Name:____________________________________________________ Date:__________________

AP Biology Lab  -  Cell Respiration

This investigation uses respirometry techniques to calculate the rate of oxygen consumption (cellular respiration) in germinating pea seeds.   The effect of temperature and whether a seed has broken dormancy are quantified and graphed.  The ideal gas law and its concepts are reviewed and applied.

Objectives

lab

Background

Each individual cell is responsible for the energy exchanges necessary to sustain its ordered structure.  Cells accomplish this task by breaking down nutrient molecules to generate ATP (adenosine triphosphate), which can then be used to run cellular processes that require energy.  This process is called cellular respiration which requires nutrient molecules and oxygen.   Carbon dioxide and water are products of the series of reactions involved in cellular respiration.

                       equation

There are several methods of indirectly measuring the rate of cellular respiration in organisms.  One method involves monitoring changes in temperature; since the process of respiration is exergonic (produces heat).  Another method is to measure either the oxygen consumption or the carbon dioxide production.  Respirometers are devices that measure these types of gas volume changes, and therefore provide information about the rate of cellular respiration.

In order to be able to use a respirometer, you will need to use the ideal gas law, which describes the relationship between temperature, pressure and volume. (PV = nrT)

During cellular respiration, two gases are changing in volume.  Oxygen gas is being consumed by the respiring cells and carbon dioxide gas is diffusing out of the cells.  The respirometer, therefore, has to be able to deal with two simultaneously changing gas volumes.  This is accomplished by introducing potassium hydroxide into the device.  KOH absorbs carbon dioxide, following this equation

CO2 + 2KOH --> K2CO3 + H2O

Potassium carbonate ( K2CO3 ) is a solid precipitate.  Any CO2 produced is immediately converted from a gas to a solid and is therefore no longer governed by gas laws.  This allows the respirometer to measure only one variable, the consumption of oxygen gas by living cells.

Assembling the Respirometers

Two sets of three respirometers will be assembled during this lab exercise.  Each set will be incubated at a different temperature.  One respirometer will contain germinated seeds, one will contain a mix of nongerminating seeds and plastic beads, and a third will contain only plastic beads. 

The purpose of the beads is to ensure that each respirometer is uniform in volume.  The respirometers will also contain a layer of cotton that has been saturated with KOH so that carbon dioxide will be absorbed.   The respirometers will be submerged in a pan of water;  water will flow from an area of high pressure to an area of low pressure.  As oxygen is used up by the respiring seeds, the gas pressure inside the respirometer will decrease and the water will flow into the pipet down its pressure gradient. 

 

 

Lab Materials:   50 germinating pea seeds,  50 dry seeds,  100 plastic beads,  3 respirometer vials, Weights for vials, 3 stoppers,  1 ml graduated pipets, sealant (Vaseline), absorbent cotton, nonabsorbent cotton, 1 round wood stick,  3 pieces of paper towel, marking pen, water bath, ice, 100 ml graduated cylinder, thermometer, masking tape, stopwatch or clock, water.    Dropper Bottle of 15% KOH

Safety – wear safety goggles.  KOH is caustic, avoid direct skin contact.  If contact occurs, flush affected area with running water for 10 minutes. 

Procedure: Day 1

1.  Label three paper towels as follows:  1a, 2a, 3a.  These numbers will correspond to the respirometers of the same numbers. checkbox
2.  Fill a graduated cylinder with 20 ml of water.  Count out 25 germinating seeds and place them into the graduated cylinder.   Record the total volume of the seeds and water in the data table.  Subtract the initial 20 ml to determine the total volume of the germinating seeds.  Pour out the water from the graduated cylinder and place the 25 germinating seeds on paper towel 1a. checkbox
3.  Fill the graduated cylinder with 20ml of water.  Count out 25 dry (nongerminating) seeds and place them into the water.  Drop plastic beads into the cylinder until the final volume is the same as from step 3.
4. Place the pea/bead mixture on paper towel 2a.  (Your goal here is to make sure each respirometer has the same volume).checkbox
5.  Fill the graduated cylinder with 20 ml of water.  Add beads to the water until the total volume equals the final volume from steps 2 & 3.  Place the beads on paper towel 3a. checkbox
6.  Assemble the respirometers.  Begin with 3 vials , rubber stoppers and 1 ML pipets.  You will also need a sealant and a marker to label the vials 1a, 2a, 3a. checkbox
7.  Insert the nontapered end of one pipet into the upper surface of one of the rubber stoppers.  It should fit tightly.  Place a layer of sealant around the junction between the pipet and the stopper so that no air can escape.  (*The pointy end of the pipet should be outside, not inside) checkbox
8.  Place a piece of absorbent cotton in the bottom of each of the weighted vials.  Push the cotton firmly into the bottom of the vial with a wooden stick or stirring rod.  Saturate the cotton in the vial with a few drops of 15% KOH, or alternatively use KOH pellets.  Use the same number of drops on each cotton ball.  *Caution, KOH is caustic.checkbox   
9.  Place a piece of nonabsorbent cotton on top of the saturated cotton in each vial.  Push the cotton to the bottom of the vial. (If using KOH pellets, only the nonabsorbent cotten is necessary to prevent peas from touching poisonous pellets.checkbox
10.  Add the peas, peas/beads, and beads to the appropriate respirometer.  Place the stoppers on each of the vials and ensure they are secured tightly.  Any leaks will cause the experiment to fail.   Set your apparatus aside for day 2. checkbox

water bathDay 2

1.  Place a strip of masking tape across the narrow width of the water bath, approximately 2/3 of the way from one end (see diagram).  Place a white paper towel in the bottom of the tub so that you can more easily read pipettes. checkbox

2.  Place respirometers 1a, 2a, 3a into the room temperature water bath so that the pipets rest on the masking tape prop.  Begin time for a total of 7 minutes – this is the equilibration period, where your respirometers will become the same temperature as the water.     
Use a thermometer to determine the water temperature:  _________ checkbox

3. At the end of 7 minutes, submerge each of the tubes entirely in the water bath.  Some water will enter the tip of the pipet, but the influx of water should stop fairly quickly.  If it does not stop, check the respirometer for leaks.   checkbox

4.  At this point, check to make sure you can read the pipets. 

The air bubble should extend from the main chamber up the tube of the pipet.  The pipet may need to be rotated so that you can see the numbers. checkbox

5.  If your respirometers float, you may need to weight them.  Some come with weights inside and some do not. You can improvise here, stainless steel dissection scissors; for instance, can serve to weight the tubes. checkbox

6.    Record the water level in each pipet onto the data table at the Time Interval 0.  checkbox

7.  Record the position of the water in each pipet at the end of 5, 10, and 15, 20 min on Data Table 2. checkbox

8.  Remove the respirometers from the water and set aside for Day 3 checkbox

Day 3

1.  Add ice to the water bath to lower the temperature to 10 Celsius.  You will need to monitor the temperature and possibly add more ice to keep this temperature constant throughout this part.  checkbox
2.  Now submerge the respirometers into the ice bath and let them sit for 7 minutes so their temperature becomes equal to the water bath temperature. checkbox
3.  Record the position of the water at 0, 5, 10, 15, 20 min. checkbox

Extension:   Remove your peas from the chamber and replace with a living organism. You could use a cricket or a mealworm for instance.   Determine the respiration rate of your living organism in both cold and warm water.  *You may be assigned to a particular temperature*

DATA & ANALYSIS

DATA TABLE 1: Calculation of Volume in Respirometers

Respirometer 1a
(germinating seeds)
Respirometer 2a
(non germinating)
Respirometer 3a
(beads only)
Initial Volume (mL) 20 20 20
Final Volume (mL)
Volume of beads/seeds

Data Table 2: Calculation of Oxygen Consumption

Respirometer 1a:  Room Temperature, Germinating Peas
Time interval (min): 0 min 5 min 10min 15 min 20 min
Reading, mL
Δ  Volume, mL (reading – time 0) N/A
Respirometer 2a: Room Temperature, Dry Pea Seeds  
Reading, mL
Δ  Volume, mL (reading – time 0) N/A
Respirometer 3a:  Room Temperature, Beads Only
Reading, mL
Δ  Volume, mL (reading – time 0) N/A
Respirometer 1b:  10°C, Germinating Pea Seeds  
Time interval (min): 0 min 5 min 10 min 15 min 20 min
Reading, mL          
Δ  Volume, mL (reading – time 0)
Respirometer 2b:  10°C, Dry Pea Seeds
Reading, mL
Δ  Volume, mL (reading – time 0) N/A
Respirometer  3b: 10°C, Beads only
Reading, mL
Δ  Volume, mL (reading – time 0) N/A
Extension:  Cricket 0 min 5 min 10 min 15 min 20 min
Reading, mL
Δ  Volume, mL (reading – time 0)

Graph:  Rate of Oxygen Consumption in Respirometers  (room temperature)

Plot the independent variable on the X axis (time), and the dependent variable on the Y axis (reading on pipet).  

Graph a line for:

Germinating Peas (room temp)
Germinating Peas (cold)

Non Germinating peas (room temp)
Non Germinating peas (cold)

Beads (room temp) 

**Use a line of BEST FIT so that you can calculate slope**

  

graph

Analysis

1.   State a hypothesis that relates to temperature that is being tested by this lab exercise.

 

2.   State a hypothesis that relates to the state of seed germination that is being tested by this lab exercise.

 

3.  Calculate the RATE of oxygen consumption for the germinating seeds in both cold and room temperature water.  Rate can be calculated by determining the SLOPE of the line from your graph above.

 

4.  In this lab exercise, what is the purpose of the ….

  1.  Beads

 

  1. KOH
  1. Respirometer

 

5.  Explain why the water moved within the pipet.