LAB 6

Photosynthesis and
Cellular Respiration

Big Idea 2: Cellular Processes: Energy

and Communication
What factors affect the rate of photosynthesis in living leaves? What factors affect
the rate of cellular respiration in multicellular organisms?

Please be sure you have read the

student intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations Summary

Pre-lab and Questions

Part A: Photosynthesis
What is a product of photosynthesis in a water plant and what factors
increase or decrease the rate of production of this product?
Part B: Cellular Respiration
How does exercise affect disposal of waste products from cellular
respiration?

Lab Investigation 6.1

Part 1 - Floating Leaf Disk

The Floating Leaf Disk Technique for Quantifying Net Oxygen
Production during Photosynthesis
Part 2 - Cellular Respiration
Cellular Respiration Leaf Disk Method Extended to Cellular
Respiration
Part 3 - Student Guided Inquiry
Student Guided Inquiry - Testing a variable that affects the Rate of
Photosynthesis.

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LAB 6 - Photosynthesis and

Cellular Respiration
Big Idea 2: Cellular Processes: Energy and Communication
What factors affect the rate of photosynthesis in living leaves? What factors
affect the rate of cellular respiration in multicellular organisms?

BACKGROUND
Living systems require free energy and matter to maintain order, to grow, and
to reproduce. Organisms use different strategies 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. In multicellular
plants, photosynthesis occurs in the chloroplasts within cells.

Chloroplast Structure
Photosynthesis the Light Reaction.
White light is composed of all the colors present in the visible spectrum. Some
light wavelengths can be used by the photosynthesis pigments present in green
plants to produce sugars. Because there are different pigments present in green
leaves and these pigments absorb different wavelengths of visible light, a large
portion of white light can be used during the light reaction of photosynthesis.

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Photosynthesis Rate (as measured by oxygen production)

Chlorophyll a
Chlorophyll b

Light Absorption

Beta-Carotene

Wavelength of Light (nm)

During the light reaction of photosynthesis, chlorophyll molecules in the
thylakoids of chloroplasts transfer electrons through a series of electron acceptors
to NADP+ (nicotinamide adenine dinucleotide phosphate) and ADP (adenosine
diphosphate). During this process NADP+ is reduced to NADPH (the reduced
form of NADP+) and ADP to ATP (adenosine triphosphate), which then transfers
energy-rich electrons to the Calvin cycle intermediates to eventually produce sugar
(glucose).

Su

nli

gh
t

CO2

ATP

tio
ns

rom

eac

yf

Li
gh
tR

En
er g

O2

H 2O

A DP
NADP+

NAD
PH

ru

c
bis

Calvin Cycle

H 2O

G3P

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The process of photosynthesis occurs in a series of enzymatic reactions that

capture light energy to build energy-rich sugars and store energy in the molecules
ATP and NADPH. The overall process is summarized in the following reaction:
water + carbon dioxide + light sugars + oxygen
6 H2O + 6 CO2 + light C6H12O6 + 6 O2
How can you quantify the rate of photosynthesis? (How many moles of O2 are
produced for every mole of glucose?) To determine the net rate of photosynthesis,
one could measure one of the following:
The production of oxygen - The difficulty in measuring the production of
oxygen from photosynthesis is the simultaneous consumption of O2 in aerobic
respiration.
The consumption of carbon dioxide - Measuring the consumption of CO2
is more generally practiced but equipment and procedures are not usually
available in most labs.
In the following photosynthesis labs, you will practice a technique called floating
leaf disk to indirectly measure the rate of photosynthesis by the accumulation of
oxygen gas. Normally the leaf disks float due to the gases in the spongy mesophyll.
A vacuum is used to remove trapped air in the spongy mesophyll layer of the leaf
and replaced by a solution of sodium bicarbonate ions that serve as a carbon dioxide
source for photosynthesis. The disks will initially sink (since the gases have been
removed by the vacuum). As photosynthesis proceeds, O2 again accumulates in
the air spaces of the spongy mesophyll, and the leaf disks will once again become
buoyant and float. In this way, the rate of photosynthesis can be indirectly measured
by the rate of rise of the leaf disks.
However, cellular respiration is also taking place simultaneously in the
mitochondria and O2 is being consumed. Therefore, the rise of the floating disks
indirectly measures the NET rate of photosynthesis occurring in the leaf tissue.

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PREPARATION
Materials and Equipment are listed with each individual lab.
Timing and Length of Labs

Pre-lab A and B are paper scenarios and do not require lab bench time.

Lab Investigation 6.1

Part 1 - Floating Leaf Technique Practice

Two lab periods are needed for all students to practice the techniques involved
in getting the vacuum and leaf disks to sink; as well as collect data, graph, and
discuss data analysis.
Part 2 - Extension of Photosynthesis Leaf Floating Technique to measure
net rate of cellular respiration
This lab could be piggybacked with Part 1 with results in the dark obtained in
less than 30 minutes. Otherwise, if started from scratch with new floating disks
and then put into the dark, it could take 60-90 minutes to complete.
Part 3 - Student Guided Inquiry Quantitative Net Rate of Photosynthesis
This activity will take a minimum of two lab periods one for design and
discussion and one for conducting the experiment and collecting data. Data analysis
could be done out of class.

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Learning objectives aligned standards and science

practices (SP):
Design and conduct an experiment to explore the effect of certain factors,
including different environmental variables, on the rate of cellular
photosynthesis and cellular respiration (4A2 and SP 6.2).
Connect and apply concepts, including the relationship between cell structure
and function (chloroplasts and mitochondria); strategies for capture, storage,
and use of free energy; diffusion of gases across cell membranes; and the
physical laws pertaining to the properties and behaviors of gases (2A2 and SP
1.4, SP 3.1).
Learn how a respirometer system can be used to measure respiration rates in
plant seeds or small invertebrates, such as insects or earthworms (2A1 and SP
6.1).

General Safety Precautions:

Safety goggles must be worn when working in close proximity to exposed
light bulbs that can easily shatter.
Keep solutions away from the electrical cords of light sources;
If temperature is the chosen variable to test, it is not necessary to heat any
solution beyond 60 C; and
Most but not all syringes are capable of withstanding the vacuum created in this
procedure without failure, however, you should test the syringes beforehand.

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Pre-lab and Questions: Part A Photosynthesis

What factors increase or decrease the rate of production of oxygen gas production
in the photosynthesis reaction in a water plant?
You are going to make predictions of the rate of photosynthesis based on the
amount of oxygen produced at an increased or decreased rate. This can be observed
by counting the number of bubbles released from the plant. Placing a water plant
like Elodea or Anacharis in a cup half full of water and a 0.2% solution of baking
soda (sodium bicarbonate, NaHCO3) gives the plant an instant source of dissolved
carbon dioxide in the water.
Pre-lab Data Table 1: Photosynthesis increase? Slow? None?
1. Original set-up
2. Super intense light
3. Artificial light 10 feet away
4. Natural room light
5. No light (dark)
6. 10% NaHCO3
7. No NaHCO3
8. 5 C
9. 60 C
10. pH 4.0

Increase

Slow or decrease

No gas released

Chart descriptions 1-10:

1. Original set up with bright light, 0.2% sodium bicarbonate
(NaHCO3).
2. Predict how results would differ if the light source was very close
to the plant and super intense;
3. What if light was further away from the plant;
4. What if the light was very dim (no artificial light, just natural
lighting in the room);
5. What if plant was put in the dark.
6. Predict what would happen if the sodium bicarbonate was much
more concentrated (10%);
7. What if no NaHCO3 was used.
8. Predict what would happen if the 0.2% sodium bicarbonate
solution was near freezing temperature (5 C)? or
9 What if the solution was a hotter temperature (no greater than
60 C)?
10. Predict what would happen in an acid solution pH by adding
drops of HCl to the 0.2% sodium bicarbonate water until the
pH was around 4.
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Questions
1. What are the gases involved before and after photosynthesis?

2. What is the gas product of photosynthesis that accumulates on

the leaves of the original set up?

3. What plant organelle carries out photosynthesis?

4. What variable change out of the 9 different scenarios in your

prediction chart increased photosynthesis?

5. Did any of the variables appear to inhibit photosynthesis?

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 6. Was this a quantitative or qualitative experiment? Does counting

bubbles actually measure the amount of gas produced?

In Lab Investigation 6.1 using the floating disk technique, you will practice a
technique that measures the accumulation of oxygen. However, it will be the NET
accumulation since respiration is going on at the same time in the mitochondria
and oxygen is being used up for cellular respiration to convert ATP to ADP and
produce energy.

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Pre-lab Part B: Cellular Respiration

How does exercise affect disposal of waste products from cellular respiration?
Carbon dioxide is one of the products of cellular respiration. Because CO2 is
slightly acidic when dissolved in water, pH indicators such as bromothymol blue
(BTB) can detect its presence.
Cellular Sue obtained a small water bottle of distilled water with 10 drops of
bromothymol blue (BTB). BTB is an indicator that is very sensitive to changes in
pH and turns blue if slightly basic (pH greater than 7.0) and starts turning green to
yellow as pH changes from base to acid (pH value below 7.0).
After blowing into the straw for five minutes, the BTB liquid gradually turned
from blue to green and finally to yellow; demonstrating the production of CO2 from
respiration

Before

10

After

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Questions:
1. After running in place for 1 minute and jumping rope for another
minute, Cellular Sue once again blew her breath through the
straw into a blue-colored solution of BTB. Do you think it took
five minutes for the solution to change color this time? Would
there be more or less CO2 in her breath compared to the first
trial before exercising?

2. How does exercise affect the amount of CO2 produced from

cellular respiration? Where in your cells is ATP being converted
for quick energy and what are the products of this reaction?

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Lab Investigation 6.1:

Part 1 - Floating Leaf Disk

The Floating Leaf Disk Technique for Quantifying Net Oxygen Production
during Photosynthesis.
In this part of the lab, you will practice the floating leaf disk technique to measure
the net rate of photosynthesis by testing a variable that you know affects photosynthesis.
For this technique, the most challenging skill is to get the disk to sink initially.
When immersed in water, oxygen bubbles are usually trapped in the air spaces of
the spongy mesophyll in the plant leaf.

By creating a vacuum in this experimental procedure, the air bubbles can be drawn
out of the spongy mesophyll, and the space is refilled by the surrounding solution.
This allows the leaf disks to sink in the experimental solution.
If the solution has bicarbonate ions and enough light, the leaf disks will begin to
produce sugars and oxygen through the process of photosynthesis. Oxygen collects
in the leaf as photosynthesis progresses, causing the leaf disks to float again. The
length of time it takes for the leaf disks to float again is a measure of the net rate of
photosynthesis.

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Bubbles of O2 Forming

CO2 in Solution

Solution

Floating Disk Technique set-up

Materials:
Baking soda* (sodium bicarbonate - NaHCO3) (1 g)
Liquid soap - Dawn* (approx. 5 mL of dish washing soap in 250 mL of water)
Plastic syringes, 60 cc (2)
Pipets (2)
Living leaves* (spinach, ivy, etc.)
Hole punch*
Clear plastic cups (2)*
Stopwatch
Light source*
*item not included

Procedures:
1. Prepare 300 mL of 0.2% bicarbonate solution for each experiment
(0.2 g/100 mL). What is the purpose of the bicarbonate solution
for the leaf disks while they are in solution (see Pre-lab)?

2. Pour the bicarbonate solution into a clear plastic cup to a depth

of about 3 cm. Label this cup With CO2. Fill a second cup
with only water to be used as a control group. Label this cup
Without CO2. Throughout the rest of the procedure, you will
be preparing material for both cups, so do everything for both
cups simultaneously.

3. Using a pipet, add one drop of a dilute liquid soap solution to

the solution in each cup. It is critical to avoid suds. If either
solution generates suds, then dilute it with more bicarbonate or
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water solution. Why use soap? The soap acts as surfactant or

wetting agent it wets the hydrophobic waxy surface of the
leaf, allowing the solution to be drawn into the leaf and enabling
the leaf disks to sink in the fluid.

4. Using a hole punch, cut 10 or more uniform leaf disks for each
cup. Avoid major leaf veins. The choice of plant material to cut
is perhaps the most critical aspect of this procedure. The leaf
surface should be smooth and not too thick.

5. Draw the gases out of the spongy mesophyll tissue and infiltrate
the leaves with the sodium bicarbonate solution by performing
the following steps:
Remove the piston or plunger from both syringes. Place
the 10 leaf disks into each syringe barrel.
Replace the plunger, but be careful NOT to crush the
leaf disks. Push in the plunger until only a small volume
of air and leaf disk remain in the barrel (less than 10%).

Pull a small volume (5 cc) of sodium bicarbonate plus

soap solution (previously prepared) up into one syringe
and a small volume of water plus soap into the other
syringe. Tap each syringe to suspend the leaf disks in
the solution. Make sure that, with the plunger inverted,
the disks are suspended in the solution. Make sure no
air remains. Move the plunger to get rid of air from the
plunger before you attempt the next step.
You now want to create a vacuum in the plunger to draw
the air out of the leaf tissue. This is the difficult step to
master. Once you learn to do this, you will be able to
complete the entire exercise successfully.
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Create the vacuum by holding a finger over the narrow

syringe opening while drawing back on the plunger (see
above figure). Hold this vacuum for about 10 seconds.
While holding the vacuum, swirl the leaf disks to suspend
them in the solution. Now release the vacuum by letting
the plunger springs back. The solution will infiltrate the
air spaces in the leaf disk, causing the leaf disk to sink in
the syringe (see above figure). You may have to repeat
this procedure two to three times in order to get the disks
to sink.

If the plunger does not spring back, you did not have
a good vacuum, and you may need a different syringe.
If you have difficulty getting your disks to
sink after three tries, it is usually because
there is not enough soap in the solution.
Try adding a few more drops of soap to the
cup and replacing the liquid in the syringe.
Placing the disks under vacuum more than
three times can damage the disks and you
may need to cut new ones.

6. Pour the disks and the solution from the

syringe into the appropriate clear plastic
cup. Disks infiltrated with the bicarbonate
solution go in the With CO2 cup, and
disks infiltrated with the water go into the
Without CO2cup (control).

7. Place both cups under the light source and

start the timer. At the end of each minute,
record the number of floating disks. Then
swirl the disks to dislodge any that stuck
against the side of the cups. Continue until
all of the disks are floating in the cup with
the bicarbonate solution.

Data Table 6.1a: Number of Floating Leaf Disks

Minutes

Cup With
CO2
Cup Without
CO2

10

11

12

13

14

15

ET50

0
0

8. To make comparisons between experiments, a standard point

of reference is needed. Repeated testing of this procedure has
shown that the point at which 50% of the leaf disks are floating
(the median or ET50, the Estimated Time it takes 50% of the
disks to float) is a reliable and repeatable point of reference for
this procedure.
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Analyzing Results
Graph your results in the following format. The point at which 50% of the
leaf disks are floating (the median) is the point of reference for this procedure.
By extrapolating from the graph, the 50% floating point is about 11.5 minutes.
Using the 50% point provides a greater degree of reliability and repeatability for this
procedure. As Steucek, et. al. (1985) described, this terms is referred to as the ET50.
How do your results and your graph compare?

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Number of Disks

10
8
6
4
2
0

5
10
Time in Minutes

15

ET50

1/ET50

One of the problems with the ET50 graph is that it is an inverse relationship to
photosynthesis. In other words, as the ET50 number of minutes goes down, the rate
of photosynthesis goes up. To correct for this representation of data, Steucek, et.
al. 1985 showed a positive slope for photosynthesis with the ET50 by calculating the
inverse or 1/ET50 for his graphs.
Keep this in mind as you analyze your results in the next Lab Investigations
2.2, 2.3.

Rate of Photosythensis
16

Rate of Photosythensis

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Part 2 - Cellular Respiration

BACKGROUND

Living systems require free energy and matter to maintain order, to grow, and
to reproduce. Organisms use different strategies 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.

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Mitochondria are located in both plant and animal eukaryotes. Respiration is

not just a process for animals. Plants consume oxygen and release carbon dioxide
the same way as humans and other animals. Their growth, maintenance and
reproduction rely on respiration as a way of breaking down the molecules produced
during photosynthesis and using them for energy.
Plant cells not only convert energy from the sun into sugars in the chloroplasts,
but they also go through the breakdown of the sugar molecules to produce energy
for growth in the mitochondria. Cellular respiration occurs at all times if the plant
cell has sufficient water and sugars. In many plants, it most often occurs at night.
Cellular respiration is the process that releases energy by breaking down glucose
and other food molecules in the present of oxygen (oxidation). Cellular respiration
requires oxygen, a sugar or other complex food molecules (carbohydrates, lipids)
and gives off CO2, water, and energy. The equation for cellular respiration is:
glucose (sugar) + oxygen gas carbon dioxide gas + water + energy
C6H12O6 + 6 O2(g) 6 CO2(g) + 6 H2O + 686Kcal/mole of glucose
The three main stages of cellular respiration are shown in the following diagram:
Glycolysis, the Krebs Cycle, and Electron Transport Chain. The energy received is
trapped in small packages of ATP. This is sometime referred to as chemiosmosis,
in which the energy available in electrons pump protons across a membrane and
provides the energy for ATP synthesis. You can see from the diagram that about
38 ATP for each glucose molecule oxidized is formed during the cycle.

Illustration by RegisFrey

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During glycolysis, one molecule of 6-carbon glucose sugar is broken in half,

producing two molecules of pyruvic acid, a 3-carbon compound. During the
Krebs cycle, pyruvic acid is broken down further into CO2 in a series of energyextracting reactions. The Krebs cycle generates high-energy electrons that are
passed to NADH and FADH2. The electrons are then passed from those carriers
to the electron transport chain. The electron transport chain uses the high-energy
electrons from the Krebs cycle to convert ADP to ATP, for a grand total of about
36-38 ATP per glucose molecule. These 36-38 ATP molecules represent about
38% of the total energy of glucose. What happens to the remaining 62%? It is
released as heat. Although 38% does not seem like much, the cells are actually
more efficient at using food than the engine of a typical automobile is at burning
gasoline.

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Procedure
Leaf Disk Method Extended to Cellular Respiration
Why do the floating disks, now filled with oxygen a product of
photosynthesis, represent only the net rate of photosynthesis?
What other process is going on in the plant cells in the mitochondria?
What would happen if the floating disks from your photosynthesis lab were
immediately placed in a dark cabinet after all 10 disks started to float? Would
you be able to estimate the rate of cellular respiration using the floating leaf
disk technique?
Try it in this lab. Either continue from Lab Investigation 6.1 Part 1, or start a
new floating leaf technique. Once the 10 leaf disks have all floated, immediately
put them in the dark and continue assessing every minute until all floating disks
have sunk. Make sure to follow the same procedures for recording the number of
floaters each minute and stirring the disks so they do not stick to the sides of the
cup.
1. Graph your results.

2. What is your ET50 for cellular respiration (sinking instead of

floating)?
3. How does it compare to net photosynthesis in the same plant?

4. Describe what is happening in the plant cell when placed in the

dark to cause the floating leaf disks to sink.

Part 3 - Student Guided Inquiry

Testing a variable that affects the Rate of Photosynthesis
During the Pre-lab, you investigated several variables that might influence the
rate of photosynthesis. Based on your findings and questions raised in that lab, you
will now design your own experiment using the floating leaf technique to assess
the net rate of photosynthesis. Here is a list of ideas to consider in the following
categories: environmental variables, plant or leaf variables, and methods variables in
using the floating disk technique:
Environmental Variables: light intensity (brightness), direction of light or angle
of insolation, light colors and different wavelengths, temperature, concentration of
CO2 (sodium bicarbonate), pH (acid or basic);
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Plant or Leaf Variables: leaf colors (amount of chlorophyll), leaf size, stomata
density, stomata distribution, light-starved leaves vs. leaves kept in bright light, type
of plant (house, desert, full-sun, succulent, etc.), leaf age, leaf variegation, role of
respiration and measuring the gross photosynthesis;
Methodology Variables in Floating Disk Technique: size of leaf disk, depth
of CO2 solution, methods of cutting disks, leaf disk overlap, soap amount, repetition
of experiment using the same disks, how long can disks stay sunk (overnight?),
methods of collecting data.

Materials:

One advantage of the leaf floating disk technique is that equipment and supplies
required are inexpensive.
If the experimental design is looking at quantifying light intensity differences,
it is highly recommended that a PAR meter (photosynthetically active radiation) be
used instead of foot-candles (which is more of an outdated, subjective measure of
luminance). A PAR meter counts photons in the PAR spectrum and will greatly
facilitate the experimental design. These can be purchased for around $35.

Procedures:

Design and conduct an experiment to test a variable that affects the Rate of
Photosynthesis.
Step 1:
Chose your driving question to investigate.
Step 2:

Fill in the ExD (Experimental Design) form on the next page

to plan your experiment. Make sure you are only testing one
variable at a time and include a control, identify your independent
and dependent variables.
Step 3:

Design data tables and collect data.

Step 4:

Analyze the data.

Step 5:

Summarize your conclusions.

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Experimental Design (ExD) Form

Complete this ExD pre-lab planning form before beginning your lab
1.

Independent variable: (What is the cause agent? What are you changing?)

2.

Dependent variable: (What is being measured?)

3.

Lab set-up:
Experimental
Groups
Number of Trials

4.

Control: (What is the experimental group being compared to?)

5.
Hypothesis: (Use an if [Independent Variable], then [Dependent Variable] format.
State the cause and effect relationship between the I.V. and the D.V. It must be testable.)

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6.

Lab title: (The effect of ____[I.V.] ____on ____[D.V.]____.)

7.

Experimental constants: (Name at least six variables NOT altered during the experiment.)

8.

Sketch of experimental set up with labels:

9.

Detailed procedure:

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Analysis and Conclusions:

1. Graph your ET50 data.

2. Make a second graph with ET50 (1/ET50) inverted to show a direct

relationship between the ET50 and the rate of photosynthesis.

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Post-lab Connections and Questions

Comparing Photosynthesis and Cellular Respiration

1. How is the energy flow in photosynthesis and cellular respiration
related?

2. Exactly how is the equation for photosynthesis different from

the equation for cellular respiration? Compare their reactants,
products and energy flow.

3. Compare photosynthesis to cellular respiration in terms of

location within the cells, and what types of organisms can
photosynthesize and what types of organisms can oxidize sugars
and other food molecules to produce ATP during cellular
respiration?

4. Compare the function ATP in the light dependent and light

independent(Calvin Cycle) reaction of photosynthesis to the
three main activities of respiration (glycolysis, Krebs Cycle, and
electron transport).

5. What is the difference on a global scale in the atmospheric

contributions of photosynthesis and cellular respiration?

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