organism you are using, but the basic procedure of calibrating the machines at the different temperatures is the same. See Appendix E: Use of the Oxygen Electrode and Data Analysis for a full explanation of this process. 1. First calibrate the oxygen machine at your starting temperature using the solution found in the tube within the water bath. Be certain that the system has reached the appropriate temperature by checking the thermometer in the water bath. When working with the electrode, it is essential that you avoid piercing the membrane at the bottom of the chamber. Always turn the stir bar off prior to emptying the chamber. Always place the tip of the plastic Pasteur pipette against the side of the chamber . 2. Once you have completed your calibration , d rain the fluid from the reaction chamber with the plastic pipette. Mix the cell culture . Add 2.0 ml to the reaction chamber with a pipette. Leave the top off the chamber at this point. Start the stirrer without adjusting the speed. Adjust the placement of the pen on the chart recorder to allow for accurate measurement (see specific procedure). Then, place the chamber stopper into the reaction cell by slowly screwing it downwards, until the fluid of the culture can be seen just moving up the capillary tube inside the stopper. Stretch a small piece of Parafilm over the top of the stopper, to prevent any casual leakage in the system out of the capillary tube. Check for the presence of an air bubble in the chamber and readjust the chamber stopper until you are confident there isn’t one. 3. Set the chart to record the change in oxygen fo r the specified time . 4. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, stop the stir bar, remove the cells, and wash the chamber three times with distilled water. 4 i. Measuring the rate of aerobic respiration in yeast. 1. Prepare a beaker containing 500 mL of tap water at approximately 40 o C . Add the contents of the provided vial, which contains 1 g of sucrose and 0.5 g of yeast . Stir well to dissolve and allow to sit at room temperature for 10 minutes . 2. Check to make sure that the spectrophotometer is set to 600 nm . Use tap water to BLANK the spectrophotometer and then, after mixing your yeast culture well, determine the absorbance. You should obtain an OD of approximately 0.80 to 1.00 . 3. As we will be comparing data obtained by different groups at different times, it will be necessary to ensure that we have standardized our experiment across days. In order to achieve this, we will need to determine the cell number in the cultures used that day. At 600 nm, an OD of 1.00 is equal to approximately 1 x 10 7 cells/mL . This information is then used to prepare a 20 mL culture of 0.75 x 10 7 cells/mL prior to running the experiment, which ensures that the number of cells utilized by all groups are approximately equal. U se the C 1 V 1 = C 2 V 2 equation (See Lab 2 if you need a refresher on this!) 4. Follow the basic procedure provided on page 35. Calibrate the oxygen machine at 20 o C . After adding your 2.0 mL of yeast culture, reposition the pen on the chart recorder to approximately 70 units by gently bubbling air into the culture. Once the chamber has been properly sealed, record the rate of oxygen consumption for 4 minutes . 5. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, suck out the cells, and wash the chamber three times with distilled water and r epeat the entire measurement procedure at the same temperature with a fresh 2 ml sample of cells from the stock culture . Average your results and record them for that temperature. 6. Adjust the water bath to 3 0 o C . Monitor with the thermometer until this temperature is reached. Re-calibrate the oxygen electrode and chart at this new temperature , using the same procedure you followed before. Do replicate determinations of oxygen utilization at this temperature, using fresh cells for each run ( swirl the flask!! ), as described previously. 7. Inform your lab instructor when you are at the end of your second trial at 3 0 o C. They will add the carbonyl cyanide m-chlorophenyl hydrazone (CCCP) into the reaction chamber for you with an injection syringe. Monitor the effects of CCCP for another 4 min . It is essential that at the end of this run you are extremely thorough when rinsing out the chamber (wash out 6 times and be sure to clean the stopper as well!), as any remaining drug may contaminate the readings obtained in your remaining trials. 8. Repeat the procedure from steps 4 & 5 at 40 o C, 50 o C, and 60 o C, performing replicate trials at each temperature. You do not need to have CCCP added at these temperatures. Remember to recalibrate the machine at each temperature. 5 ii. Measuring the rate of aerobic respiration in Euglena . 1. You need to determine the concentration of the liquid culture of Euglena sp . using the haemocytometer. To do so, place a coverslip on the haemocytometer and add a drop of Euglena culture and a drop of iodine to immobilize/fix the cells. Focus on the haemocytometer using a bright field microscope at 40x magnification. Count the 4 corners and the middle square as shown below. If cells are found on the borders, be consistent and count only those that are on the same two of the four lines. Add the 5 areas and mulitply by 50,000 to obtain the number of cells/mL. An image of what you will see under the microscope is shown below. 2. Use this information and the C 1 V 1 = C 2 V 2 equation (See Lab 2 if you need a refresher on this!) to prepare a 20 mL culture at a concentration of 1.5 x 10 7 cells/mL. 3 . Follow the same procedure as in Part i above (Steps 4-8) using your Euglena culture. The only alteration is that you must remember to leave your culture in the dark, and that you must wrap the reaction chamber with the provided black cloth. This ensures that the cells will only be utilizing aerobic respiration, rather than photosynthesis. iii. Measuring the rate of photosynthesis in Euglena 1. Use the same culture as provided for Part ii steps 1 & 2 to prepare a 20 mL culture at a concentration of 1.5 x 10 7 cells/mL. 2 . Follow the basic procedure provided on page 35. Calibrate the oxygen machine at 20 o C . After adding your 2.0 mL of Euglena culture, reposition the pen on the chart recorder to approximately 30 units by gently bubbling nitrogen into the culture. Once the chamber has been properly sealed, immediately turn on the desk lamp and place it as close to the reaction chamber as possible. Record the rate of oxygen production for 3 minutes in the light . 3. After 3 minutes, immediately turn off the lamp, swing it out of the way, and cover the reaction chamber securely with the provided black cloth. Record the rate of oxygen utilization for 2 minutes in the dark. 4. At the conclusion of the run, stop the chart from recording, remove the chamber stopper, stop the stir bar, remove the cells, and wash the chamber three times with distilled water and r epeat the entire measurement procedure at the same temperature with a fresh 2 ml 6 sample of cells from the stock culture . Record the average of your trials (average light and average dark) for later analysis. 5. Adjust the water bath to 3 0 o C . Monitor with the thermometer until this temperature is reached. Re-calibrate the oxygen electrode and chart at this new temperature , using the same procedure you followed before. Do replicate determinations of oxygen production and utilization at this temperature using fresh cells for each run ( swirl the flask!! ), as described previously. 6. Once you have completed your second trial at 3 0 o C, set up a third trial, and i nform your lab instructor . They will add the CCCP into the reaction chamber for you with an injection syringe. Monitor the effects of CCCP for another 3 min in the light. It is essential that at the end of this run you are extremely thorough when rinsing out the chamber (wash out 6 times and be sure to clean the stopper as well!), as any remaining drug may contaminate the readings obtained in your remaining trials. 7. Repeat the procedure at 40 o C, and 50 o C, performing replicate trials at each temperature. You do not need to have CCCP added at these temperatures. Remember to recalibrate the machine at each temperature. iv. Microscopy of Euglena and yeast cells It is essential in any study which utilizes live cells for them to be accessed to ensure that they appear healthy. A wet mount of each of the cultures should be prepared and accessed by phase contrast microscopy at 400x magnification. Do the cells for each culture demonstrate the expected shape? Are the Euglena generally active and demonstrating motility? Can you find any actively dividing yeast in the culture? Please view the Oxygen Electrode video prior to the Lab 4A Tutorial . A more in depth description of the use of the oxygen electrode, as well as the procedure for data analysis is found in Appendix E: Use of the Oxygen Electrode and Data Analysis . Further explanation on interpretation will be provided in the Lab 4A Tutorial. Lab 4 Report Lab 4A will be combined with 4B into a formal lab report worth 10% of your final grade and due by 11:59 PM on November 12 th . You should review the General Lab Report Guidelines, as well as the Lab 4 Report Requirements available in the Lab 4 folder on Nexus. 7
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
APPENDIX E: Use of the Oxygen Electrode and Data Analysis In Lab 4A, we will measure the rates of oxygen consumption and/or evolution as a function of temperature using an oxygen electrode (shown in Figure 1 ). At the bottom of the reaction chamber is a bare, platinum electrode covered by a porous Teflon membrane. A small polarizing current is passed through the electrode. Oxygen diffuses across the membrane and is reduced at the electrode tip to form water. The higher the concentration of oxygen in the solution being measured, the higher the rate of oxygen diffusion across the electrode membrane, and the greater the pen deflection on a chart recorder which is attached to the electrode. Conversely, as oxygen is utilized, the concentration of oxygen in the solution will decrease, resulting in a negative slope on the chart recorder. Figure 1: The reaction chamber of the oxygen electrode. In (A), the reaction chamber and controls can be observed. Note the tubes extending from the chamber, which allows for the circulation of water of a given temperature around the chamber. In (B), the inside of the chamber can be seen. The oxygen electrode is in the middle of the orange O-ring. The following procedure describes how to properly calibrate the oxygen electrode: 1. The water bath has been set to 20 o C and a vial of tap water placed within for use during your experiment. 2. Examine the oxygen electrode apparatus. You will notice that the reaction chamber is full of water. There should be a small magnetic stir bar visible at the bottom of the chamber. The chamber has a plastic stopper loosely inserted in the top. The power switch on the blue cabinet should be “on”. A B 3. Remove the plastic stopper, and suck the water out of the reaction chamber with the plastic pipette provided. Discard this water. BE CAREFUL THAT YOU DO NOT PUNCTURE THE MEMBRANE THAT IS COVERING THE ELECTRODE AT THE BOTTOM OF THE CHAMBER . Add 3-4 mls of fresh distilled water from a squeeze bottle, and then remove this water with the plastic pipette. Repeat several times to ensure that the chamber is clean, and free of any residual chemicals from any previous experiment. 4. Suck all the water out of the chamber. Retrieve the tube of water that you have suspended in the water bath, and shake it well to thoroughly aerate it. Add 2.0 ml to the reaction chamber with a pipette. Leave the top of the reaction chamber off at this point . Switch on the stirring motor, and adjust it to a low or moderate speed (about setting 5). 5. With this air-saturated mixture, the pen should be near the maximum side (the right-hand side) of the chart. Using the black “sensitivity knob” on the oxygen electrode control box , set the pen to 95 units on the chart, which will represent the maximum amount of oxygen for your experiment (See Figure 2 ). Figure 2: Oxygen probe and chart recorder. Use the “Sensitivity” knob (shown in A) to adjust the chart to 95 units (shown in B) 6. With the reaction chamber still open , carefully bubble nitrogen gas into the solution using the tubing/pipette tip assembly which is attached to the nitrogen gas cylinder. You must place a finger over the “blow hole” in the tubing in order to get the gas to flow through the pipette tip. The nitrogen gas will displace oxygen, and the pen will move slowly towards zero on the chart. As the pen begins to level out towards the left-hand side of the chart, adjust the pen such that the arbitrary “zero oxygen” level is at 5 units on the chart paper; the control knob to do this is marked ” 0 ”, and is on the right side of the chart recorder. The system is now calibrated for all experiments performed at this temperature . See Figure 3 on the following page. 2 95 units = maximum oxygen = 100% A BUse to adjust to 95 units units Figure 3: Use the chart recorder “0” knob to adjust to 5 units after reading is stabilized following the addition of nitrogen gas to the chamber . At this point the oxygen probe is calibrated and ready for use. Remember to turn off the stir bar prior to emptying the chamber. Add 2 mL of your culture and adjust the pen on the scale according to your procedure through the addition of “oxygen” (air) or nitrogen gas. When sealing the chamber, you must ensure that all excess air is removed. This can be ensured by observing the culture in the capillary tube of the plug (see Figure 4 ) and sealing the top appropriately with Parafilm TM . You can then perform your readings for the time indicated in your procedure. Remember to cover the chamber with the black cloth (visible in Figure 1 ) if required. Figure 4: Inappropriate versus appropriate sealing of the reaction chamber. If the plug has not been sufficiently inserted, an air bubble will be apparent underneath (A). If the plug has been depressed sufficiently, liquid can be observed within the capillary tube of the plug (B). 35 units = minimum oxygen = 0% Use to adjust to 5 units units A BAir bubble Liquid in capillary tube You have arbitrarily set a distance of 90 chart units to represent the total amount (or 100% ) of oxygen present in the reaction chamber, at 20 o C . To convert this total pen deflection to µg oxygen/min you will use the maximum solubility values for oxygen found in Table 1 below to first calculate the amount of oxygen/unit. TABLE 1: Solubility of oxygen in pure water as a function of temperature at sea level. Temperature ( o C) Oxygen ( µ g/mL) Temperature ( o C) Oxygen ( µ g/mL) 10 11.29 40 6.46 20 9.10 50 5.76 30 7.58 60 5.37 For example, at 60 o C the amount of oxygen/unit is: O 2 / unit ( in 2 ml ) = (max. O 2 / ml) x 2 ml total units = (5.37 μ g/ml) x 2 ml 90 units = 0.119 μ g/unit Remember, that this will need to be determined for each temperature for your analysis. We can then determine the rate using the change in units/min obtained from the chart reader (see Figure 5 for an example). As each group has standardized their data by ensuring that their culture contained the same number of cells, we can simply average the determined units/min and use the formula shown below: observed rate ( μ g/min/ml) = units/min x max O 2 ( μ g) /unit 2 ml Figure 5: Sample of data obtained using the oxygen electrode. 4
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
LAB 4B: Observation of the Generation of Proton Motive Force through pH Gradients Chloroplasts are the intracellular organelles in plants which contain all the necessary pigments, enzymes, and intermediates to support the reactions of photosynthesis . These reactions can be broadly divided into two processes: the Hill reactions and the Calvin cycle . In the Hill reactions, chlorophyll molecules are oxidized by light, water is hydrolyzed, oxygen is produced, and the electrons and hydrogen ions are transported down a series of transmembrane electron carriers in the thylakoid membranes . The electrons terminate their travels by reducing NADP + to NADPH (a soluble electron carrier), which is then consumed by the Calvin Cycle in the stroma of the chloroplast. (See Figure 3 on Page 34 ) The electron transport proteins in the thylakoid membrane are asymmetrically arranged, and some of them can transport both hydrogen ions (protons) and electrons whereas some can transport only electrons. The arrangement of the thylakoid electron transport chain promotes the pumping of protons from the stroma into the thylakoid space . This translocation of hydrogen ions causes the lumen of the thylakoid sacs to be acidic (approximately pH 3.5) whereas the stroma is alkaline (approximately pH 8.0). In 1961 Peter Mitchell and Jennifer Moyle proposed the chemiosmotic hypothesis . In essence, the hypothesis states that photosynthetic electron transport results in the accumulation of protons in the lumen of the thylakoids, and that this steep proton gradient is capable of generating ATP. They suggested that the proton motive force (PMF) drives protons through large thylakoid membrane complexes, called ATP synthases , from the lumen of the thylakoids back towards the stroma. For every three protons that pass through the ATP synthase, approximately one ATP is generated. While protons are only translocated into the lumen of the chloroplast during the Hill reactions, protons will move through ATP synthase whenever there is a concentration gradient present, regardless of whether light is present or not. In this lab, we will isolate chloroplasts from spinach leaves and illuminate them with a strong light source in order to examine if the changes in pH that we observe are consistent with the chemiosmotic hypothesis . PROCEDURES i. Isolating chloroplasts from spinach leaves 1. At your bench, there will be 35 g of spinach leaves floating on the surface of a bowl of water. These leaves were left in the cold and dark overnight to reduce the starch content, and have been sitting for a couple of hours under a light source to activate the chloroplasts. Remove the leaves from the water. 2. Rip the leaves into small pieces, and then transfer the pieces to a chilled blender cup, and add 100 ml of homogenizing medium . 3. Blend at top speed for three 10 second bursts . Remove the blender cup, and pour the contents through three layers of cheesecloth into a chilled 250 ml beaker. Squeeze the cheesecloth gently to wring out as much fluid as you can. 4. Distribute the filtered homogenate equally into two 50 ml centrifuge tubes. Remember to balance your tubes by redistributing the homogenate as needed and using the triple-beam balance prior to each centrifugation! 5. Centrifuge at 500 x g for 2 minutes at 4 o C . 6. Transfer the supernatants to two clean 50 ml centrifuge tubes. Discard the pellets . Centrifuge the supernatants at 2000 x g for 10 minutes at 4 o C to pellet the chloroplasts. 7. Discard the supernatants . Add 1.5 ml of 0.01 M NaCl to each tube. Resuspend the chloroplast pellets gently, by sucking them up and down into a Pasteur pipette. Then add a further 6 ml of 0.01 M NaCl to each tube. Place the tubes in an ice bath, and let them incubate for 10 minutes . This treatment swells the chloroplasts slightly, and makes the outer membrane leaky, so that any pH changes in the stroma can be detected in the external medium. 8. Centrifuge the chloroplast suspension at 10,000 x g for 5 minutes at 4 o C . Discard the supernatants . Resuspend the pellets by adding 2 ml of 0.01 M NaCl to each tube and sucking them up and down gently with a Pasteur pipette. Combine the re-suspended pellets into one tube, and store them on ice. This is your osmotically swollen chloroplast preparation. ii. Chlorophyll determination Before doing the pH assay, it is necessary to ensure that the concentration of chlorophyll is 0.5 mg chlorophyll/ml . This will allow us to standardize our data across multiple trials. 1. Add 0.1 ml of the swollen chloroplast suspension to 19.9 ml of 80% acetone in a graduated cylinder. Cap the cylinder with Parafilm® and shake vigorously. 2. Pour a small volume of the acetone extract into a glass spectrophotometer cuvette ( acetone will dissolve plastic!!) and measure the absorbance of the solution at a wavelength of 654 nm . Use 80% acetone as a blank. 3. Take the absorbance value you have observed, and multiply it by 5.6 . The product gives you the concentration of chlorophyll in your suspension in mg/mL. 4. Dilute the chloroplast suspension ( NOT THE CHLOROPLASTS IN ACETONE ) as needed (use the formula C 1 V 1 = C 2 V 2 ) with enough cold 0.01 M NaCl to bring the final chlorophyll concentration to 0.5 mg/ml. You will need to make sufficient chloroplast suspension for a minimum of two trials, so make 15 mL in case you need extra . 5. Return the tube to the ice bath. Cover the diluted chloroplasts with aluminum foil until you are ready to do the pH assays. 2 iii. pH assays Important: Do not make the beakers for this section until you need them. (if they sit too long, they may not work) The solution should be a slight green colour, not blue. 1. The pH meter should already be turned on and in “standby” mode. The pH electrode will be sitting in a standard pH solution of pH 7.0. Please note that when removing the pH electrode from any solution, rinse it well with a squeeze bottle of distilled water and remove any residual water with a Kim Wipe before placing it into another solution. Refer to the pH meter meter operating instructions found in Appendix F . 2. To two 10 ml beakers, add the following: 1. A small (“flea”) magnetic stir bar (be sure not to dump this down the sink after the assay) 2. 1.1 ml 0.048 M NaCl 3. 0.6 ml phenazine metosulphate (PMS) ( TOXIC!!-be careful ) 4. 3.6 ml distilled water 3. Place the beaker on a stir plate. Turn on the stir plate and get the stir bar moving at a moderate speed. Carefully lower the pH electrode into the solution. 4. Add 3.7 ml of your chloroplast suspension using the Gilson pipet provided. Turn on the light source , and position it as close as you can to the beaker. 5. Record the change in pH every 10 seconds for 2 minutes in total using the data table provided on the bench. Then turn off the light , and use the garbage bag provided to make a lightproof curtain over the box. Again, record the change in pH every 10 seconds for 2 minutes . Remove the curtain, and turn the light source back on . Continue to record the change in pH every 10 seconds for a final 2 minutes . 6. Do a second experiment by repeating steps 2-4 above with another beaker and a fresh preparation of chloroplasts. Using the second data table, record the pH at 10 second intervals for 2 minutes, with the light on . Add 20 microliters of carbonyl cyanide m- chlorophenyl hydrazone (CCCP) (a proton ionophore) using the Gilson micropipette provided. Continue to record the change in pH at 10 second intervals for another 2 minutes, with the light on . Prior to the Lab 4B Tutorial please view the pH Assay video. You may wish to refer to Appendix F: Use of the pH Meter as well. Lab 4 Report Labs 4A and 4B will require the submission of a formal lab report worth 10% of your final grade. The Lab 4 Report Requirements are available in the Lab 4 folder. You should review the General Lab Report Guidelines as well. Data analysis will be discussed during the Lab 4B Tutorial. 3
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
APPENDIX F: Instructions for the Accumet AB15 pH Meter The pH probe is relatively simple to use. Refer to the images in Figure 1 below when following the procedure for its use. Figure 1: The Accumet TM AB15 pH probe. The blue collar is indicated with an arrow in image (A), which shows the probe in the storage solution. (B) shows an active pH meter readout, with the control buttons on the right of the screen. Ready pH probe: Prior to use , rotate the blue or purple ring near the top of the probe fully clockwise to open the probe to the atmosphere. At the end of the lab , turn the ring fully counterclockwise to close the probe . Calibration: 1. rinse and blot probe, place it in the first calibration buffer – pH 7.0 2. press stdby to activate meter 3. press std (standardization) twice in fairly quick succession. The first will access the Standardize screen – check that the appropriate buffer group is selected – it should include 4,A B 7 and 10. The second will begin calibration – the meter will automatically recognize the buffer. When the meter accepts the buffer, it will briefly display the percent slope associated with the electrode’s performance before automatically returning to the Measure screen . If the electrode is within the range of 90-100% efficiency, the GOOD ELECTRODE message will appear. If the electrode is outside this range, the meter will display the ELECTRODE ERROR message, and will not return to the Measure screen until the user presses enter. If this message appears, inform your Instructor. (See back of sheet for troubleshooting help.) When the stable icon appears the meter should read the pH value of the buffer you are using to calibrate it with. Press stdby before removing the probe from the buffer, then rinse the probe and blot dry. 4. repeat steps 1 to 3 with the second buffer – either pH 4 or 10 Measurements : 1. Rinse and blot probe, place it in the test solution and wait for the stable icon to appear before recording the value. Remember to place the meter in stdby before removing the probe from the solution, then rinse the probe and blot dry. 2. Store probe in electrode storage solution when measurements are complete. Remember to close the ring at the top of the probe. BAD ELECTRODE ERROR When the meter is flashing ELECTRODE ERROR, follow the steps below: 1. press Enter 2. press Setup twice (pH buffer range will be displayed – it should include 4, 7 and 10) 3. press Enter to clear the previous measurement 4. begin calibration procedure again, starting at step 1 2
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
Data for Lab 4A (U2020F) Yeast (4A part i) units/min Temp Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 20 1 2.5 1.5 1 2 2.5 30 3 4.5 2.5 3.5 3.5 4 40 6 6.5 5.5 7.5 6 6.5 50 5.5 7 6.5 7 6.5 7.5 60 2 1.5 2 2.5 1.5 1.5 Euglena (4A part ii) units/min Temp Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 20 2 4 0 2 2.5 2 30 6 8 6 7 9 7.5 40 11 8 12 12 14 14.5 50 8 4 8 6 6 7 60 3.5 3 2 3 3 2 Euglena Light Conditions (4A part iii) units/min Temp Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 20 10 8 17 11 14 26 30 28 25 27.5 19 26 27.5 40 32 29 33.5 26 35 33.5 50 15.5 16.5 10 20 14 12.5 Euglena Dark Conditions (4A part iii) units/min Temp Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 20 0 0.5 0.5 2 1 0 30 0.5 1.5 1.5 2.5 1.5 2.5 40 1.5 2 3 4 3.5 4 50 1 1 2 3 2.5 1
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
Lab 4B Data U2020F Light on/off: No final 2 minutes in the light will be included in your report. Light on/+CCCP Time Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 0 5.97 6.3 6.42 6.2 6.22 6.06 10 6.48 6.38 6.43 6.33 6.24 6.43 20 6.72 6.41 6.5 6.4 6.26 6.52 30 6.84 6.46 6.54 6.54 6.3 6.64 40 6.91 6.52 6.59 6.66 6.36 6.67 50 6.97 6.57 6.63 6.74 6.43 6.72 60 6.97 6.72 6.66 6.8 6.51 6.78 70 7.01 6.77 6.69 6.86 6.57 6.82 80 7.01 6.82 6.72 6.86 6.63 6.85 90 7.01 6.85 6.75 6.92 6.68 6.87 100 6.96 6.88 6.77 6.92 6.73 6.89 110 6.96 6.91 6.79 6.92 6.76 6.91 120 6.96 6.92 6.81 6.93 6.78 6.91 130 6.86 6.92 6.79 6.78 6.78 6.94 140 6.8 6.89 6.77 6.6 6.77 6.83 150 6.75 6.85 6.75 6.52 6.75 6.77 160 6.73 6.82 6.75 6.45 6.72 6.72 170 6.71 6.79 6.74 6.39 6.7 6.68 180 6.69 6.77 6.73 6.37 6.68 6.65 190 6.67 6.76 6.73 6.34 6.66 6.64 200 6.67 6.75 6.72 6.33 6.65 6.62 210 6.67 6.73 6.72 6.32 6.64 6.6 220 6.66 6.72 6.71 6.31 6.63 6.6 230 6.65 6.71 6.71 6.31 6.62 6.6 240 6.64 6.71 6.71 6.31 6.61 6.59 Time Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 0 6.26 6.65 6.29 5.85 5.66 6.25 10 6.31 6.68 6.34 5.88 5.84 6.27 20 6.56 6.71 6.49 6.14 5.95 6.38 30 6.71 6.75 6.58 6.32 6.06 6.5 40 6.81 6.8 6.69 6.43 6.15 6.72 50 6.89 6.85 6.72 6.52 6.24 6.91 60 6.92 6.89 6.77 6.61 6.32 6.91 70 6.95 6.92 6.8 6.66 6.4 6.91 80 6.97 6.94 6.83 6.7 6.46 6.91 90 6.97 6.96 6.85 6.73 6.51 6.91 100 6.97 6.97 6.86 6.76 6.57 6.92 110 6.98 6.98 6.88 6.78 6.61 6.92 120 6.98 6.99 6.89 6.79 6.65 6.92 130 6.86 6.97 6.9 6.65 6.68 6.88 140 6.7 6.91 6.87 6.49 6.71 6.76 150 6.61 6.85 6.86 6.39 6.74 6.62 160 6.56 6.81 6.85 6.32 6.72 6.56 170 6.52 6.77 6.83 6.28 6.68 6.53 180 6.5 6.75 6.82 6.25 6.66 6.51 190 6.48 6.72 6.82 6.23 6.63 6.49 200 6.48 6.7 6.82 6.21 6.62 6.48 210 6.47 6.68 6.81 6.2 6.6 6.48 220 6.47 6.67 6.81 6.2 6.59 6.46 230 6.45 6.66 6.8 6.19 6.58 6.46 240 6.45 6.65 6.8 6.19 6.57 6.46
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
Lab 4A: Determination of the Rates and Optimal Temperature for Aerobic Respiration & Photosynthesis Sherry Hebert BIOL-3221L-U2020F Gerd Guenther/Science Photo Library/Getty Images Lab Procedure Overview ● Basics of utilizing the oxygen electrode: see Video ● Processes examined ● Species used and specific procedures Aerobic Respiration: Glycolysis & the TCA Cycle Aerobic Respiration: ETC Oxygen is the terminal electron acceptor: amount in the medium decreases! https://courses.lumenlearning.com/boundless-biology/ chapter/oxidative-phosphorylation/ Photosynthesis: Hill Reactions Oxygen is produced by PSII: amount in the medium increases! Photosynthesis Dark conditions ≠ dark reactions! ● Hill reactions (“light reactions”) and the Calvin cycle (“dark reactions”) are interdependent – While the Calvin cycle does not directly require light, it cannot proceed in the absence of the products of the Hill reactions Effects of Temperature ● Metabolic processes in the cell are largely enzyme-dependent ● Increasing temperature increases the kinetic energy: reactions occur more quickly ● Past the optimum temperature proteins become denatured: so steep decrease in activity Yeast ( Sacchromyces ) ● Unicellular fungi containing mitochondria, but no chloroplasts: measure aerobic respiration only ● Determine cell number using spectrophotometry: – O.D. of 1 ≈ 1 x 10 7 cells – Use culture at 0.75 x 10 7 cells/mL Euglena ● Flagellated protozoan ● Contains mitochondria & chloroplasts: measure both aerobic respiration & photosynthesis © Carolina Biological Supply Euglena ● Problem: if it both uses and produces oxygen, how do we interpret data from the probe? Euglena ● Problem: if it both uses and produces oxygen, how do we interpret data from the probe? – In the dark, only aerobic respiration (oxygen use) can occur – Cover with black cloth in aerobic respiration experiment (Part B) Euglena ● Problem: if it both uses and produces oxygen, how do we interpret data from the probe? – In the light, both processes occur: provides us with the apparent rate of photosynthesis – Must also determine rate of oxygen use by covering with cloth: actual rate = │ rate light │ + │ rate dark │ Euglena ● Determine cell number by counting with haemocytometer: add 5 areas and multiply by 50,000 = cells/mL – Use culture at 1 .5 x 10 7 cells/mL – Remember to account for different cell densities when comparing Euglena and yeast! Data Analysis: Amount of oxygen/unit ● Determine the µ g oxygen/unit in 2 mL sample: O 2 / unit ( in 2 ml ) = (max. O 2 / ml) x 2 ml total units From Table 1 of Appendix E Changes with temp! Based on maximum and minimum set on chart recorder 95 units – 5 units = 90 units Data Analysis: Amount of oxygen/unit ● Determine the µ g oxygen/unit in 2 mL sample: O 2 / unit ( in 2 ml ) = (max. O 2 / ml) x 2 ml total units = (5.37 μ g/ml) x 2 ml 90 units = 0.119 μ g/unit ● Use the chart to determine the change in oxygen (# units/min) At 60 O C Data Analysis: Δ oxygen (units/min) Data Analysis: Amount of oxygen/min/mL = ( µ g O 2 /unit in 2 mL )(units/min) 2 mL Same in each experiment at a given temp From chart: will be provided for report You may need to be able to read the chart for your exam!Sample volume Lab Report ● Lab 4 is to be submitted as a formal lab report (see the General Lab Report Guidelines AND the Lab 4 Report Requirements ) ● Your lab report must be submitted through Nexus by 11:59 PM on November 12 th as a pdf file ● It is worth 10% of your final grade! Start Now! ● Introduction : max 1000 words – Same general guidelines for information to be included. – Why is oxygen a good measure of aerobic respiration and photosynthesis? Why use alterations in pH? – How do our readouts relate to the biological processes we are studying? Start Now! ● Methods : no word limit – Same guidelines for preparation ● Results : no word limit – Follow the Lab 4 Report Requirements – For Part A: Data will be available to do your analysis as of Thursday midday Avoid Plagiarism! ● Ensure you are familiar with the rules and regulations on plagiarism ● You cannot utilize the same or similar phrasing as any other source! This includes the lab manual. ● All information from another source must be given in your own words and be appropriately referenced .
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
Lab 4B: Observation of the Generation of Proton Motive Force through pH Gradients Sherry Hebert BIOL-3221L-U2020F Lab Procedure Overview ● Preparation of spinach chloroplasts ● Determination of chlorophyll concentration and dilution ● pH assay Part i: Chloroplast preparation Tear 35g of spinach leaves and place in a blender with 100 mL of homogenizing medium Blend at top speed, 3x 10 seconds Part i: Chloroplast preparation Filter with 3 layers of cheesecloth, squeezing to collect as much liquid as possible. Split into two 50mL centrifuge tubes Part i: Chloroplast preparation Spin 500x g for 2 minutes. Supernatant Keep for next step Pellet Discard Part i: Chloroplast preparation Centrifuge supernatants 2000x g for 10 minutes. Supernatant Discard Pellet Chloroplasts! Part i: Chloroplast preparation ● Gently resuspend chloroplasts in 6 mL 0.01M NaCl. ● Incubate on ice for 10 minutes. Results in swelling of outer membrane. ● Centrifuge 10,000x g 5 minutes. ● Resuspend in 2 mL 0.01M NaCl. Part ii: Chlorophyll determination ● Add 0.1 mL of chloroplasts to 19.9 mL acetone, cover with Parafilm TM and shake ● Pour into cuvette and read at 654 nm (use acetone as a blank) Part ii: Chlorophyll determination ● Multiply absorbance by 5.6 to obtain the concentration in mg/mL ● Use C 1 V 1 = C 2 V 2 to obtain a 15 mL solution (diluted with 0.01M NaCl) of 0.5 mg chlorophyll/mL: chloroplast suspension for your assay! Part iii: pH Assay ● Combine 1.1 mL 0.048 M NaCl, 0.6 mL PMS, and 3.6 mL distilled water in each of two 10mL beakers with “flea” stir bars ● Position probe in beaker Part iii: pH Assay ● Add 3.7 mL of prepared suspension in beaker with the stir bar on. ● Turn on the light and monitor pH every 10 seconds for 2 minutes ● Continue another 2 minutes with the light off and cover in place ● Repeat assay, but add CCCP instead of turning the light off Data Analysis ● Simply determine the average and standard deviation for each time point during the two trials ● Create graph with both trials using curved connecting lines: – include error bars – clearly indicate when the light was turned off/CCCP was added Photosynthesis: Hill Reactions Protons are pumped into the thylakoid during light conditions. Protons diffuse out through ATP synthase in both light and dark conditions! Lab Report ● Lab 4 is to be submitted as a formal lab report (see the General Lab Report Guidelines AND the Lab 4 Report Requirements ) ● Your lab report must be submitted through Nexus by 11:59 PM on November 12 th as a pdf file ● It is worth 10% of your final grade! Lab Report ● Results : no word limit – All results will be available by Thursday midday – Follow the Lab 4 Report Requirements! ● Discussion : 1000 words max – As per Guidelines and Requirements – Focus on agreement between theory and results obtained – Improvements? New hypotheses to test? Lab Report ● Abstract : 250 words max – Don’t forget to include specifics ● References : – Need minimum of 7 references (including lab manual) – Use acceptable format – Ensure you reference all information appropriately
Lab Report 4 on Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis.
BIOL3221L Lab 4 Report Requirements Worth :10 % of Final Grade Submission Deadline :Your lab 4 report is due on Thursday, November 12 th at 11:59 PM.Lab reports will be submitted through the lab Nexus site in the Assignments folder. A late penalty of 5%/day will apply to reports received after the deadline. NO reports will be accepted after Thursday, November 19 th (one week late). Guidelines : Be certain to read the General Guidelines for Lab Reports, which can be found in the Lab Manual & Info folder on Nexus, in order to familiarize yourself with the expectations for your report. Failure to follow these guidelines will result in deductions. The number of marks and which will be assigned, as well as the word limits (if applicable), for each section are indicated below. The specific requirements for data inclusion are also provided. Again, ifyou are uncertain about any of the requirements for your report, please contact your instructor for clarification. Plagiarism or academic dishonesty in any form will not be tolerated. Be certain to familiarize yourself with the university regulations. Total Marks (100 marks) Title Page (0.5 marks) Abstract (7marks; 250 word max) Introduction (20 marks; 1000 word max ) Include an introductory paragraph. You must state your general hypothesis/purpose in the introductory paragraph. Explain, with references, the general theory behind cellular respiration and photosynthesis, focusing on the specific processes you are investigating. Explain why you are using the techniques you are using. Why is oxygen consumption/evol ution a useful means of monitoring these processes? Why is temperature important in these processes? Why are we monitoring pH? Provide sufficient information in this section for your reader to understand why you performed the experiments you did and how they work. Conclude your introduction with abrief summary of the methods you will employ and the purpose of your work. State your specific hypotheses to be tested in your concluding paragraph. Methods (10 marks; no word limit ) Results (30 marks; no word limit ) Please include the following results using the data posted on Nexus: -Table of rates of oxygen/min/mL with individual trials, average, & standard deviation for yeast respiration -Table of rates of oxygen/min/mL with individual trials, average, & standard deviation for Euglena respiration -Figure of average cellular respiration rates for yeast and Euglena .You should include error bars at each temperature. Use a curved line to connect the data points for each species -Table of rates of oxygen/min/mL with average, & standard deviation for observed and actual rates of photosynthesis. You will need to determine the actual rate for each trial before determining the average and standard deviation -Figure of average observed and actual photosynthesis rates. You should include error bars at each temperature. Again, use acurved connecting line. -Include a statement(s) in the text of your results which incorporates the observations provided to you for the addition of CCCP in the oxygen electrode experiments. -Graph of class data for pH over time. Include dark and CCCP conditions on the same graph, each with curved connecting lines. You do not need to include atable of values for this experiment. *Again you may choose to organize this data in anumber of ways, and may include more than one experiment in a single table. However, remember to include information in alogical sequence which provides for anarrative. Also, the figures/tables support the text, not the other way around! Discussion (30 marks; 1000 word max ) Remember to avoid simply reciting your results (already done) and instead to present your findings in the context of what they mean and how they relate to one another. You should also provide references to information which supports your observations and conclusions about the processes you are investigating. Address the validity of your experiments, and means of improving or extending them. Are there further hypotheses you could test? What is the overall significance of your findings? References (2.5 marks) You should include a minimum of 7 references (including the lab manual). Again, you may need to utilize websites to find appropriate information. Try to find reliable sources and be certain to reference appropriately.