Cellular Respiration Lab By: emily michaud

Cellular Respiration Introduction

In order for organisms to function, they need energy. Carbohydrates are the most preferred energy source for organisms. Glucose can be broken apart releasing energy stored in its bonds. The energy released from the breakdown of glucose through metabolic processes is what supplies organisms with the energy needed to function. This is what allows animals to move and for plants to grow. In order to make the energy stored in these bonds usable, glucose is broken down and converted into ATP, the main energy molecule of the cell. This process is known as cellular respiration. The energy produced usually is not needed immediately and instead combines ADP with phosphate ions to form ATP. The ATP can then be used for processes in the cells that require energy. This transfer of energy occurs in two stages, glycolysis and aerobic respiration. In glycolysis a small amount of ATP is produced when glucose is broken down to pyruvate. In the second stage the pyruvate either produces a large amount of ATP by passing through aerobic respiration or produces a small amount of ATP through anaerobic fermentation. When oxygen is not available for cellular respiration pyruvate is converted to either lactate or ethanol and carbon dioxide. In eukaryotes, aerobic respiration occurs in the mitochondria while in prokaryotic cells, which do not have membrane bound organelles, this occurs in the folds of their cell membranes.

ATP provides essential energy needed to perform all cellular functions. The general equation for cellular respiration as a whole is:

Any heterotrophic cells, such as animal cells, obtain the carbohydrates that undergo cellular respiration from the food organisms eat, which contain important molecules such as proteins, lipids, and for cellular respiration, carbohydrates. Autotrophic cells, such as plant cells, obtain the carbohydrates needed to perform cellular respiration from photosynthesis. Plants undergo photosynthesis using water, carbon dioxide, and energy in the sun to produce oxygen and glucose in their chloroplasts. Cellular respiration is then performed when cells use glucose and oxygen to produce carbon dioxide, water, and ATP.

Heterotroph Cellular Respiration
Autotroph Cellular Respiration

Processes of glucose metabolism

Glycolysis is the first process of cellular respiration. It occurs outside the mitochondria in the cytosol. Glycolysis does not require oxygen, and therefore is referred to as anaerobic. Glycolysis produces two molecules of pyruvate and two molecules of ATP. The pyruvate, if there is oxyegen available, will move into the mitochondria and go through the Krebs Cycle and the Electron Transport System and eventually ends up producing 36 ATP molecules. If there is no oxygen available, the pyruvate will move on to fermentation. Fermentation releases only two ATP molecules. In lactate fermentation pyruvate is reduced to lactate, and in alcohol fermentation it is reduced to alcohol.

Cellular respiration lab

In this lab we were able to study the rate of cellular respiration among different organisms in different temperatures to see how cellular respiration works in different organisms and how it is effected by the change in temperature. We had 6 vials in total, 2 sets of 3 that we filled with germinating peas, nongerminating peas, and glass beads. The glass beads served as the control for the experiment as they did not perform cellular respiration. These respirometers included a graduated pipet that could measure the change in oxygen volume in the vial in order to tell how much oxygen was being used for cellular respiration. Potassium hydrooxide was placed in the bottom of the vials absorbed into a cotton ball. The purpose of using potassium hydroxide in this lab is to accurately measure the rate of oxygen consumption directly by the change in gas volume in the respirometers. Carbon dioxide is a byproduct of cellular respiration, and so we would not be able to measure a change in the volume of oxygen in the vials if carbon dioxide was being released keeping the volume of gases relatively stable. KOH is able to combine with CO2 to form a solid called potassium carbonate, removing CO2 from the vial so that the change in volume of gas in the respirometer will be directly related to the amount of oxygen consumed, and so the rate of cellular respiration can be measured. The first three vials were placed in room temperature water and the last three were placed in an ice bath. In the different temperature waterbaths we were able to see how the rate of cellular reproduction was affected by temperature. We were also able to see how cellular respiration of germinating peas differs from non-germinating peas. The experiment was performed again with crickets and worms and again we were able to see the rate at which these organisms performed cellular respiration and how temperature affected them.

Hypothesis

For this experiment I hypothesized that if the peas or insects were placed in warm water, then they will respire more than the peas and organisms placed in the cold water and will therefore show a greater oxygen consumption rate as the cold water will slow down cellular respiration. I hypothesized this because I believed the cold water would slow down cellular respiration in the organisms due to enzymes slowing down in the cold water and the metabolism of the cells slowing down. I also believed that the germinating peas would respire more than the dry peas and I believed the cricket would respire more than the worm. I hypothesized that the cricket would show the greatest rate of oxygen consumption and respire more than the germinating peas as well because they are more complex organisms that need energy to move around.

Procedure

Safety

Gloves, goggles, and apron should be worn while performing this lab. Potassium Hydroxide is corrosive

steps

1. Set up an ice waterbath in a large tray making sure to keep the tray filled with ice at all times.

  • Add a thermometer
  • Chill the water to less than 10°C and make sure to maintain this temperature throughout the experiment
  • If possible add a Styrofoam pad under the ice waterbath to insulate it from the benchtop

2. Set up a waterbath with room temperature water in another large tray

  • Keep water out in room temperature to have it adjust to room temperature and add it to the tray
  • Add thermometer

3. Obtain six vials with steel washers on the bottom. Number the vials 1 through 6 with a glass marking pen or whatever is available to you

4. Fill a 100 mL graduated cylinder with 50 mL of water

  • Add 10 germinating peas to the graduated cylinder and take a reading of the displaced water. This is the volume of the germinating peas. Record the volume
  • Decant the water, remove the peas and place the on a paper towel pat the peas dry and set aside

5. Refill the graduated cylinder with 50 mL of water

  • Add 10 dried non-germinating peas to the graduated cylinder
  • Add glass beads until the water level is the same as that of the germinating peas ( so they have the same volume)
  • Remove the peas and beads and place them on a paper towel; pat the peas and beads dry and set aside

6. Refill the graduated cylinder with 50 mL of water

  • Add glass beads to the graduated cylinder until until the water level is the same as that of the germinating peas (so they have the same volume)
  • Remove the beads and place them on a paper towel; pat the beads dry and set aside

7. Repeat steps 3 through 6 with more germinating peas, non-germinating peas and beads, and beads. Set this aside for vials 4-6

8. Place a small absorbent cotton ball in each of the six vials and push each down to the bottom using a pipet or pencil tip BE SURE to use the absorbent cotton balls and NOT the non-absorbent rayon

9. Use a pipet to add 1 mL of 15% potassium hydroxide (KOH) to the absorbent cotton without getting any liquid on the sides of the vial. Then add a piece of non-absorbent rayon that is slightly smaller than that of the cotton ball and place it on top of the KOH soaked cotton (keeps the toxic KOH away from the peas)

  • The KOH is used to combine with the carbon dioxide released through cellular respiration to form a solid so that the gas volume change can be directly measured to the rate of oxygen consumption

10. Using the first set of germinating peas, non-germinating peas and glass beads, and glass beads, place them in vials 1-3 respectively

11. Using the second set of germinating peas, non germinating peas and glass beads, and glass beads, place them in vials 4-6 respectively

12. Insert the non-tapered end of a graduated pipet into the wide end of the stopper so that the tapered end of the pipet is furthest from the stopper and so that the piper extends just beyond the bottom of the stopper (Figure 1)

Figure 1

13. Firmly insert the stopper into the vial. The seal that has been created between the stopper and the vial should be sufficient enough to prevent the pipet from easily moving up and down in the stopper and to prevent leaking

  • Place a washer over the pipet tip and guide it down the pipet until it rests on the stopper
  • Repeat this step for the remaining 5 vials. The set of respirometers should look like those shown in figure 2 below with a steel washer around the pipet
Figure 2

14. Place vials 1-3 in the room temperature waterbath with the pipet tips resting on the edge of the tray (Figure 3)

  • Place vials 4-6 in the chilled waterbath in the same manner
  • Allow all respirometers to equilibrate for 10 minuets
Figure 3

15. Add one drop of food coloring to the exposed tip of each respirometer and wait one minute

  • Turn each of the respirometers so that the graduation marks on the pipets are facing up
  • Carefully immerse all six respirometers in their waterbaths. DO NOT touch the respirometers once the experiment has started!
  • Let the respirometer equilibriate for another 5 minutes before preceding to step 16

16. Read all of the respirometers to the nearest 0.01 mL and take the temperature of each waterbath. Record the initial readings and he temperature of each waterbath

17. Take additional readings of the temperature of the two waterbaths and the readings of the food coloring in the respirometers every five minutes for 30 minutes and record the readings and temperatures

18. When all the readings have been taken, calculate the difference and the corrected difference for each result and record each value

  • Difference: (initial reading at time 0)-(reading at time x)
  • Corrected Difference: (initial pea reading at time 0- pea seed reading at time x)-(initial bead reading at time 0- bead reading at time x)
  • The corrected difference is important and helps to make the data more accurate by canceling out any readings influenced by outside factors

17. On graph paper graph your results from the corrected difference column for the germinating peas and the dry peas, in both the room temperature and chilled waterbaths. Plot the time in minutes

18. Repeat steps 1-17 now with a cricket in vial 1, a worm in vial 2, and glass beads in vial three

  • Instead of using displacement to make sure the volume of the cricket and worm are the same, find the mass using a scale or triple beam balance and add beads until the worm, the cricket, and the glass beads vials are all the same mass

Data

Table 1: germinating & non-germinating Pea respiration

Table 2: Cricket & worm respiration

Respiration lab results Graph

Analysis

Comparing the data tables

In this lab we took periodic measurements of the change in the oxygen volume in the respirometers in order to see the rate of cellular respiration of all of our samples in the experiment. As seen in the data section above, we were able to measure the placement of the food coloring that we initially put into the respirometer at the beginning of the experiment and by comparing it to its initial position we were able to figure out how much oxygen had been consumed every time interval. These measurements were recorded in the charts above. In the first experiment with the germinating peas, non-germinating peas and beads, and glass beads in the 21°C waterbath, looking at the data it is clear to see that the dry peas and glass beads did not show any change in oxygen consumption, while the germinating peas did consume oxygen shown as the measurements of the food dye got closer to the vial, meaning that oxygen was being consumed and cellular respiration was occurring. The glass beads were the control group in this experiment and because they are not living things they will not perform any cellular respiration, which is why no oxygen was consumed and there was no change in measurements. The dry peas, while alive, are not growing, so they do not require much energy and will therefore show relatively no change in oxygen consumption as they do not really perform cellular respiration. The germinating peas, which are still growing and preparing for reproduction, need a lot of energy in order to do this, and so they will have a high rate of oxygen consumption as they must undergo cellular respiration quickly in order to provide their growing cells with energy in the form of ATP. In the second experiment, in which we compared the cellular respiration rates of worms and crickets. As shown in the data table for the worms, crickets, and glass peas at room temperature, 21°C, the glass beads as expected showed no change in oxygen consumption as they do not perform cellular respiration, and the cricket consumed much more oxygen than the worm, which did not consume much oxygen at all. This means that in this experiment that the crickets had the greatest rate of respiration. The worms did not consume a lot of oxygen because they are very simple organisms that do not require that much energy. The worms mainly burrowed into the cotton balls and did not move very much, which is why they did not need a lot of energy and therefore did not consume much oxygen. The crickets continued to move and jump around in the vial and therefore needed a lot of energy in order to function. When comparing the data in experiments one and two, the germinating seeds seemed to have a greater rate of cellular respiration than the crickets because they showed a greater rate of oxygen consumption. While their rates of oxygen consumption were close, the germinating seeds had a greater rate. This is most likely because it must take more energy to grow and prepare for reproduction and so the germinating peas had to perform cellular reproduction more rapidly.

The affect of temperature on cellular respiration

The data can also be used to look at the affect temperature had on the experiments. The warm, room temperature water seemed to allow respiration to occur normally, while the cold water slowed down respiration. Although the cold water did affect the respirometers and falsified some of the data, it can still be seen that the rate of respiration in the germinating peas has slowed down in the cold water as well as with the cricket and the worm. The reason cold water causes the rate of respiration to decrease is the metabolism slows down and therefore doesn't require as much oxygen. Enzymes important in the process of cellular respiration are not able to perform as effectively and slow down in the cold water, therefore cellular respiration is decreased as well. At a cold temperature, the enzyme used to regulate respiration slows the process.

Analyzing the graph

The data on the graph was graphed according to the corrected differences of the data in the table, showing how much oxygen was consumed over time. When analyzing the graph it is very clear to see that the blue line for the germinating peas at 21°C is the specimen that had the greatest rate of respiration. If we were to draw a line of best fit for the blue line, it would be an increasing linear line, showing that the amount of oxygen consumed increased in a steady way. This makes sense because the germinating peas consume the most energy of all the organisms and will therefore consume more oxygen for cellular respiration. The green line, the cricket at 21°C, also increases steadily but does not go as high as the blue line meaning that the cricket did not consume as much oxygen as the germinating peas. The dry peas at 21°C did not show any change in oxygen consumption and the food coloring never made it to any of the numbers on the respirometer, and are therefore not included on the graph. The purple line for the worm at 21°C consumed a very minute amount of oxygen and therefore does not have any change. While the data for the cold water was skewed, it can still be seen that the oxygen consumed in the cold water is less than in warm water for all the experiments, which shows how cold water slows the rate of cellular respiration and so not as much oxygen is consumed.

Conclusion

The lab proved many important concepts relating to cellular respiration. From this lab I have concluded that organisms placed in a cold environment will show a lesser rate of cellular respiration than those in an average temperature environment. The lab showed that temperature and respiration rates are proportional to each other. As the temperature becomes lower, so does the rate of respiration. Because of this, the experiments placed in the cold water had a much lower rate of cellular respiration than the experiments placed in the room temperature water. This conclusion supports my hypothesis that if the peas or insects were placed in warm water, then they would respire more than the peas and organisms placed in the cold water and therefore would show a greater oxygen consumption rate as the cold water slows down cellular respiration. I have also concluded that germinating peas consume more oxygen than non-germinating peas. The non-germinating peas consumed far less oxygen because the germinating peas need a larger amount of oxygen to be consumed so that the seeds will continue to grow, survive, and get ready for reproduction. This supports my conclusion that germinating peas would respire at a greater rate than non-germinating peas. I have concluded that crickets consume more oxygen and therefore have a higher rate of cellular respiration than worms because they are much more active organisms and would therefore require more energy. This also supported my hypothesis that the crickets would have a higher rate of oxygen consumption than the worms. The last thing I have concluded from this lab is that the germinating peas have the highest rate of cellular respiration. This is because they require energy for growing, surviving, and preparing their seeds for reproduction, which needs a lot of energy and therefore the germinating peas will undergo cellular respiration at a high rate. This conclusion refuted my hypothesis that the cricket would have the highest rate of cellular respiration.

Errors/Changes

A large variety of errors could have occurred throughout this experiment to cause the data to be skewed and produce false results. The volume of peas and beads could have been measured incorrectly and produced a unbalanced experiment. Because we were very careful in these measurements, however, I do not believe this was any problem in my lab. There also could have been varying amounts of KOH and cotton. This again is not a major problem and most likely did not affect our experiment. One of the most important errors that could have occurred in this lab are leaks in the respirometers. The vials may have been poorly sealed and would have allowed water to enter the respirometer, which would greatly skew the data. Air may have been allowed to creep into the vial as well. Errors that we are aware likely took place in our lab is that we were not able to properly equilibriate the vials for the required amount of time because of the time restraints in class. Not allowing the vials to equilibriate is what caused the readings in the cold water to move away from the vial and pour the food coloring out of the respirometers rather than towards the respirometer as oxygen was consumed. Because the cold water was so freezing, not allowing the experiments to equilibriate long enough caused the water to expand and push the food coloring out of the respirometer, where it was driven to sink in the cold water. This is why the cold water data for our lab is so skewed and unreliable, as well as why it does not make a lot of sense. Not properly equilibriating the vials, especially those in the cold water, cause the experiment to perform unexpected results due to the error. Another error that may have occurred is that our group may have read the pipettes either too soon or too late or completely misread the pipettes giving false data that does not match up with the rest of the experiment. Another error due to time constraints is that with the worm and cricket experiment we were not able to monitor it for the full 30 minutes, and instead only monitored it for 12 minutes. This makes it difficult to compare the first experiment to the second experiment as the crickets and the worms were not given as much time as the peas for cellular respiration. This may have caused the crickets to look like that respire a lot less than the peas only because it was not given as much time to perform cellular respiration. Another possible error is if KOH came into contact with the sides of the vials. This could have affected the respiration data by poisoning the peas, causing them to die and reducing the amount of peas performing cellular respiration. The worms in experiment two burrowed down into the cotton with the KOH and killed themselves, another error that produced false results for the worm as they could not perform cellular respiration if they were killed, therefore the results came out to look like they did not respire at all. Another error we faced was that the worm and the cricket most likely did not have the same volume, as we were not able to calculate the volume, so the data may have been slightly off if their measurements were unequal. The temperature change of the cold water bath was another possible error in this lab as we did not have more ice to keep adding to the containers, so the temperature of the ice bath increased over time. This could have caused respiration to increase when the cold water began to warm up. One final error could have come with the mathematical calculations or with a faulty calculator that could have given false data in the tables.

Future Experimentation

Cellular respiration is a process in which cells use glucose and oxygen to produce carbon dioxide, water, and ATP. However, not all the energy produced in cellular respiration is converted to ATP, and instead some is released as heat energy. Therefore, one possible future experiment to measure the rate of cellular respiration between different organisms and to measure the effect temperature has on cellular respiration would be to measure the energy given off by each experiment as they undergo cellular respiration. This experiment could help enhance our knowledge about cellular respiration and give us more readings to help us better understand the results for this lab and the new lab. In this experiment, we could put germinating peas, non-germinating peas and beads, and glass beads in vials for the first experiment and a worm, cricket, and glass beads in the vials for the second experiment. The vials would be closed with a stopper with a thermometer stuck into the vial. Next, one set of the germinating peas, non-germinating peas, and beads will be placed in room temperature water, while the others will be put in chilled water. We would read the initial temperature of the container and over periods of time we could record how the temperature inside the vials has changed. The same process would follow for the worm, cricket, and beads as well. This way, by measuring the change in temperature of the vials we will be able to tell which organism has the highest rate of respiration because it will give off the most heat. We will also be able to tell the affect temperature has on respiration by comparing the temperature change of the room temperature submerged vials to the freezing water vials, seeing how much more heat is released in the room temperature vials.

Credits:

Created with images by kaibara87 - "Magnolia petal cells"

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