Cellular Respiration and Photosynthesis Labs

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Cellular Respiration and Photosynthesis Labs

Cellular Respiration Lab

Background Information:

Living systems require free energy and matter to maintain order, to grow, and to reproduce. Energy deficiencies are not only detrimental to individual organisms, but they cause disruptions at the population and ecosystem levels as well. Organisms employ various strategies that have been conserved through evolution to capture, use, and store free energy. Autotrophic organisms capture free energy from the environment through photosynthesis and chemosynthesis, whereas heterotrophic organisms harvest free energy from carbon compounds produced by other organisms. The process of cellular respiration harvests the energy in carbon compounds to produce ATP that powers most of the vital cellular processes. In eukaryotes, respiration occurs in the mitochondria within cells.

If sufficient oxygen is available, glucose may be oxidized completely in a series of enzyme-mediated steps, as summarized by the following reaction:

C6H12O6 + 6O2 6CO2 + 6H2O + energy

Morespecifically:

C6H12O6 + 6O26CO2 + 6H2O +686 kilocalories of energy

Mole of glucose oxidized

The chemical oxidation of glucose has important implications to the measurement of respiration. From the equation, if glucose is the energy source, then for every molecule of oxygen consumed, one molecule of carbon dioxide is produced.

In this procedure, you will learn how to calculate the rate of cellular respiration by using a respirometer system.These measure relative volume (changes in pressure) as oxygen is consumed by germinating plant seeds. A number of physical laws relating to gases are important to the understanding of how the apparatus that you will use in this experiment works. The laws are summarized in the general gas law that states:

PV = nRT

Where:

P = pressure of the gas

V = volume of the gas

n= number of moles of gas

R = the gas constant (its value is fixed)

T= temperature of the gas (in K)

This law implies the following important concepts about gases:

If temperature and pressure are kept constant, than the volume of the gas is directly proportional to the number of molecules of the gas.
If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas present.
If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume.
If the temperature changes and the number gas molecules is kept constant, then either pressure or volume (or both) will change in direct proportion to the temperature.

It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure.

In this experiment, as oxygen gas is consumed during respiration, it is normally replaced by CO2 gas at a ratio of one molecule of CO2 for each molecule of O2. Thus, you would expect no change in gas volume to result from this experiment. However, in the following procedure the CO2 produced is removed by potassium hydroxide (KOH). KOH reacts with CO2 to form the solid potassium carbonate (K2CO3) through the following reaction:

CO2 + 2KOH K2CO3 + H2O

Thus, as O2 is consumed, the overall gas volume in the respirometer decreases. The change in volume can be used to determine the rate of cellular respiration. Because respirometers are sensitive to changes in gas volume, they are also sensitive to changes in temperature and air pressure; thus, you need to use a control respirometer. What would be a good control for this procedure? Talk with another student for a minute, and come up with at least one possible control you could use.

Learning Objectives:

To learn how a respirometer system can be used to measure respiration rates in plant seeds or small invertebrates, such as insects or earthworms.
To conduct an experiment to explore the effect of certain factors, including environmental variables, on the rate of cellular respiration.
To connect and apply concepts, including the relationship between cell structure and function (mitochondria); enzymatic activity; diffusion of gases across cell membranes; and the physical laws pertaining to the properties and behaviors of gases.

General Safety Precautions:

KOH is caustic. Use appropriate safety measure when using.

Procedures:

The class will test the difference in the rate of cellular respiration when germinating peas and non-germinating peas are put in different temperatures—half the class will test room temperature and half the class will test 10oC.

Based upon the experiment assigned to you, please write the following information in your lab notebook:

Purpose of your experiment
Independent Variable(s)
Dependent Variable
How you will measure your Dependent Variable
Hypothesis

Procedure for Both Test Groups:

Measuring the Volume of Germinating Peas: Obtain a 100=mL graduated cylinder and fill it with 50 mL of tap water. Drop in 25 germinating peas and determine the amount of water that was displaced (which is equivalent to the volume of peas). Record the volume of 25 germinating peas in your lab notebook. Remove these peas and place them on a paper towel. These will be used in one or your respirometers.
Measuring Beads for Control Respirometer: Refill the graduated cylinder with 50 mL of tap water. Determine how many glass beads would be required to attain a volume equivalent to that of the germinating peas. Remove these beads and place them on a paper towel. These will be used as one of your control respirometers.
Assembling the Respirometers:
Place a wad of absorbent cotton in the bottom of each of your dry vials and use a dropper to saturate the cotton with 15% KOH. Do not get KOH on the sides of the respirometer. Place a small wad of dry cotton on top of the KOH-soaked absorbent cotton. It is important that the amounts of cotton and KOH be the same for each respirometer.
Place germinating peas in one vial, insert the stopper that is fitted with the calibrated pipette. Place a washer on the pipette.
Repeat Step B for remaining beads, germinating peas, and/or dry peas.
Equilibrating the Respirometers and Taking Data:
Make a sling of masking tape attached to each side of each of the water baths to hold the pipettes out of the water during an equilibration period of seven minutes.
After 7 minutes, immerse respirometers entirely in their water baths for a waiting period of another three minutes. Water will enter the pipettes for a short distance and then stop. If the water continues to move into a pipette, check for leaks in the respirometer. Work swiftly and arrange the pipettes so that they can be read through the water at the beginning of the experiment. They should not be shifted during the experiment. Hands should be kept out of the water bath after the experiment has started. Make sure a constant temperature is maintained.
After 3 minutes, record to the nearest 0.01 mL the initial position of the water in each pipette. This recording is time 0. Take readings of the water’s position in each pipette every 5 minutes for 20 minutes.
Collect Class Data for Germinating and Non-Germinating Peas at Room Temperature and 10oC.

Analyzing your Data:
Calculate amount of O2 Consumption in mL over time in the following manner:
Determine the total distance the bubble has moved at each time interval by taking the Total Change in Volume for Each Time Interval = (initial reading at time 0) – (reading at time X)
Using the values you obtained for the control respirometer, correct for any changes in volume that you measure that may be attributed to changes in temperature and air pressure.
Corrected Total Change in Volume for Each Time Interval = (Total Change in Volume of Peas at Each Time Interval) – (Total Change in Volume of Beads at Each Time Interval)
Perform appropriate statistical analysis.
Graph your data.

Special Directions for Testing Germinating vs Non-Germinating Seeds at Room Temperature:

Before setting up respirometers, be sure to have water bath at room temperature.
In addition to assembling your germinating peas and beads respirometers, you need to assemble a dry pea respirometer.
Fill graduated cylinder with 50 mL of tap water.
Add 25 dry (non-germinating) peas to the graduated cylinder.
Add bead to graduated cylinder so total volume equals the volume of the germinating peas.
Assemble respirometer as described above, being sure to put beads closest to the cotton and dry peas at the top.
Complete “Equilibrating the Respirometers and Taking Data” as described above.

Special Directions for Testing Germinating vs Non-Germinating Seeds at 10oC:

Before setting up respirometers, add ice to the water bath to achieve and maintain a 10oC temperature.
In addition to assembling your germinating peas and beads respirometers, you need to assemble a dry pea respirometer.
Fill graduated cylinder with 50 mL of tap water.
Add 25 dry (non-germinating) peas to the graduated cylinder.
Add bead to graduated cylinder so total volume equals the volume of the germinating peas.
Assemble respirometer as described above, being sure to put beads closest to the cotton and dry peas at the top.
Complete “Equilibrating the Respirometers and Taking Data”as described above.

Photosynthesis Lab

Background Information:

Photosynthesis is a process that converts the light energy into chemical energy. Plants cannot store light energy as light, but they can store chemical energy in the form of glucose or starch. Plants can then use the stored energy when they need it, or we (and other animals) can use the stored energy when we eat plants.

Light is a part of a continuum of electromagnetic radiation or energy waves. Human eyes respond to a certain range of wavelengths of electromagnetic radiation: we call radiation within this range visible light. The shorter the wavelengths are, the greater the energy of the radiation. Ultraviolet radiation, x-rays, and gamma rays have shorter wavelengths and more energy than visible light. These high-energy radiations can harm living tissues. Electromagnetic radiation with lower energy (longer wavelengths) than visible light are called infrared radiation and radio waves.

Molecules can absorb electromagnetic energy. The energy absorption is specific; a certain molecule will absorb energy of certain wavelengths. Molecules that absorb wavelengths in the visible light range are called pigments. Plant pigment molecules are part of complexes called photosystems, which are found in the chloroplasts of palisade mesophyll spongy mesophyll leaf cells. These chloroplasts have two different pigment systems, photosystem I and photosystem II. Photosystem I contains a specialized chlorophyll amoleculue called p680 (its absorption spectrum peaks at 680 nm).

When light is absorbed by leaf pigments, electrons within each photosystem are boosted to a higher energy level. The energy absorbed by photosystem II excited electrons which reduce NADP+ to NADPH. The high-energy products, ATP and NADPH, are then used in a series of reactions that convert CO2 gas from the atmosphere to glucose.

In this experiment, photosynthesis will be detected by the donation of a high-energy electron to a dye molecule. The dye is 2,6-dichlorophenol-indophenol (DPIP), and will take the place of NADP+ as the electron acceptor in photosynthesis. Unreduced DPIP is blue. When it is reduced by a high energy electron, DPIP changes from blue to colorless. Therefore, photosynthesis can be detected by following the color change of DPIP. In this particular activity, you will extract chloroplasts from spinach leaves and incubate them with DPIP in the presence of light. You will measure the change in the color of the solution over time using a spectrophotometer.

Learning Objectives:

To conduct an experiment that uses a pigment as an electron acceptor and a spectrophotometer to measure the rate of photosynthesis.
To explain why the rate of photosynthesis varies under different environmental conditions.
To connect and apply concepts, including the relationship between cell structure and function (chloroplast); enzymatic activity; oxidation-reduction reactions; and the absorption of visible light by pigments.

Procedures:
The class will test the effect of light and using boiled vsunboiled chloroplasts on the rate of photosynthesis.

Based upon the experiment assigned to you, please write the following information in your lab notebook:

Purpose of your experiment
Independent Variable(s)
Dependent Variable
How you will measure your Dependent Variable
Hypothesis

Procedure for Both Test Groups:

Turn on spectrophotometer to warm up the instrument and set the wavelength to 605 nm by adjusting the wavelength control knob.
Set up an incubation area that includes a light, fish bowl filled with water, and test tube rack. The water in the fish bowl acts as a heat sink by absorbing most of the light’s infrared radiation while having little effect on the light’s visible radiation.
Set-Up Labeled Cuvettes as described in the table below, but do NOT add chloroplasts to CUVETTES 2-5 until you are ready to take readings.
Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts to CUVETTE 1. Cover the top of the cuvette with your thumb and invert two times to mix. Insert CUVETTE 1 into the sample holder and adjust the instrument to 100% transmittance by adjusting the light-control knob. CUVETTE 1 is the blank to be used to recalibrate the instrument between readings. In other words, you will measure the light transmitted through this tube. For each reading, make sure that the cuvettes are inserted into the sample holder so that they face the same way as in the previous reading.
Once you add 3 drops of chloroplasts to your experimental and control cuvettes, immediately take your initial reading for % transmittance. Record the initial reading as time 0.
Take and record additional readings for % transmittance of each experimental/control cuvette at 5, 10, and 15 minutes.
Collect class data for multiple trials.
Perform appropriate statistical analysis and graph your data.

Solutions / AMOUNT OF SOLUTIONS IN EACH CUVETTE
1
Blank
(No DPIP) / 2
Unboiled Chloroplasts Dark / 3
Unboiled Chloroplasts Light / 4
Boiled Chloroplasts / 5
No Chloroplasts
Phosphate Buffer / 1 mL / 1 mL / 1 mL / 1 mL / 1 mL
Distilled Water / 4 mL / 3 mL / 3 mL / 3 mL / 3 mL + 3 drops
DPIP / ---- / 1 mL / 1 mL / 1 mL / 1 mL
Unboiled Chloroplasts / 3 drops / 3 drops / 3 drops / ---- / ----
Boiled Chloroplasts / 3 drops

To make CUVETTE 2 your “Dark” condition, wrap aluminum foil around the walls and bottom of the cuvette. Additionally, make a cover for the cuvette. Every time you take a reading for this cuvette, quickly slide the cuvette out of this sleeve, place in the spectrophotometer, and then immediately return it.