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From the respiration figure it is possible to see that during cellular respiration oxygen is used while carbon dioxide is being produced. This relationship is direct in a quantitative sense, since for every mole of oxygen used one mole of carbon dioxide is produced. Thus, if respiration proceeds in a closed system the volume of gas will remain constant even though the composition of the gas changes. If the liberated carbon dioxide is removed then the volume of gas in a closed system will decrease. Thus, the amount of the decrease over time will reflect the amount of carbon dioxide liberated as well as the amount of oxygen used during respiration. We will make use of this information to measure the overall rate of respiration in plant material (germinating seeds) and animal material (flour beetle larvae).
Keeping this information in mind. several simple respirometers were set up. Each respirometers consists of a test tube with the test material , pellets of KOH suspended above the material on glass wool, a set of tubes which join the test tube to a u-tube with fluid and a 1 cc syringe.
Germination
One of the reasons cellular respiration in germinating plants is not well understood or documented is because the biochemical changes that occur within the seed during this time are not themselves well understood. Scientists have tried to study the physiological changes taking place during germination using techniques like labeled metabolites to study carbon dioxide release, but this work has largely been unsuccessful since the impermability of the seed coat prevents current research chemicals from reaching the inner layers of the embryonic plant where the main metabolic activity is. Furthermore, the fact that the embryo and nutritive layers of seeds also display dramatically different physiologies makes studying germination at the molecular level difficult. With the different affinities for carbon dioxide and oxygen that various seed structures exhibit (e.g. phosphoenol pyruvate (PEP) carboxylase), it becomes almost impossible to know what is going on where with regard to cellular respiration in germinating seeds (Botha, Potegieter, & Botha, 1992), and researchers are left trying to find manifestations of metabolic activity at the gross morphological level.
Lab 5 Cellular Respiration
Introduction
Cellular respiration is the procedure of changing the chemical energy of organic molecules into a type that can be used by organisms. Glucose may be oxidized completely if an adequate amount of oxygen is present.
Equation For Cellular Respiration
C6H12O6 + 6O2 -> 6CO2 + 6H2O + energy
Carbon dioxide is formed as oxygen is used. The pressure due to C02 might cancel out any change due to the consumption of oxygen. To get rid of this problem, a chemical will be added that will selectively take out C02. Potassium hydroxide will chemically react with carbon dioxide by the following equation:
C02 + 2 KOH -> K2CO3+ H20
A respirometer is the system used to measure cellular respiration. Pressure changes in the respirometer are directly relative to a change in the amount of gas in the respirometer, as long as the volume and the temperature of the respirometer do not change. To judge the consumption of oxygen in two different respirometers you must reach equilibrium in both respirometers.
A number of physical laws relating to gases are important to the understanding of how the equipment that you will use in this exercise works. The laws are summarized in general gas law that states: PV = nRT Where:
P--the pressure of the gas
V--the volume of the gas
n--the number of molecules of gas
R--the gas constant
T= the temperature of the gas
Hypothesis
In this experiment, the rate of cellular respiration in the germinating peas, in both water baths, will be much higher than that of the beads and non-germinating peas. The cooler temperatures in the other water bath should cause the rate to be much slower in all three respirometers.
Materials
A Water bath, thermometer, masking tape, washers, beads, germinating peas, non-germinating peas, beakers, graduated cylinder, ice, paper, and pencil are needed for this lab.
Methods
Begin the experiment by setting up two water baths, one at room temperature and the other at 10 degrees Celsius. Next, find the volume of germinating peas, non- germinating peas and bead, and beads alone. Repeat these steps for another set of peas and beads. Assemble the six respirometers, placing enough KOH pellets to cover the bottoms of the respirometers. Put non-absorbent cotton balls in each respirometer above the KOH pellets and then add the peas and beads. Place one set of respirometers in the room temperature water bath and the other set into the 10 degree water bath. Slightly elevate the respirometers, supporting them with masking tape, for 5 minutes while they equilibrate. Then lower the respirometers into the water bath and take a reading at 5, 10, 15, 20, 25, and 30 minute time intervals. Record the data into the table.
Data:
Questions
1. In this activity, you are investigating both the effect of germination versus non-germination and warm temperature versus cold temperature on respiration rate. Germinating peas should consume more oxygen than non-germinating peas. Peas germinating at warm temperatures should consume more oxygen than peas germinating at cold temperatures.
2. This activity uses a number of controls. Identify at least three of the control, and describe the purpose of each control.
Water baths held at constant temperature
Volume of KOH is the equal in every tube
Equilibration time is identical for all respirometers
3. Graph the results from the corrected difference column for the germinating peas and dry peas at both room temperature and 10 degrees Celsius.

4. Describe and explain the relationship between the amount of oxygen consumed and time. The amount of oxygen consumed was greatest in germinating peas in warm water. The oxygen consumption increased over time in germinating peas.
5. Complete the following table:

6. Why is it necessary to correct the readings from the peas with the readings from the beads?
To show the actual rate at which cellular respiration occurs in the peas. The beads were the control variable.
7. Explain the effect of germination (versus non-germination) on peas seed respiration.
Germination, the seeds are growing and need to respirate to grow.
8.Explain the results shown in the sample graph in your lab manual. As the temperature increased, enzymes denatured so germination was inhibited.
9. What is the purpose of KOH in this experiment?
KOH pellets absorb carbon dioxide and form an insoluble precipitate
10. Why did the vial have to be completely sealed around the stopper?
The stopper at the top of the vial had to be completely sealed so that no gas could leak out of the vial and so that no water would be able to enter into the vial.
11. If you used the same experimental design to compare the rates of respiration of a 25 g reptile and a 25 g mammal, at 100 degrees Celsius, what results would you expect? Explain your reasoning.
I would expect the respiration to be higher in the mammal since they are warm blooded.
12. If respiration in a small mammal were studied at both room temperature 21 degrees Celsius and 10 degrees Celsius, what results would you predict? Explain your reasoning.
Respiration would be higher at 21 degrees because the animal would have to keep his body temperature up.
13. Explain why water moved into the respirometer pipettes.
While the peas underwent cellular respiration, they consumed oxygen and released carbon dioxide. The carbon dioxide reacted with the KOH resulting in a decrease in the volume of gas in the pipette and the vial. Because the pipette tip was exposed to the water bath, water moved into the pipette.
13. Design an experiment to examine the rates of cellular respiration in peas that have been germinating for 0, 24, 48 and 72 hours. What results would you expect? Why?
Set up four respirometers which have one of the following-Seeds that have not begun to germinate; Seeds that have been germinating for one day; Seeds that have been germinating for two days; Seeds that have been germinating for three days. It is expected that there will be no oxygen used by the seeds that have not germinated yet. The seeds that had been germinating for three days would consume the most oxygen.

Error Analysis:
The seals on the respirators may not have been completely air-tight. The use of KOH pellets, instead of liquid, may have caused errors in the carbon dioxide absorbed. The temperature may have been slightly off in the water baths.

Conclusion:
Oxygen consumption in the respirometers with germinating peas was greater than that in respirometers with non-germinating peas. Respiration rate was also affected by temperature. Respiration occurred at a faster rate in the respirometers in the warm water bath.
Experiment to show the release of heat energy by germinating seeds
Seeds are dormant stages of living organisms, and contain embryo plants, ready to grow when conditions are right. Most of the time, they appear to be doing nothing much, but they are respiring only slowly because they do not need much energy, and need to conserve the food reserves they contain.

However, before they start to grow into plants (and continue the life cycle by flowering and producing seeds again), seeds must germinate. In order to do this, seeds absorb water which they need in order to mobilise their food reserves using enzymes (basically the same process as digestion in animals), then they speed up their rate of respiration quite dramatically. They then use the energy released in order to sprout roots to absorb more water and minerals, and grow a shoot which takes the leaves above ground, so as to make food by photosynthesis. Of course this usually happens under the ground, but it is not necessarily so.

Dried peas (as packaged for food) are alive, and can be encouraged to germinate by soaking them in water and keeping them in airy conditions in fairly warm temperatures (room temperature is warm compared with soil!). Other seeds, such as wheat grains will also do.

The effect of respiring seeds in releasing heat can be shown by placing them into a vacuum flask - as used for keeping foods and drinks hot, or cold.

The sprouting seeds can be divided into 3 portions:
1 left as they are, i.e. alive
2 boiled to kill them, then cooled
3 placed in concentrated disinfectant solution

Each portion is then placed into dilute disinfectant solution, to kill any bacteria or fungi on their surface, and poured into a vacuum flask. A thermometer is placed into each mass of seeds, held with a stand and clamp, and the mouth is \"sealed\" with a plug of cotton wool.
The temperature is then taken in each flask, and repeated (say twice a day) over a period of a few days. It is a good idea to record room temperature at the same time.
Use the table below to record the results, and then plot them as a graph.

• Are Seeds Alive? (AP Lab #5 - Cellular Respiration)
Using oxygen and carbon dioxide gas sensors, students can compare germinating and non-germinating pea seeds to observe that germinating seeds (as well as plants) do use oxygen and give off carbon dioxide as they perform cellular respiration. (40 minutes for basic lab; 80 minutes with addition of temperature variable)

Cellular Respiration
Introduction
The reactions within cells which result in the synthesis of ATP using energy stored in glucose are referred to as cellular respiration. Aerobic respiration requires oxygen as the final electron acceptor. Fermentation does not require oxygen.
The equation for aerobic respiration is below.
C6H12O6 + 6O2  6CO2 + 6 H2O + 36 or 38 ATP
In aerobic respiration (equation above) glucose is completely broken down to CO2 + H2O but during fermentation, it is only partially broken down. Much of the energy originally available in glucose remains in the products produced. Plant and fungal cells produce alcohol as a result of fermentation and animal cells produce lactic acid. The equation for alcohol fermentation is below.
C6H12O6  2CO2 + 2C2H5OH + 2 ATP
Notice from the above equations that aerobic respiration produces much more ATP per glucose molecule than fermentation.
Fermentation
We will investigate fermentation by measuring the amount of carbon dioxide produced by yeast. The rate of cellular respiration is proportional to the amount of CO2 produced (see the equation for fermentation above). In this experiment, we will measure the rate of cellular respiration using four different food sources.
Fill each of four small test tubes two-thirds full with the solutions listed below. Each tube should be filled to exactly the same level.
Tube 1 - glucose (a monosaccharide)
Tube 2 - fructose (a monosaccharide)
Tube 3 - sucrose (a disaccharide)
Tube 4 - starch
Tube 5 - distilled water
Use a dropper to finish filling tube 1 with a thoroughly-mixed yeast suspension. Be sure to mix the yeast suspension immediately before adding it to the tubes. The tube should be filled as full as possible while holding it over a sink. Carefully invert a larger tube and place it over the smaller tube containing the yeast suspension and glucose. Push the smaller tube all the way into the larger tube using your finger or a pencil and then invert both tubes so that the opening of the larger tube is up. Repeat this procedure for the other four tubes.

Below: Tubes containing yeast and a sugar solution are inverted so that CO2 produced by the yeast can collect.

Click on the image to view an enlargement. Press \"back\" to return here.

Place the five test tubes in a 37 degree incubator and record the time.
Time at the start of incubation: _________
The tubes should be checked every 5 minutes to observed the size of the gas bubble that accumulates in the small tube. Proceed to the Aerobic Respiration part of this exercise while waiting for this experiment to complete.


The level of the liquid can be seen through the sides of the test tubes.
Click on the image to view an enlargement. Press \"back\" to return here.


The experiment should be stopped when the gas bubble in any of the tubes is approximately two thirds the length of the tube. Record the time when the experiment is terminated.
Time when yeast is removed from the incubator: _________
After the tubes are removed from the incubator, hold each tube over a sink and quickly invert them as shown below. Use your finger or a pencil to keep the small tube in place while inverting so that the liquid inside the small tube remains in the small tube. Lift the larger tube off of the smaller tube and set the smaller tube in a test tube rack. Repeat this procedure with the other tubes.

The size of the gas bubble produced by the yeast can be measuring the amount of liquid remaining in the tube and subtracting it from the total volume of the tube. Measure the amount of liquid in each of the tubes with a graduated cylinder and record that value in the table below.
Measure the total volume of one of the small tubes with a graduated cylinder. With this number you can calculate the volume of gas produced. by each tube. Perform these calculation and enter the values in the table below.
Total volume of a smaller tube: _______
Tube Contents Milliliters of
liquid remaining Milliliters of
CO2 produced Milliliters of CO2
produced per minute
1 glucose
2 fructose
3 sucrose
4 starch
5 distilled water
Which food source produced in the highest rate of cellular respiration? Which food source produced the slowest rate? Explain your results for tube 5 (distilled water).
Below: The level of liquid in each of the tubes below is indicated with a blue line. Notice that each of the sugars (glucose, fructose, and sucrose) produced approximately the same amount of CO2. Sucrose is expected to produce CO2 at a slower rate because it is a disaccharide and must first be converted to glucose by the cell. Fructose is easily converted to glucose by yeast cells. Yeast cells in water produced little CO2 because they do not have a source of sugar.

Aerobic Respiration
We will measure the rate of aerobic cellular respiration in beans by measuring the volume of O2 consumed using the apparatus shown below. The apparatus consists of three stoppered test tubes with a graduated pipette inserted into each stopper. A colored liquid is placed in each of the pipettes. When the volume of gas in the test tube changes, the liquid in the pipette will move.

The assembled respirometer apparatus.
Click on the photograph to view an enlargement.
Oxygen consumption cannot be measured simply by putting beans in the test tubes because beans are also producing CO2. Any change in gas volume will be due to both O2 consumption and CO2 production. In order to minimize the confounding effect of CO2, KOH will be added to the tubes. It reacts with CO2 to form solid potassium carbonate. The solid will not have a measurable increase in the volume inside the tubes.
CO2 + 2KOH K2CO3 + H2O
Temperature will also affect the measurement of gas consumption because gasses expand when they warm and contract when they cool. This effect will be minimized by keeping the respirometer tubes immersed in water at room temperature. Water temperature changes slowly, so the water will minimize temperature fluctuations inside the tubes.
Obtain the materials for assembling the respirometer apparatus. A tank containing water at room temperature will be needed to hold the three respirometer tubes. Three large test tubes with stoppers and graduated pipettes will also be needed.
Push a small wad of cotton to the bottom of each respirometer tube. The cotton should occupy approximately 2 cm of space on the bottom of the tube. Use a dropper to add 15% KOH solution to the cotton in each tube. Use enough KOH to saturate the cotton but not enough to pour out of the test tube. Use the same amount of KOH in each tube. Be careful not to let KOH come in contact with the sides of the test tube because it will kill the bean seeds.
Push a small wad of dry cotton on top of the KOH-saturated cotton in each tube. This will prevent KOH from coming in contact with the bean seeds and killing them.
Add 50 germinating soybeans to one of the tubes. Add 50 nongerminated soybeans to a second tube. The third tube will remain empty to measure the effect of temperature changes.
Assemble each apparatus and place the tubes in the water tank. After the respirometer tube is inserted into the water tank, use a dropper to push a drop of a colored liquid into the tip of each of the graduated pipettes. Try to force the marker into the region past the tip where its position can be read using the calibrations on the pipette.
Record the time that you insert the respirometer tubes into the water. You will be ready to begin the experiment after the tubes have been in the water for 10 minutes.
A valve in the stopper of each tube should be kept open to allow air into the test tubes before the experiment begins. Do not close this valve until you are ready to take your first reading after 10 minutes.
Allow the respirometer to stand for approximately 10 minutes, then close each of the three valves. Check the fluid mark to be sure it is in a region where calibrations occur.
Record the position of the fluid every 10 minutes for a total of forty minutes. Record your data in the table below. Do not disassemble the apparatus yet. It will be needed in the experiment below.
Effect of Temperature
After you take your last reading, remove the respirometer that does not contain any bean seeds and place it in a beaker of cold water to observe movement of the fluid in the pipette. Next, put the tube in a beaker of warm water. What happened in each case? What is the function of using a respirometer without any bean seeds in this experiment?
Tube Initial reading
(after 10 min.) Reading after
20 min. Reading after
30 min. Final reading
(after 40 min.) Change in volume (ml)
(initial - final) Correction for
temperature change
Germinated beans
Nongerminated beans
No beans XXXXXX
In your lab report, you should be able to explain what two gasses are involved in cellular respiration and why only one of the gasses affects the measurements here. In order to answer this question, you should 1) review the equation for cellular respiration and 2) review the discussion of KOH above. You should also explain how temperature could affect your results and how it was corrected.

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