Grade Level: High School, 9-12 and Advanced Placement Biology

The aim of this exercise is to demonstrate the process of cellular respiration using peas. In addition, you will learn how to collect data, to plot those data in a graphical form to observe the relationship between the Y- and X- variables, and to interpret any relationship observed.

So, how are the two related? Energy in the form of ATP and carbon dioxide are released from the conversion and interaction of glucose and oxygen. Breathing allows us to acquire oxygen while disposing of the carbon dioxide generated from cellular respiration as demonstrated by the equation below.

C6H12O6 + 6O2 -----> CO2 + H 2O + 686 kilocalories of energy/mole of glucose

dioxide)

Thus, if we want to measure the respiration of an organism we can measure the rate of oxygen consumption or the production of carbon dioxide or both. In our experiment, we will measure the relative change in the rate of oxygen consumption between germinating and non-germinating peas at three different temperatures. In brief, the respirometer is a tube with a rubber stopper that has an opening for a graduated pipette tip. When the system is fully assembled and submerged in a water bath, the water enters the pipette tip until a point in which the pressure inside and outside of the respirometer is equal. In general, the system follows the Ideal Gas Law and the equation below:

PV = nRT.

Each component of the gas is represented by P (pressure), V (volume), n (number of molecules of gas), and T (temperature). R is the universal gas constant and is given by 8.31 J/(mol*K).

The law states the following.

Gases move from regions of high pressure to low pressure.

When pressure and temperature are kept constant, the volume of gas is directly related to the number of molecules of gas.

When temperature and volume are kept constant, the pressure of gas is directly related to the number of molecules of gas.

When the number of molecules of gas and the temperature are kept constant, the pressure is inversely related to the volume.

When the temperature varies and the number of gas molecules is kept constant, the pressure or volume or both is directly related to the temperature.

When you assemble the respirometer, you should have a layer of cotton with potassium hydroxide (KOH) and peas in that order (see Figure 1). Because the KOH absorbs gaseous CO2 to produce a solid potassium carbonate (K2CO3), the setup will allow you to measure the rate of oxygen consumption during cellular respiration.

Germinating Peas

Heat-treated Peas (Non-germinating peas)

Respirometers (Test tube, Rubber stopper, Graduated pipette tip)

Shallow Pans

Thermometers

Hot Plate

Ice

Set up a room temperature, warm- and cold-water bath in two beakers.

What is the temperature of the room temperature bath? __________

What is the temperature of the warm water bath? __________

What is the temperature of the cold water bath? __________

Fill a 100 ml graduated cylinder with 50 ml of water. Gently place 25 germinating peas into the cylinder and record the displacement of water. Dry the peas on a paper towel. These peas will be placed in the first tube and be labeled respirometer 1.

How much water does 25 germinating peas displace? __________

Refill a 100 ml graduated cylinder with 50 ml of water. Place 25 heat-treated (non-germinating) peas in the graduated cylinder, and then gently add glass beads until the same volume of water from step 2 is displaced. Remove both peas and beads and dry on a paper towel. These peas and beads will go in a second tube and be labeled as respirometer 2.

Create a second and third set of respirometers by repeating steps 2 and 3. These respirometers will be labeled respirometer 3, 4, 5, and 6, respectively.

For all six test tubes + contents, place an equal amount of cotton into each test tube.

Add an equal amount of 15 % KOH to each test tube. (Note: Do not get KOH solution onto the inside wall of the tube) Then place a small wad of cotton over the KOH-soaked cotton.

Add the peas and beads to each respirometer.

Place the rubber stopper with the graduated pipette tip snuggly onto the tube.

Submerge respirometers 1 and 2 into the room temperature bath and allow your system to equilibrate for 7 minutes. Make sure that the peas + beads are completely covered by the water but the pipette tip rest on the edge of water bath container. Submerging the respirometer reduces the temperature changes in the tube.

Submerge respirometers 3 and 4 into the warm water bath for 7 minutes. Again check to see if the peas + beads are completely covered by the water with the pipette tip on the edge of the container.

Repeat this for respirometers 5 and 6 with a cold water bath and let the system equilibrate for 7 minutes.

After the 7 minutes, submerge the entire system (i.e. pipette tip) into the water. The water will move into the pipette tip until a point when the internal and external pressure is equivalent. (Note: If your respirometer has a leak, the water will continue moving into the pipette instead of maintaining its position.)

Adjust the pipette tip so that you can read the marks without having to move the respirometers after the experiment starts.

Let the respirometers equilibrate for another 3 minutes and then record the position of the water. This is your initial or time 0 reading.

Record the position of the water every 5 minutes for the next 20 minutes in the tables provided below.

Table 1. Oxygen consumption of room temperature-treated peas

Table 2. Oxygen consumption of warm water-treated peas

Table 3. Oxygen consumption of cold water-treated peas

Congratulations! You’ve completed the experiment. Now it’s time to graph your data.

Presenting data as a graph or figure is of utmost importance in science. Graphs allow us to see trends in our data that would otherwise look unorganized. In this exercise, you will graph the data you collected with the pea experiment above. The Y-axis is the response variable and the X-axis is the independent variable; that is, the Y-variable changes in response to changes in the X-variable.

So, what variable that you’ve measured goes on the Y-axis? __________

What variable goes on the X-axis? __________

If you said that the distance (moved by the water) goes on the Y-axis and time goes on the X-axis, you are correct. When you graph these data, you should be able to calculate the rate of oxygen consumption from the slope of the line for each of the treatments. Remember that you are graphing the difference between the initial reading and the reading at time X. Time X refers to the time at 5 minutes, 10 minutes, etc. For example, if you wanted to know the distance the water moved after 20 minutes, then you would subtract the value for initial position of the water by the value recorded for position of the water at 20 minutes.

What should your graph look like? First, each axis should be labeled correctly with the proper units for each variable. Second, your graph should have a title that describes what is being presented so that anyone not familiar with your experiment can tell what is going on. Third, your figure should have a legend because you are plotting several lines onto one sheet of graph paper to observe how each line compares to the other.

In which direction does the water in the pipette tip move. Why do you think this is the case? (Hint: Use your knowledge of the Ideal Gas Law to answer this question)

What is the rate of oxygen consumption for each of the treatments? What is the unit for rate of oxygen consumption? (Hint: How did you calculate the slope?)

How did temperature influence the rate of oxygen consumption?

Did you see a difference in the rate of oxygen consumption between germinating and non-germinating peas? If so, what could explain that difference?

Given the set up of your system, what two concepts of the Ideal Gas Law does your apparatus (i.e. respirometer) follow? (Hint: You keep the temperature of each treatment group constant by placing the respirometers in their respective water bathes. What else is held constant in your set up? Refer to the procedure if you are unsure.)