Pea Seedling Respiration

Henrik Kibak x4144
Brenda Beech

This lab set up is still a draft since precise equiment description may change...

 

Overview: The net equation for respiration is C6H12O6 + 6O2 = 6CO2 + 6H2O. Using respirometers set up to remove carbon dioxide, we will measure the change in the volume of the gas surrounding the peas and assume that it represents oxygen consumption.

Background:

15% Potassium Hydroxide is a strong base! We will use gloves and goggles during the lab set up today.

 

Checklist of Tasks:

Setting up your data tables:

Setting up the two baths:

  • Obtain a bucket of ice from cold room.
  • Using ice, set up a 10° C bath and a Room Temperature bath that can each hold three respirometers.

Setting up the six respirometers - part I (controlling the volume):

  • Organize the numbering of these respirometer tubes to align with the data table on the next page.
  • Using a 100 mL graduated cylinder, add carefully add 50 mL H2O and then drop in 25 germinating peas to measure the volume of the peas. Record the displacement volume (X - 50 = volume of peas). Remove these peas and place them on a paper towel. They will be used in respirometer #1.
  • Measure and record the volume of 25 dried peas using the same technique. Add enough glass beads to equal the volume of the germinating peas. Remove the peas and beads and store them on a separate paper towel. These beads and peas will be used in respirometer #2.
  • Using the same technique, find the number of beads required to displace the same volume as the germinating peas. Remove these beads and place them on a third paper towel. These beads will be used in respirometer #3.
  • Now do all of this again for the second set of three respirometers.
  • One set will be used in the 10° C bath, and the other in the RT bath.

Setting up the six respirometers - part II (the CO2 trap):

  • Place a small wad of cotton in the bottom of the vial that you will use for the respirometer..
  • Using a dropper, carefully saturate the cotton with 15% KOH without getting the sides of the vial wet. Keep track of how many drops you used.
  • Place another small wad of dry cotton on top of the wetted cotton to protect the seeds from the KOH.
  • Try to have the amounts of cotton and KOH be the same for each vial.
  • Assemble the respirometers according to the list below:
  Treatment Chip or Pea Volume Temperature
Respirometer #1 Germinating Peas   RT
Respirometer #2 Dry Peas plus inert chips   RT
Respirometer #3 Inert chips   RT
Respirometer #4 Germinating Peas   10° C
Respirometer #5 Dry Peas plus inert chips   10° C
Respirometer #6 Inert chips   10° C
  • Insert the 1mL calibrated serological pipette and weigh down the vials so that they will sink. Be sure to orient the graduations so that you can see all three when they are laying down.
  • Using masking tape, prepare a sling to hold the tips of the respirometers out of the water during the equilibration period.
  • After the respirometers have rested in water (tips out) for 7 minutes, immerse all six respirometers entirely in their water baths. Water should enter the pipette a short distance and then stop (unless there is a leak).
  • Arrange the respirometers so that they can be read through the water. After 3 minutes of further equilibration begin your recordings. This is T=0:00:00. Record the position of the water in the pipette to the nearest 0.01 mL.
  • Do not move the respirometers during the experiment and make sure a constant temperature is maintained by gently adding ice to a far corner of the bath.
  • It is important to record the data in all six respirometers as the data from the inert respirometers (#3 & #6) will be used to correct the data in the other four.

Data Table

Pea Seedling Respirometer Data Table
Measurement of Oxygen Consumption Using a Volumetric Method
Temp Time Inert Chips Germinating Peas Dry Peas & Chips
Reading Change Reading Change Correction Reading Change Correction

Room Temp

(= _____ ° C)

 T=00:00                
T=05:00                
T=10:00                
T=15:00                
T=20:00                
T=25:00                
T=30:00                
10° C T=00:00                
T=05:00                
T=10:00                
T=15:00                
T=20:00                
T=25:00                
T=30:00                
  • At the end of the experiment, weigh the pea seedlings and the dry peas from the RT vials.

Clean Up: KOH waste beaker, can save peas for a student who wants them, tray for beads to rinse and dry.

 

Assignment (Assignment as WORD Document):

  1. Record measurements carefully into Data Table and calculate corrected values.
  2. Using corrected values plot four curves: Germinating seeds at 10° C and RT, Dry Seeds at 10° C and RT.
  3. Write a paragraph interpreting the data and curves.
  4. Calculate the mL O2 consumed per hour per gram of peas.
  5. How much O2 would 100 kg of peas consume per hour?
  6. What proportion of air is O2?
  7. How many liters of air per hour would be necessary to supply the germinating peas in #5?
  8. How many moles of O2 per hour does #5 represent?
  9. How many moles of glucose do 100 kg of peas consume per hour?
  10. How many grams of glucose do 100 kg of peas consume per hour?

 

 

 

Group equipment:   Class equipment:
  • waterbaths (2)
  • respirometers (6)
  • 100 mL graduated cylinder (1)
  • weights to submerge respirometers
  • thermometer
  • timer
  • forceps
  
  • digital balance
Group materials:   Class materials:
  • Plastic beads or inert chips (~70 mL)
  • Germinating pea seeds (50)
  • Dry pea seeds (50)
  • cotton
  • 15% (g/v) KOH and dropper
  • masking tape or large rubber bands
  
  • ice