Lab Manual - Teacher Edition

College Biology for AP Courses

Lab Manual
Teacher Version

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This content was originally authored through a collaboration with the Texas Education Agency (TEA). It is
presented here with modifications, including updates to align with the 2019 Course and Exam Description
for AP Biology. These resources are available to all verified instructors free of charge at the following
hyperlink: [Link]
TO THE STUDENT: 5
LAB 1: LAB SAFETY, SCIENTIFIC METHOD, AND MEASUREMENTS 10
LAB 2: INTRODUCTION TO MICROSCOPY 26
LAB 3: MACROMOLECULES IN COMMON FOOD 41
LAB 4: PROKARYOTIC AND EUKARYOTIC CELLS 59
LAB 5: SUBCELLULAR STRUCTURES 76
LAB 6: DIFFUSION AND OSMOSIS 96
LAB 7: FACTORS AFFECTING THE ENZYMATIC ACTIVITY OF LACTASE 112
LAB 8: CELLULAR RESPIRATION 130
LAB 9: FERMENTATION IN YEAST 150
LAB 10: PLANT PIGMENTS 162
LAB 11: THE LIGHT REACTION OF PHOTOSYNTHESIS 177
LAB 12: PHOTOSYNTHESIS: THE DARK REACTIONS 194
LAB 13: MITOSIS AND MEIOSIS 211
LAB 14: MENDELIAN GENETICS 228
LAB 15: DNA RESTRICTION ANALYSIS 241
LAB 16: BACTERIAL TRANSFORMATION 256
LAB 17: HARDY-WEINBERG EQUATION 272
LAB 18: NATURAL SELECTION SIMULATION 280
LAB 19: PLANT TRANSPIRATION 289
LAB 20: BIODIVERSITY IN LEAF LITTER 301

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To the Student:
Congratulations on being accepted into, and having the courage to take, an Advanced
Placement biology class! You are about to delve deep into some very detailed biology concepts.
This lab manual aims to help you better understand these concepts through hands-on
experiences in the laboratory. In addition, it will challenge you to critically think about biology
concepts, scientific methods, and experimental design as part of its inquiry-based framework.
Inquiry-based learning involves challenging yourself to learn through self-discovery. Instead of
simply presenting you with facts to memorize, this manual encourages you to ask questions
about the material that you will then answer through your own exploration. By creating your
own hypotheses and then planning and carrying out your own experiments on a variety of
topics in the lab manual, you will hopefully learn biology by satisfying your own curiosity.
In this AP lab manual, the inquiry-based structure includes the following components:
1. Pre-assessment section. This section contains a list of questions that you should answer
before starting each activity. These are meant to get you thinking about the main
concepts of each lab. The pre-assessment questions are designed to connect the
concepts in each lab to your experiences in daily life. Whether you realize it or not, you
observe biology constantly in the world around you. Therefore, you are likely familiar
with more biology topics than you realize! The pre-assessment questions are meant to
tap into the biology knowledge you already have and apply it to what you will learn in
each lab. As a result, your answers to these questions may not be graded and you will
benefit greatly by discussing your answers as a class. This also allows your teacher to
measure how familiar you and your classmates are with the material.

2. Structured Inquiry. In this section, you will be introduced to an experimental system by

doing a well-laid out experiment with detailed steps. This section is meant to guide you
in using the equipment in a “safer” activity before planning and performing an entire
experiment. However, you will still be posing questions, predictions, and hypotheses in
the structured inquiry. You will also critically think about how to achieve the most
accurate and reliable results during the structured inquiry in preparation for creating
your own experiments in the guided inquiry.

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 3. Guided Inquiry. In the guided inquiry, you will use the familiarity you gained during the
structured inquiry to perform your own self-investigation. The experimental setup of the
guided inquiries is often identical to that used in the structured inquiry. Therefore, you
will be working with equipment and methods that you have already tried in the
structured inquiry. This time, you will pick a variable to study, create a hypothesis, and
fully design an experiment to test your hypothesis. You will determine which equipment
and methods you should use to collect accurate and precise data.
Once you have planned your experiment, be sure to have your plan approved by your teacher,
who will also ensure that your plan is safe and appropriate for the equipment available.
Finally, you will analyze your own data and make conclusions based on your experimental
evidence. If time allows, you will then refine and re-run your experiments or test additional
hypotheses that you find interesting. In many ways, the guided inquiry step is meant to engage
you in the same processes that scientists have used to discover information about our world
and universe!

Components of Structured and Guided Inquiry Sections

To ensure that an inquiry-based approach is implemented in each activity, both the structured
and guided inquiries also contain each of the following steps at least once:
Hypothesize/Predict: This is where you will be creating hypotheses, which are questions or
predictions about what will happen during an experiment. Be sure that your hypotheses are
clear, specific, and t
estable.
Good hypothesis: The volume of water in a container will be higher when a 2-gram mass is
added compared to when a 1-gram mass is added.
Poor hypothesis: The volume of water in this experiment will increase as larger objects are
added.
Good hypothesis: The speed of a vehicle traveling down the 30 ramp will be lower than the
speed of the vehicle traveling down the 60 ramp.
Poor hypothesis: The vehicle will travel fast down the ramp with the greater amount of slant.
Student-led Planning: Each inquiry contains at least one step where you and your lab partners
will plan how to properly conduct your experiment. During the Structured Inquiry, you will
generally plan proper techniques for getting the best results possible using the available
equipment and described methods. As with many things in life, two or more heads are often
better than one, and you and your group members should come to a consensus on a plan

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before proceeding. This will lay the groundwork for the Guided Inquiry; you and your group will
need to plan an entire experiment in this step.
Critical Analysis: This step typically occurs near the end of each inquiry. Here you will critically
analyze your results, judge their validity, and explain why your hypotheses were supported or
not supported by your results. You will also suggest ways that your experimental methods could
have been improved to get more accurate or precise data as well as determine new questions to
ask related to your results.

A Note About Your Notebook

As part of the challenge of taking an AP course, this lab manual does not contain data tables
where you record your findings. Therefore, you will be required to design your own tables,
answer assessments, and do any other note-taking in a separate notebook. You should use the
same notebook for biology lab throughout the year. This will allow you to easily refer back to
previous labs when you need to reference earlier content. Do not put non-biology content in
your biology notebook, as your teacher may collect and grade your notebook throughout the
year.

Components of a Lab
Main introduction:
Each lab contains an introductory section under the title. This introduces the “big picture”
concepts of the lab as well as how they connect to everyday life. They will also introduce the
pioneering physicists and experiments that led to our current knowledge of each lab topic.
Relevant equations that you will use in the labs are also introduced here, including definitions of
their variables. Many of the labs involve measuring the value of these variables so that you can
later perform your own calculations. Please read the lab and activity introductions carefully
before your lab period. Then, before the lab starts, ask your teacher about any concepts of
which you are unsure.
In this lab you will learn:
This section presents learning objectives for the lab. These are the “take away points” that you
should be able to explain or perform after doing the activities. It is helpful to read these
objectives before each lab to prime yourself for what you will learn. It is then helpful to reread
these at the end of each lab to ensure that you have achieved all of the learning objectives.
Activities:
Each lab is divided into 2-3 activities. Please note that your teacher may or may not have you
perform all activities in a given lab, so pay close attention to your teacher’s instructions
throughout the lab.

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Safety precautions:
These bullet points list important safety issues that will prevent injury to yourself or your
classmates during the lab activities. Each activity has its own safety precautions section. Please
read and understand all safety precautions before beginning each activity!
For this activity you will need section:
This section lists all of the materials needed for each activity. Before you start the lab, make sure
that you can identify all items on this list. Also, pay close attention to your teacher’s
instructions, as you may be using different equipment for these labs than those on this list.
Activity introduction:
These are short introductions relevant to specific activities. As with the main introduction, the
activity introductions may contain formulas, equations, or other background information
needed to successfully carry out and understand the activities. As with the main introduction,
please read these introductions carefully before your lab period. Then, before the lab starts, ask
your teacher about any concepts of which you are unsure.
Process steps:
These are the steps you will perform to carry out the activities. Please read through all of the
process steps and setup diagrams before starting Step 1. Ask your teacher if there are any steps
you don’t understand prior to starting. This will help you perform the activities correctly the first
time, preventing the need to redo activities or having to leave your laboratory period with
unusable data.
Assessments:
The assessment sections provide questions that test your knowledge of the lab material. Your
teacher will instruct you on how to submit answers to the assessments for grading.
College Board® (CB) Standard Alignment:
College Board® standards are summarized in a table format at the beginning of each lab. The
College Board’s® AP Biology Course and Exam Description was used to provide this information.
In addition, standards tags are found on the assessments, allowing you to quickly identify which
standard is addressed by each question.

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 Lab 1: Lab Safety, Scientific Method, and Measurements
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED.
SCIENCE PRACTICES
Science Practice Skills
SP 2 Visual Representations
2.D Represent relationships within biology molecules,
including mathematical models, diagrams, and flow
Analyze visual representations of biological concepts
charts.
and processes
3.A Identify or pose a testable question based on an
observation, data, or model.
3.B State the null and alternative hypotheses, or
SP 3 Questions and Methods predict the result of an experiment.
3.C Identify experimental procedures that are aligned
Determine scientific questions and methods. to the questions, including variables and controls.
3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E. Propose a new/next investigation.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations.
Perform statistical tests and mathematical calculations
to analyze and interpret data.

Many dangers are associated with working in the lab, so you must be aware of lab
safety. Lab safety is the knowledge of how to safely work in the lab by following safety
procedures. You need to know the location of various kinds of safety equipment, the hazards of
the laboratory, and what to do when you encounter hazards. The Occupational Safety and
Health Administration (OSHA) is a federal agency whose mission is to enforce safety and health
laws, including the handling of chemicals in the lab. Hazards and handling information can be
found on the material safety data sheets for any materials used in a lab. You should also be
aware of good laboratory practices, such as how to properly maintain your lab notebook, which
will help make it easier to write your final report. Maintaining the equipment properly will help
it last longer and remain available to others who will use the equipment after you.
People are naturally curious about the world around them. The scientific method is a
method of inquiry—a way of investigating the world around us. It allows us to consider a
problem or a situation, make observations, ask questions about those observations, and
generate a statement that can be tested about what we believe to be true (a testable
hypothesis). Then, we test the hypothesis using an experiment. The results of the experiment, if
it is well-designed, will either reject or support the hypothesis. Scientists often avoid saying that
a hypothesis or theory is ever proven, as it implies that there is no need for further study.
Therefore, we always refer to hypotheses in this lab manual as being supported by data, not
proven by data.

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 To help us understand the world around us, we have developed standards and tools of
measurement to determine quantities such as mass and volume. Although we use the British
System in the United States, scientists use the International System (SI), or metric system. There
are standards in weight (gram), volume (liter), and length (meter). To convert one measure to
another, there are simple conversion factors, due to the metric system being based on multiples
of ten. An easy way to remember the order of units to determine the conversion factor is to use
this mnemonic (memory) device: King Henry died unusually, drinking chocolate milk. King = Kilo,
Henry = Hecto, Died = Deca, Unusually = Unit, Drinking = Deci, Chocolate = Centi, and Milk =
Milli (see Table 1.1). For example, let’s say a problem asked you to measure the length of a
pencil using a metric ruler, and you found it to be 7 cm. The ruler allows you to measure in
centimeters (cm), but the question needs you to give the answer in meters (m). To convert from
centimeters to meters, you need a conversion factor, such as that shown in Table 1.1. There are
100 cms in a meter. Therefore, the calculation would be

Table 1.1: Conversion Table

King Henry Died Unusually Drinking Chocolate Milk

Unit
Kilo Hecto Deca Deci Centi Milli
Meter (length)
10x10x10x 10x10x 10x 10x 10x10x 10x10x10x
Liter (liquid
LARGER LARGER LARGER SMALLER SMALLER SMALLER
volume)
than a unit than a unit than a unit than a unit than a unit than a unit
Gram
1 kilo = 1 hecto = 1 deca = 10 deci = 100 centi = 1,000 milli =
(mass/weight)
1,000 units 100 units 10 units 1 unit 1 unit 1 unit
1 unit

hm = dam = dm = cm = mm =
hectometer decameter decimeter centimeter millimeter
km = kilometer m = meter
hL = daL = dL = cL = mL =
kL =kiloliter L = liter
hectoliter decaliter deciliter centiliter milliliter
kg = kilogram g = gram
hg = da = dg = cg = mg =
hectogram decagram decigram centigram milligram

You will perform unit conversions during this lab.

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Instructor Introduction Notes
The following are recommendations for introducing this lab’s background content to your
students:
● If your students have not had a lab class in a long time, or if this is their first time being
in a lab, it may be helpful to begin the first lab by discussing the importance of lab safety
prior to showing the lab safety video referenced in the cited link.
● Examples of points to emphasize include common lab safety mistakes that lead to injury,
such as failure to use personal protection equipment including goggles, lab coats, and
gloves while conducting experiments. It would be useful to underscore the importance
of advance preparation by reading the lab material ahead of time to ensure that
students know what to expect for a given class period.
● Additional safety points to consider include the location of items such as fire
extinguishers, fire blankets, fire safety valves, and first-aid kits.
● Upon finishing with the discussion of lab safety, introduce the major content of the lab
for the day by discussing the scientific method and its importance in conducting sound
science. Outline the steps and ask, after each step, why students believe that particular
step is important. This will help you grasp their understanding of the topic before
proceeding.
● A simple diagram such as a flow chart, drawn on the board listing each of the steps of
the scientific method, may also help students visualize how each step of the method
leads to the next.
● Because some students may have issues performing the conversion calculations, a
diagram of a chart outlining the metric system units of measurement and their
corresponding conversion factors should serve as a reference point. This may be
provided to the students separately as a handout. It would be beneficial to perform a
few conversion problems with the students, prior to letting them conduct the lab, to
underscore the importance of this skill in analyzing data and writing lab reports.

In this lab, you will learn

● How to safely work in the lab,
● How to use the scientific method, and
● How to measure weight and volume.

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Activity 1: Pre-Assessment
1. Answer the following question in your notebook. Name one piece of lab safety
equipment. What is it used for?
2. Answer the following question in your notebook. What is one hazard of the lab, and
why is it hazardous?
3. Discuss the answers to questions 1 and 2 with your class.

Activity 1: Lab Safety

Safety Precautions
● Take care as you move around the lab.
● Wear all appropriate safety equipment.
For this activity, you will need the following:
● Access to the internet or a downloaded version of the safety video
● Lab safety equipment
● Lab safety agreement

Activity 1: Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Review the tenets of lab safety with your students. Consider having students handle this
activity individually or in pairs.
● Assist students in locating lab safety items. This may be the first time students have been
in your lab room, so covering the location of safety items is important. You should also
demonstrate the proper usage of each item including the first-aid kit, emergency exits,
shower, fire extinguisher, eyewash, waste containers (broken glass waste), safety glasses
bin, gloves, and any other safety equipment and supplies in the lab.
● Demonstrate for the students how to wear and remove the personal protection
equipment (e.g., goggles, lab coats, gloves). Students should also know where to
properly dispose of lab waste.
● Remind students of the importance of abiding by all safety rules of the lab during each
class period.

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 ● Lead the students in a group discussion on lab safety after the activity to grasp their
understanding of the topic. It may be useful to give a small quiz on the topic to grasp
their understanding of the topic before proceeding.
● Collect the signed lab safety agreements after completion of this activity.
Step 1: Locate the following safety equipment and supplies in the lab: first-aid kit, emergency
exits, shower, fire extinguisher, eye-wash, waste containers (broken-glass waste), safety glasses
bin, gloves, and any other safety equipment and supplies in the lab.
Step 2: Draw a diagram of the lab showing the location of the safety equipment and supplies.
Step 3: Watch the lab safety video, either the one linked here or another of your choosing.
Step 4: Review the personal lab safety equipment shown in Figure 1.1.
Step 5: Record and sign the lab safety agreement, which states: “I have been trained in lab
safety, and I agree to follow the rules of the lab.”

Figure 1.1: Basic personal lab safety equipment.

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Assessments
1. Describe what you would do in each of the following situations. [SP 1, Concept
Explanation; SP 3, Questions and Methods]
a. There is a chemical spill in the lab.
b. A fire starts in the lab.
c. A beaker breaks in the lab.
2. List personal safety equipment needed to work in the lab. [SP 3, Questions and
Methods]
3. Name one rule that you learned from the video. [SP 3, Questions and Methods]

[Answer Key]
1.
a. Alert the teacher to the type of spill.
b. Alert the teacher and use the fire extinguisher to put out the fire.
c. Alert the teacher, clean up the glass with a broom and dustpan, and dispose of
the glass into a broken-glass box.
2. Goggles, gloves, lab coat, long pants, and closed-toe shoes. Other answers can include
long sleeves, hair ties, etc.
3. Answers will vary but could include not eating or drinking in the lab and washing your
hands before leaving the lab.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook. When doing an experiment, why is it
beneficial to alter only one experimental condition at a time?
2. Answer the following question in your notebook. How can you ensure that your
experiment results aren’t influenced by any outside conditions?
3. Discuss the answers to questions 1 and 2 with your class.

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Activity 2: The Scientific Method
A simple experiment will contain two types of variables: independent and dependent.
The independent variable is the variable being tested and manipulated by the experimenter. The
dependent variable is the variable being analyzed (counted or measured) for how it responds to
changes in the independent variable. Experiments also need controls, which are additional
setups that have predictable results. If the controls show the predicted response, this indicates
that the experiment was set up and run properly and that there are no outside factors
influencing the experiment. A positive control confirms that the experiment can detect a
change in the dependent variable when the independent variable changes. Conversely, a
negative control confirms that the experiment does not detect a change in the dependent
variable when the independent variable does not change. For example, consider a test for the
presence of sugar. The test yields a blue color when sugar is present but does not change color
when sugar is absent. A positive control for this test would be to test several sugars and ensure
that they always produce a blue color. A negative control would be to test several non-sugars,
(e.g. water or proteins) and ensure that they never produce a blue color. In this way, positive
and negative controls ensure that the experiment will generate reliable results. However, all
experiments should be done multiple times (replicated) to reduce the influence of human error
when performing them.
Safety Precautions
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
● Wash your hands with soap and water after completion of the activity.
For this activity, you will need the following:
● Water
● Sugar or salt
● Beakers
● Ice
● Hot plate
● Thermometer
For this activity, you will work in pairs.

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Activity 2 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 2:
● Remind students of the importance of lab safety and that they should wear appropriate
clothing and shoes to work in the lab. Additionally, because a hot plate is being used for
this activity, it is important to underscore the importance of the proper use of this piece
of equipment to avoid burn injuries and possible fire hazards.
● Provide students with instructions on the proper handling of mercury thermometers (if
being used in this activity), as well as what to do in the event that one breaks during lab.
● Have extra materials available while prepping for this experiment, as students working in
pairs may require a bucket or other carrying container for their materials (e.g., ice).
● The first part of this activity requires students to examine materials available in the lab
for which they are to design a test for a hypothesis, so have a variety of materials for the
students to use. If any potential safety hazards are associated with the materials, discuss
these hazards with the students prior to letting them conduct the experiments and/or
handle the materials.
● Discussing the proper way to state or format a hypothesis is important to this exercise.
This information could also be reviewed in your initial discussion for the lab. Remind
students of the steps of the scientific method and encourage them to follow these steps
when performing this exercise. Also, remind students that they should have controls for
their experiment and that they should also identify their independent and dependent
variables for their experiments.
The following are recommendations for the Structured Inquiry and for the Guided Inquiry for
Activity 2:
● Students can remain in groups of two while doing the Guided Inquiry project, but you
may wish to have each student write his or her own redesign of the experiment. This is
to ensure that each student contributes equally to each group.
● Check the students’ hypothesis and initial experimental design during the Structured
Inquiry before allowing them to proceed with performing their experiment. This will
help to ensure that they are performing an experiment that can actually be conducted in
your lab, given the available equipment and time you have allotted for this activity.
● Offer students advice on how to determine what controls to use for their experiment as
well as how to identify their independent and dependent variables. Remember that
students may not otherwise include all vital components of an experiment due to their
inexperience.

17
 ● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 4, clarify to students how to analyze their results. Often students don’t
understand the importance of why they must perform experiments multiple times or
have multiple variables and controls. Remind them of the importance of the
reproducibility of data when trying to analyze results for scientific significance. Instruct
students on how to determine if any human error could have been introduced into the
experiment that could have skewed the results. Remind them that they should come up
with a completely new hypothesis based on the results of their first study, if needed.
● Provide a grading rubric for this activity with criteria describing each of the mandatory
parts of this activity to help guide students to include the proper components in their
experiment.

Structured Inquiry
Step 1: Hypothesize/Predict. Examine the materials available in the lab. Think of a question you
could examine about the available materials (e.g., does sugar dissolve faster in warm water than
in ice-cold water?). Formulate a clear, specific hypothesis that you could test during the lab
period. Record your hypothesis in the data table.
Step 2: Student-Led Planning. Design an experiment to test your hypothesis. Set up a data table
in your notebook to record your data. Write out the steps of your experiment, and devise
positive and negative controls. Discuss your plan with your teacher before proceeding.
Step 3: After your teacher approves, implement your experiment and record the data in your
data table.
Step 4: Critical Analysis. Does your data support your hypothesis? Why or why not? What
methods could you use to improve your results? Discuss with your partner, and then write your
answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict. Redesign the experiment you previously performed in the
Structured Inquiry. Create a more specific hypothesis, or create a follow-up hypothesis that you
can test based on the results of the Structured Inquiry.
Step 2: Student-Led Planning. Create a plan for your experiment, including controls and data
tables. As before, your teacher should approve your plan before you start your experiment.
Step 3: After your teacher approves, execute your experiment and record the data in your data
table.

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Step 4: Critical Analysis. Does your data support your hypothesis? Why or why not? How did
you improve your experiment over what you did in the Structured Inquiry? How could you
further improve your results? Discuss with your partner, and then write your answers in your
notebook.

Assessments
1. What is the purpose of the scientific method? [SP 3, Questions and Methods]
2. You observe that plants sitting in one location in your house grow faster than plants
sitting in another location. Create a hypothesis to test why this occurs. Then, describe an
experiment that would test your hypothesis that includes controls. [SP 3, Questions and
Methods]
[Answer Key]
1. The purpose of the scientific method is to understand the world around us using a
systematic method of inquiry.
2. Answers will vary. Sample answer: The plants in the first location grow better because
they have more available light than the plants in the second location. To test this
hypothesis, half of the plants could be switched to the opposite location while the other
plants should remain in their locations to serve as controls during the experiment.

Activity 3: Pre-Assessment
1. Answer the following questions in your notebook. What are the conversions for the
following measurements?
a. 50 ml = ___ L
b. 20 g = ___ mg
c. 700 μl = ___ ml
d. 1 gal = ___ pt
e. 2000 ml = ___ L
2. Answer the following questions in your notebook. Visit the weighing station. What is
the weight of a weighing boat? Some balances have a method of subtracting it by
pressing the “zero” or “tare” button. Weigh three items. Record the results in your
notebook.
3. Discuss the answers to questions 1 and 2 with your class.

19
Activity 3: Measurement
In this lab, you will need to use instruments to make your measurements. There are
instruments to use for each type of substance (liquid or solid). Some of the instruments to
measure volume are graduated cylinders (shown in Figure 1.4) and pipettes (shown in Figure 1.2
and Figure 1.3). Some instruments to measure solids are balances and scales (a scale is shown in
Figure 1.5). You may also need to adjust the instrument or container you are using based on the
amount of substance you need to measure.

Figure 1.2: Serological pipettes are used to measure volumes of 1 to 50 ml.

Figure 1.3: Micropipettes are used for measuring micro quantities of liquid from 1 to 1,000 μl.

20
 Figure 1.4: Graduated cylinders are used to measure volumes from 10 ml to 1 L.

Figure 1.5: A scientific scale is used to find the mass of solids.

Also, when recording your measurements in a data table, it is important to be aware of

significant figures. The number of significant figures is the number of digits needed to express
the accuracy of a calculation. The rules for significant figures are presented in Figure 1.5.

21
 To use these rules, let’s look at an example. An experiment requires you to measure the
weight of 10 marbles. The scale shows a weight of 32.46547 g. All the digits in this
measurement are significant figures. However, in the number 32.465470, how do we know if
the last 0 is significant or not? There are three rules on how to determine significant figures:
● Non-zero digits are always significant.
● Any zeros between two significant digits are significant.
● A final zero or trailing zeros in the decimal portion only are significant.
So, based on this rule, the final 0 in 32.465470 is significant, as is the first zero in 32.046570.
However, the 0 in 0.324657 would not be significant because it is simply a placeholder.
Safety Precautions
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
● Wash your hands with soap and water after completion of the activity.
For this activity, you will need the following:
● A balance
● A weighing boat
● A graduated cylinder
● Weighable objects
For this activity, you will work in pairs.

Activity 3 Instructor Preparation and Teaching Notes

The following are recommendations for the Structured Inquiry for Activity 3:
● Because the first part of this activity requires students to predict the mass of various
objects, have a variety of materials for the students to use. If any potential safety
hazards are associated with the materials, discuss these hazards with the students prior
to letting them conduct the experiments and/or handle the materials.
● Demonstrate the proper technique for using the balance/scale by using a weighing boat
as well as a graduated cylinder and then zeroing the scale prior to filling your chosen
items with a substance. Instruct the students on how to record the data from the scale
in their lab notebooks. This demonstration will help students who have not previously
used the balance/scale.

22
 ● Demonstrate how to use all other required materials, including the micropipette and
pipettes, as well as how to properly read the meniscus of liquid in a graduated cylinder
when performing measurements.
● Perform a few sample calculations using the provided equations in this lab activity. This
will help clarify any inquiries regarding calculations.
● Clarify what significant figures are by providing students with an overview on this topic
as well, prior to letting them conduct their exercises and record their data.
The following are recommendations for the Structured Inquiry and for the Guided Inquiry for
Activity 3:
● Continue to have students work in pairs while doing the Guided Inquiry project. You may
wish to have each student write his or her own hypotheses and calculations separately.
● Check the students’ devised plan before they proceed with determining how to calculate
the density of the objects they are testing.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 5, clarify to students how to analyze their results. Often students don’t
understand the importance of why they must perform experiments multiple times or
have multiple variables or [Link] them of the importance of the
reproducibility of data when trying to analyze results for scientific significance. Instruct
them on how to determine if any human error could have been introduced into the
experiment that could have skewed the results. Remind students that they should come
up with a completely new hypothesis based on the results of their first study, if needed.
● Provide a grading rubric for this activity with criteria describing each of its mandatory
parts to help guide students to include the proper components in their experiment.

Structured Inquiry
Step 1: Create a data table for your measurements, and show your calculations in your
notebook.
Step 2: Hypothesize/Predict. Examine one of the objects. Predict the object’s mass, and write
the predicted mass in your notebook.
Step 3: Student-Led Planning. Listen to your teacher’s instructions on how to use the balance.
Discuss with your partner how you should determine the weight of the object and how many
significant figures your measurement should have. Write your ideas in your notebook.
Step 4: Determine the weight of the object using the scale. Repeat the measurement twice
more, and record all results in your notebook.

23
Step 5: Critical Analysis. How much did your measurements vary among the three trials? Were
you able to accurately measure the number of significant figures stated in Step 3? Why or why
not? Could you have improved your methods to get more accurate measurements or obtain a
greater number of significant figures? Why or why not? Discuss with your partner, and then
write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: Volume is a measure of how much space matter occupies, and it is
used to calculate density (d),

,
where m is the mass of the object and v is the object’s volume. Pick another object available in
your lab and predict whether it has a higher or lower density than the object you used during
the Structured Inquiry. Write your prediction in your notebook.
Step 2: Student-Led Planning: How can you calculate the density of your two objects? Devise a
plan for finding the density of both objects and present it to your teacher for approval. Create
the data tables you need and decide how many times you should measure each object as well
as how many significant figures you will use.
Step 3: After your teacher approves, gather the measurements you need to calculate the
density of both objects. Write your final densities in your notebook.
Step 4: Critical Analysis: How did the object’s size influence its mass and its volume? What
would you have to do to increase the mass, volume, and density of an object? How could you
increase the accuracy of your measurements? Discuss with your partner and then write your
answers in your notebook.

Assessments
1. Why does the weight of the weighing boat need to be subtracted when measuring the
weight of an object sitting in the weighing boat? [SP 5, Statistical Tests and Data
Analysis]
2. What is the most likely instrument used to measure the following volumes? [SP 5,
Statistical Tests and Data Analysis]
a. 200 ml
b. 50 μl
c. 1 L

24
 3. Calculate the density of an object that is 3 g and that displaces water in a graduated
cylinder by 3 ml. [SP 5, Statistical Tests and Data Analysis]
[Answer Key]
1. The weighing boat is not part of the object and therefore should be excluded from the
weight of the object.
2.
a. graduated cylinder
b. 100 μl pipette
c. 1 L graduated cylinder
3. 1 g/ml

25
 Lab 2: Introduction to Microscopy
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED.
REQUIRED COURSE CONTENT
Topic Enduring Understandings Learning Objective Essential Knowledge
[Link]
synthesize protein.
SYI-1.D.2 Ribosomes are
found in all cell types,
reflecting common
ancestry of all known life.
SYI-1.D.3 Endoplasmic
SYI-1.D Describe the reticulum occurs in two
SYI-1 Living systems are structure and/or function forms, smooth and rough.
2.1 Cell Structure: organized in a hierarchy of subcellular SYI-1.D.4 Chemical
Subcellular Components of structural levels that components and modification of newly
interact. organelles. synthesized proteins
occurs in the Golgi, a
series of
membrane-bound sacs.
Other membrane-bound
organelles include
mitochondria,
chloroplasts, lysosomes,
and vacuoles.
SYI-1.E Explain how SYI-1.E.1 Organelles,
SYI-1 Living systems are
subcellular components including the ER,
2.2 Cell Structure and organized in a hierarchy
and organelles work mitochondria, lysosomes,
Function of structural levels that
together to contribute to and vacuoles interact to
interact.
the function of the cell. support cellular function.
ENE-1.B. Explain the
ENE-1 The highly complex ENE 1.B.1 Surface
effect of surface
organization of living area-to-volume ratios
area-to-volume ratios on
2.3 Cell Size systems requires constant affect the ability of cells
the exchange of materials
input of energy and the to exchange chemicals
between cells or
exchange of and energy with the
organisms and the
macromolecules. environment.
environment.

26
 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and models models in applied contexts.
presented in written format.
2.A Describe characteristics of a biological concept,
process, or model represented visually.
SP 2 Visual Representations
2.B Explain relationships between different
characteristics of biological concepts, processes, or
Analyze visual representations of biological concepts
models represented visually in applied contexts.
and processes.
2.D Represent relationships within biological models,
including diagrams.
3.D Make observations, or collect data from
SP 3 Questions and Methods representations of laboratory setups or results.
3.E Propose a new/next investigation based on
Determine scientific questions and methods. evidence from an experiment or an evaluation or the
methods.
SP 6 Argumentation
6.B Support a claim with evidence from biological
Develop and justify scientific arguments using principles, concepts, processes, and/or data.
evidence.

Arguably, the invention of the microscope was a defining moment of biology in the 17th
and 18th centuries. Before its invention, scientists did not have a tool with which to see objects
and structures below the resolution limit of the human eye. The ability to magnify objects
allowed scientists to see the cellular and subcellular structure of animals and plants. Because of
the microscope, scientists were able to develop many foundational theories of biology,
including the cell theory (where all living things are made up of cells) and the germ theory
(where infectious diseases are caused by microscopic organisms).

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Explain with the simple diagram of a magnifying lens how images are magnified. Show
students how to trace rays. Introduce the terms real object, the specimen, and its virtual
image, lens, focus, and light-bending effect of glass. Emphasize that this is a simple
explanation and that a microscope is more complicated because of the number of lenses
in the optical path.
● Lead a discussion on handling the microscope with care stressing that this is an
expensive and delicate instrument. Review that rough handling of the microscope may
result in the misalignment of the optical path and loss of the quality of the images.

27
 ● Review the cell theory. Remind students that, with few notable exceptions, cells’
dimensions fall within a narrow range although living organisms vary widely in sizes. Ask
students, what are the constraints on cell size?
● Ask students for current applications of light microscopy. Students should mention basic
biological research and diagnosis. Remind them that they will use their skills in
microscopy in almost all future biology classes.
● Students may not be aware that microscopy has many applications in fields other than
biology such as material science or inspection of computer chips. Ask students to
suggest other applications. Students will realize that microscopy is a widely used current
technique and not a relic from the past.
● Review the three main factors in microscopy: magnification, contrast, and resolution.
● Explain the concept of total (final) magnification and remind students that all their
drawings should have a title and a final magnification. Don’t allow students to limit
themselves to taking pictures. They may take pictures if they wish to do so, but it is no
substitute for the exercise of sketching one’s own observations.

In this lab, you will learn

● to identify the parts of a microscope
● to effectively use a microscope to conduct observations

Activity 1: Pre-Assessment
Answer the following questions in your notebook.
1. A microscope has two sets of lenses. What do you think is the purpose of each lens?
2. Why do you think scientists use microscopes? Think of useful functions for the
microscope and discuss your ideas with your partner.

Activity 1: Parts of a Microscope

A microscope magnifies the image of an object through a series of lenses. The
condenser lens focuses the light from the microscope’s lamp onto the specimen. The light then
passes through the object and is refracted by the objective lens. The objective lens is the more
powerful lens of a microscope and is closest to the object. The light then travels to the ocular
lens, which focuses the image onto the user’s eye. Usually, the power of the ocular lens is fixed
for a given microscope.

28
 Different microscopes can magnify objects hundreds, thousands, or even millions of
times. Magnification refers to how much larger the image is compared to the original object. As
magnification increases, the image becomes larger. The total magnification is the power of the
objective multiplied by the power of the ocular lenses. Resolution refers to the ability of a
microscope to distinguish two points on the image. As resolution increases, objects that are
closer together appear as separate points. Contrast refers to the ability of a microscope to
distinguish an object from its background. The higher the contrast, the greater the difference in
intensity between an object and the background.
Safety Precautions
● Handle microscopes and lenses with care.
● Do not drop or crush slides. Alert your teacher immediately to any broken glass so it can
be properly disposed of.
For this activity, you will need the following:
● Compound microscope
● Slide with the letter e; if you do not have premade slides, cuttings from a newspaper can
be taped to a slide
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Review good practices in handling a microscope. Students may have received vague
instructions in previous classes and can be reluctant to abandon bad habits.
● Remind students to clear working space for the microscope and the supplies which will
be necessary to perform observations. The microscope should be positioned on a flat
surface with no notebook or other items underneath it.
● Tell students that using a microscope correctly will allow them to work better and faster
in the lab and learn more from their observations. They should not be satisfied with
viewing blurry images with poor contrast.
● Demonstrate proper focusing of the microscope and ask students to write down the
steps as you perform them:
1. Once the slide with the specimen is secured with clamps or stage clips, raise the
stage to its uppermost position. While looking through the ocular, bring the
specimen into focus. Make the point that one should always start at the highest or
lowest position of the stage, but not in a middle position. This approach avoids the
hit-or-miss focusing technique. It is preferable to start with the slide close to the
objective and focus down to avoid hitting the slide. This is especially the case when
working with a higher magnification objective, X400.

29
 2. Explain that a microscope with parfocal lenses will allow them to focus at higher
magnification with little adjustment. The microscope available to them may not
necessarily have parfocal lenses.
3. Tell students that they can move to higher magnification after they are comfortable
working with the low objective lens.

● Stress that the coarse adjustment knob is used with the lower magnification objectives,
typically X4 and X10. Only the fine adjustment knob should be used with high power,
X40. Use of the coarse adjustment knob with a high magnification objective easily
results in smashing the objective on the slide. It will scratch the objective lens and
spread glass shards on the stage of the microscope.
● Stress the importance of bringing down the stage before removing a slide to avoid
bumping into the objective lens.
● Emphasize that only lens paper is to be used. Paper towels or tissues will scratch the
lenses.
Tips for Instructor
● Check students individually to make sure that each can focus a microscope. When
students work in pairs, frequently one student actually does all the focusing.
● Lens tissue and lens cleaner should be readily available. The lens tissue should be
lightly moistened with cleaning fluid. Do not wipe an optical surface with a dry lens
tissue as particles can scratch the lens.
● Focusing the microscope by looking on the side and lowering the objective lens
towards the slide is considered poor practice and should be avoided because it does
not teach students how to bring the specimen in sharp focus.
● There is no need to clean lenses routinely. Clean lenses only if the specimen appears
cloudy or sample contaminates the lens. For example, students may use too much
liquid in a wet mount and cloud the objective lens.
The following are recommendations for the Guided Inquiry for Activity 1:
● Demonstrate refraction to students by asking them to observe a drinking straw in a
glass of water.
● Explain that refraction refers to the bending of a light ray as it crosses a boundary
between two different media. Explain that the magnification of a microscope is due
to the bending of light by the magnifying lenses in the light path. Students may also
be familiar with Snell’s law from physics class, but it is not required to understand
the concept of refraction.
● Ask students: how many boundaries does light typically cross when a wet mount
specimen is observed? They should answer glass, water, and air.
● Ask students why the letter C or the letter A would not have been a good choice to
observe the specimen’s change in orientation under the microscope.

30
Tips
● Have ready diagrams of the light paths through a compound microscope and an
inverted microscope as references.
● Review the paths of light and the changes in the light beam when moving the
parts of the microscope before the lab session.
Misconception of Students
● Students often will notice that a specimen appears upside-down, but not that it
is also flipped left-to-right.

Structured Inquiry
Step 1: When moving a microscope, grasp the neck firmly with one hand and place your other
hand under the microscope’s base. Do not bang the microscope on the desk. Once you have set
down your microscope, turn the revolving nosepiece so that the lowest power objective is
pointed at the stage.
Step 2: Throughout this lab, handle slides by their edges and do not touch the specimen area to
avoid smudges. Place the slide with the letter e on the stage. Make sure it is oriented so that
you can read the letter e correctly with the naked eye while standing at the microscope.
Step 3: You will now observe the slide containing the letter e. Center the specimen on the stage
and look through the ocular lens. Adjust the coarse and fine focus knobs until the letter comes
into focus. Be careful while focusing so that the slide does not touch the lenses.
Step 4: Hypothesize/Predict. From your observations of the letter e, what do you think is the
function of each part of the microscope? Write the function of each in your notebook.
Step 5: Student-Led Planning. Using your textbook and other resources, research each part of
the microscope. Identify each part of the microscope in Figure 2.1. Label each part of the
microscope in Table 2.1. Write the function of each part in Table 2.1.
Step 6: Critical Analysis. You place a sample on the stage of a microscope and you look through
the ocular lens. All you see is black in the field of view. What would you adjust to fix this
problem?
Step 7: Microscope Care and Maintenance. When you are finished using a microscope, make
sure the lowest power objective is in place and take the slide off the stage. Use only lens paper
to wipe the lenses if they are dirty. Support the microscope by the base when carrying it as
described in Step 1. If you are continuing with the other activities in this lab, keep the
microscope out.

31
Guided Inquiry
Step 1: Find the light source on the microscope. Turn on the light source and trace the path of
light from the source to the ocular lens.
Step 2: Hypothesize/Predict. Knowing the function of each part of the microscope, draw how
the light path of the microscope bends and changes as it travels from the light source to the
observer on Figure 2.1.
Step 3: Student-Led Planning. Using your textbook and other resources, research how light
travels through the microscope. Use a piece of paper to block the light path through each
component of the microscope to confirm that it is part of the light path.
Step 4: Critical Analysis. Sometimes, scientists want to study living cells that are kept in dishes.
The dishes are very deep and filled with media. The cells are on the bottom of the plate. In
order to see these cells on the microscope, the microscope has to be inverted so that the
objective lens is below the sample instead of above it. Draw a diagram of how you would design
the light path of an inverted microscope.

Figure 2.1: Can you identify all of the parts of the microscope?

32
Assessments
1. What are three precautions you must take to prevent damage to the microscope? [SP 3,
Questions and Methods]
2. A scientist uses a 40x objective to observe a specimen using a 10x ocular lens. What is
the total magnification of the object? [SP 3, Questions and Methods]
3. A magnifying glass is also used to magnify objects. How is a compound microscope
different from a magnifying glass? [SP 3, Questions and Methods; SP 1, Concept
Explanation]
4. Draw how Figure 2.2 (below) would appear under a compound microscope. [SP 3,
Questions and Methods; SP 2, Visual Representations]

[Answer Key]
1. Always carry the microscope by the base. Always place the slide on the stage with the
lowest objective in place. Always use lens paper to clean the lenses.
2. 400X
3. A compound microscope uses two sets of lenses to magnify the object.
4. Under a typical compound microscope, the fish will appear backwards and upside-down.

33
Activity 2: Pre-Assessment
1. Answer the following question in your notebook. So far, you have only viewed a slide at the
lowest power of the microscope. How do you think the field of view will change as you
move to higher powers on the microscope?
2. Answer the following question in your notebook. The microscope makes small organisms
appear larger so that scientists can more carefully observe them. How can you measure the
size of small organisms on the slide of a microscope?

Activity 2: The Field of View

When you first place a specimen on the microscope’s stage, the image you see in the
ocular lens will likely appear blurry. This is because light rays from the object are not reaching
your eye at the same time. Focusing is used to improve the image. Focusing moves the stage up
and down, which changes the point at which the light rays from the object converge. When the
stage is at a position so that the light rays exactly converge on the retina of your eye, the image
will appear sharp and clear.
When focusing the specimen, you must be careful! The distance between the objective
and the specimen is very small, and it is possible to crush the specimen against the lens if you
move the stage too far. The proper distance between the specimen and the object is called the
working distance.
When you look at a specimen through the microscope’s lenses, you will likely not see the
entire specimen at one time. Because the microscope enlarges the image of the object, only a
small portion of the object will be visible at any given time. The field of view refers to the
portion of the object that is seen through the ocular lenses.
Safety Precautions
● Handle microscopes and lenses with care.
● Do not drop or crush slides.
For this activity, you will need the following:
● Compound microscope
● Slide with the letter e; if you do not have premade slides, cuttings from a newspaper can
be taped to a slide
● Slide with a stage micrometer or a clear millimeter ruler taped across the center of the
slide
For this activity, you will work in pairs.

34
Activity 2 Instructor Preparation and Teaching Notes
The following are recommendations for the Structured Inquiry for Activity 2:
● Explain that a microscope must have good resolution for observation. Compare it to
the familiar notion of poor resolution images or videos on the internet. Optimal
resolution depends both on the quality of the optical lenses and on careful focusing.
● Ask the students to observe how the amount of light entering the objective lens
varies with the magnification of the lens.
● Remind students to lower light intensity when they switch from high magnification
to low magnification because a low magnification lens allows more light in and it will
have a blinding effect.
The following are recommendations for the Guided Inquiry for Activity 2:
● Measure the number of e’s that fit in the field of view at each magnification before class
to have a ballpark figure to compare to students’ results. Allow for some margin of error
in the students’ measurements.
● Ask students to show you and explain all their calculations when comparing the size of
the field of view at different magnifications. Make sure that they understand the relation
between field of view and magnification and don’t just copy another group’s calculation.
● Be prepared to go over the calculation on the board after students have completed their
own calculations.
● Ask students which magnifications are suitable to observe swimming living organisms.
Students should understand that lower magnifications are more suitable because
organisms would rapidly disappear from the field of view.

Structured Inquiry
Step 1: Turn the revolving nosepiece of the microscope so that the lowest power objective is
pointed at the stage.
Step 2: Place the slide with the letter e on the stage. Make sure it is oriented so that you can
read the letter e correctly with the naked eye while standing at the microscope.
Step 3: Hypothesize/Predict. How do you think the letter e will appear under different
magnifications? Draw your predictions in your lab notebook.
Step 4: Student-Led Planning. Change the magnification by turning the revolving nosepiece.
Draw your observations in your lab notebook.
Step 5: Critical Analysis. How do your observations compare to your predictions? Why do you
suppose the letter e looks different under the microscope and at different magnifications?

35
Guided Inquiry
Step 1: Hypothesize/Predict. From your observations of the letter e, hypothesize how large the
field of view is under the low power objective. To do this, measure the letter e, in millimeters,
and then calculate how many letter e’s would fit in the field of view. From that hypothesize the
size of the field of view, in mm. Will the size of the field of view change when you change
magnifications? Based on the change in magnification, hypothesize the size of the field of view
under the high-power objective.
Step 2: Student-Led Planning. With your partner, formulate a plan to measure the field of view
using either the stage micrometer or the slide with the attached ruler. Observe the micrometer
or ruler under the microscope to understand how it works before measuring. Then take three
measurements of the field of view under low power. Repeat under high power. Write your
results in your notebook.
Step 3: Critical Analysis. How did your estimated size of the field of views from Step 1 compare
to the measurements you made using the micrometer or ruler? How does the size of the field of
view change as the magnification changes?

Assessments
1. When you take a picture on your phone, does the field of view get larger or smaller as
you increase the magnification? How does this compare to the microscope? Explain. [SP
3, Questions and Methods; SP 1, Concept Explanation]
2. As you increased in power on the microscope, the image became darker. Why do you
suppose this is so? [SP 3, Questions and Methods; SP 2, Concept Explanation]
[Answer Key]
1. The field of view gets smaller in both the microscope and the camera as you increase the
magnification. This is because less of the image can fit on the screen when the image is
magnified.
2. Because you are looking at a smaller part of the field, less total light enters the lens at
higher powers.

Activity 3: Pre-Assessment
1. Answer the following questions in your notebook. When you focused the slides in the
previous activities, what part of the microscope was moving? Why do you think this
changes the focus?
2. Answer the following questions in your notebook. Review the definition of life. What
are characteristics found in all living things?
3. Discuss the answers to questions 1 and 2 with the class.

36
Activity 3: Depth of Field and Wet Mount of Specimens
As you work with a microscope, you will notice that even when an object is in focus, you
can continue to adjust the focus and see new things in the lenses. This is because most
specimens are very thick. As you move up and down with the stage, different planes of the
object come into focus. The depth of field refers to how thick each plane is. It tells you how
much of a specimen is in focus at any given time. Although you may think that a higher depth of
field is more useful because it allows you to see more objects, most scientists try to make the
depth of field as narrow as possible. This prevents light from other focal planes from interfering
with the observations.
The specimens you use on your microscope can either be wet-mounted or dry-mounted.
A wet-mount refers to living tissues that are placed on a slide with an aqueous solution to keep
them wet. Usually, a coverslip is placed on top of the specimen to flatten the specimen onto the
slide. A dry-mount refers to preserved tissue that has been fixed and stained on a slide. This
technique allows you to preserve specimens for a long time, and it also allows you to add
chemicals to increase the contrast of a specimen from its background.
Safety Precautions
● Handle glass slides with care.
● Dispose of specimens as necessary.
● Dispose of coverslips in a broken-glass container.
For this activity, you will need the following:
● Compound microscope
● Slides with crossed colored fibers or crossed strands of hair
● Clean slides
● Coverslips
● Elodea, onion skin, pond water, or other samples
For this activity, you will work in pairs.

Activity 3 Instructor Preparation and Teaching Notes

The following are recommendations for the Structured Inquiry for Activity 3:
● Remind students to compare depth of field at different magnifications. Students should
notice that the depth of field decreases at higher magnification.
● Ask students how changing focus can add to visualization of specimens because students
may not be aware that many structures lie in different planes.

37
 ● Mention cells in plant vascular tissue and the striations of a skeletal muscle as examples
of structures that do not lie on the same plane and are best observed by changing focus.
If possible, have slides that demonstrate this point.
The following are recommendations the Guided Inquiry for Activity 3:
● Demonstrate the preparation of a wet mount because students may not be able to
visualize the procedure from the written explanation.
● Emphasize the need to avoid the formation of air bubbles. Tell students to ignore a few
small air bubbles on the slide. What they need to avoid are large bubbles which interfere
with observation.
● Demonstrate how to dab excess mounting fluid – water or stain – with a tissue to avoid
contaminating the objective lens.
Tips
● Tell students that they may use the edge of the coverslip or even the contours of an air
bubble to focus the microscope.
● Show students how gently tapping the coverslip with the eraser side of a pencil can
chase air bubbles to the edges of the coverslip in a wet mount.
Misconceptions of Students
● Students may think that the highest amount of light is always best for observation.
● Lowering light intensity improves viewing of living organisms in several ways.
o It increases contrast while viewing unstained specimens and allows observation
of internal structures.
o Some organisms exhibit negative phototaxis and will swim away from the light.
o The heat from the focused light beam often kills organisms and no movement
will be observed.

Structured Inquiry
Step 1: Hypothesize/Predict. Look at the slide with crossed fibers or hair. How do you think you
can use the microscope to determine which fiber is closest to the top of the slide and which
fiber is closest to the bottom?
Step 2: Place the slide with crossed fibers or hair on the stage. Center the slide and focus it.
Step 3: Student-Led Planning. Turn the fine focus knob up and down. With your partner, decide
how to determine which fiber is on top and which fiber is on the bottom of the slide. Record
your results in your notebook.
Step 4: Critical Analysis. Take a look at the slide again without the microscope. With your naked
eye, do you think the results of your analysis make sense?

38
Guided Inquiry
Step 1: Hypothesize/Predict. Based on the microscopy you have performed so far, what would
be the challenges of viewing living organisms under the microscope?
Step 2: Student-Led Planning. You will now observe pond water or samples of living organisms
provided by your teacher. Place a small drop of water from near the bottom of the sample jar
on a slide. Place a cover slip at an angle so that one edge touches the drop. Slowly lower the
coverslip onto the specimen. Avoid trapping air bubbles under the coverslip. Record drawings of
your observations in your lab notebook. Make sure to record the magnification for each
drawing.
Step 3: Critical Analysis: What were some of the differences you observed between your
specimens? What were some of the challenges in viewing living organisms that move under the
microscope? Be prepared to present your findings to the class.

Assessments
1. A microscope can focus on many fields of different depths. However, thick samples have
to be sliced into very thin slices in order to be viewed under the microscope. Why do
you suppose this is the case? [SP 3, Questions and Methods; SP 1, Concept Explanation]
2. Elodea plants appear as solid green objects to the naked eye, but appear mostly clear
under a microscope. Explain this phenomenon. [SP 1, Concept Explanation]
3. A scientist takes a sample from a pond and examines it under their microscope. He
thoroughly examines the wet mount and finds no living thing on the slide. He concludes
that the pond is unable to support life. Is this a valid conclusion? Why or why not? [SP 1,
Concept Explanation; SP 6, Argumentation]
4. Before the cell theory was developed, two previous theories were prevalent at different
historical theories:
a. The Miasma Theory, where diseases were thought to be spread by miasma, a
poisonous, foul-smelling vapor that can travel through the air.
b. The Theory of Spontaneous Generation, where living things could be generated
from non-living matter, such as how maggots seem to appear spontaneously on
meat left out in the open.
Explain how the invention of the microscope would lead to the replacement of both of these
theories with the cell theory. [SP 1, Concept Explanation]

39
[Answer Key]
1. A thick slice lets less light through. A very thick slice will appear completely dark under a
microscope.
2. The resolution of the naked eye is not high enough to distinguish between two
subcellular structures, such as chloroplasts, which are the source of the green pigment.
Under the microscope, we see that the green pigment is enclosed in the chloroplasts
while the rest of the cell is colorless cytoplasm.
3. This is not a valid conclusion. The sample may not be representative of life in the rest of
the pond. Also, there may be organisms that are too small to be resolved with the
scientist’s microscope.
4. The microscope would allow scientists to observe cells from patients affected by a
disease and the changes in the cell’s appearance when the cause of the disease is no
longer present.
a. With a microscope, scientists would be able to analyze infection sites, mucus,
etc. and see that it contains microscopic disease-causing organisms. They could
also analyze putrid air and would find that it also contains these organisms,
showing that it is not the air, itself, that causes disease.
b. With a microscope, scientists could examine a piece of meat left out in the open
and would see fly eggs that hatch into maggots, showing that all organisms can
only appear from other organisms.

40
 Lab 3: Macromolecules in Common Food
This investigation addresses the following required course content and science practices from
the AP CED:
REQUIRED COURSE CONTENT
Topic Enduring Understanding Learning Objective Essential Knowledge
ENE-1.A.1. Organisms
exchange matter with
the environment to
ENE-1 The highly
ENE-1.A Describe the grow, reproduce, and
complex organization of
composition of maintain organization.
living systems requires
1.2 Elements of Life macromolecules ENE-1.A.2. Atoms and
constant input of energy
required by living molecules from the
and the exchange of
organisms. environment, including
macromolecules.
carbon and nitrogen,
are necessary to build
new molecules.
SYI-1.B Describe the
properties of SYI-1.B.1. Hydrolysis
SYI-1 Living systems are
monomers and the and dehydration
1.3 Introduction to organized in a hierarchy
types of bonds that synthesis cleave and
Biological Molecules of structural levels that
connect monomers in form covalent bonds
interact.
biological between monomers.
macromolecules.
SYI-1.B.2 Structure and
SYI-B. Describe the
function of polymers,
properties of
SYI-1 Living systems are including proteins,
monomers and the
1.4 Properties of organized in a hierarchy carbohydrates, and
types of bonds that
Biological Molecules of structural levels that lipids, are derived from
connect monomers in
interact. the way their
biological
monomers are
macromolecules.
assembled.
SYI-1.C Explain how a
SYI-1.C.1 Directionality
SYI-1 Living systems are change in the subunits
1.5 Structure and of the subcomponents
organized in a hierarchy of a polymer may lead
Function of Biological influences structure
of structural levels that to changes in
Macromolecules and function of
interact. structure and function
polymers.
of the macromolecule.

41
 SCIENCE PRACTICES
Science Practice Skills
1.A Describe biological concepts and/or
SP 1 Concept Explanation
processes.
1.B Explain biological concepts and/or processes.
Explain biological concepts, processes, and
1.C Explain biological concepts, processes, and/or
models presented in written format.
models in applied contexts.
2.A Describe characteristics of a biological
concept, process, or model represented visually.
2.B. Explain relationships between different
characteristics of biological concepts, processes,
SP 2 Visual Representations
or models represented visually in both theoretical
and applied contexts.
Analyze visual representations of biological
2.C Explain how biological concepts or processes
concepts and processes.
represented visually relate to larger biological
principles, concepts, processes, or theories.
2.D Represent relationships within biological
molecules, including diagrams.
3.A Identify or pose a testable question.
3.B Predict the results of an experiment.
SP 3 Questions and Methods 3.C Identify experimental procedures that are
aligned to the investigative question.
Determine scientific questions and methods. 3.D Make observations or collect data from
representations or laboratory setups or results.
3.E Propose a new investigation.
SP 4 Representing and Describing Data
4.B Describe data from a table or graph.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.D Use data to evaluate predictions.
Perform statistical tests and mathematical
calculations to analyze and interpret data.
6.A Make a scientific claim.
6.B Support a claim evidence from biological
SP 6 Argumentation principles, concepts, processes, and/or data
6.D Explain the relationship between
Develop and justify scientific arguments using experimental results and larger biological
evidence. concepts, processes, or theories.
6.E Predict the causes or effects of a change in, or
disruption to, components in a biological system.

42
 Macromolecules are important components in all living things, ranging from the smallest
virus to the largest tree. Biologists typically define macromolecules as large, organic molecules
that include carbohydrates, proteins, lipids and nucleic acids. Plants can produce all of these
macromolecules using energy that has been converted from sunlight. The raw materials to build
the building blocks of macromolecules come from simple molecules in the atmosphere and
minerals from the soil. Bacteria, fungi, protists, and animals are not typically capable of
obtaining the building blocks of macromolecules in this way. They must resort to eating other
organisms for food and then rebuild the macromolecules they need within their cells.

Food labels are one way of understanding the types of macromolecules found in foods.
The information on the labels is gathered by performing chemical tests to determine the
presence and quantity of macromolecules in the food. Food processing companies and food
agencies that monitor food use sophisticated equipment to test for macromolecules.
Fortunately, there are a variety of simple macromolecule tests that were developed many years
ago, before the advent of the instruments used today, that you can access. In this lab, you will
use these simple chemical tests to investigate the macromolecular composition of different
foods. Three of these tests are called colorimetric tests because they use a characteristic change
in color to identify the molecules. A fourth test we will use depends on the solubility properties
of a molecule to determine its presence.

So, how do these tests indicate the presence of the molecules you will be looking for?
The Benedict’s solution used to detect glucose contains an inorganic compound called copper
citrate, which appears blue in solution due to the copper ion being in the Cu2+ state. Glucose is
one of many sugars that contain an aldehyde group as part of its chemical structure. The
aldehyde groups in carbohydrates are called reducing agents because they tend to cause
chemical reduction by losing electrons and becoming oxidized. In the presence of copper
citrate, glucose’s aldehyde group reduces Cu2+ into Cu+. Cu+ in the Benedict’s solution then
combines with oxygen to form a brick-red precipitate, as shown in Figure 3.1. No change in color
indicates a negative result (–) and the absence of glucose while the change to brick red indicates
a positive (+) result and the presence of glucose.

Figure 3.1: Reduction of copper by glucose.

43
 The test of polysaccharides is highly specific for starch. It distinguishes starch from other
types of polysaccharides as well as other types of molecules. Much of the starch found in our
foods is a form called amylose, which has what are called alpha 1,4 bonds between the glucose
subunits, as shown in Figure 3.2. In the presence of a potassium iodide solution, hydrogen
bonds in the starch cause the starch to wrap around the electrically charged potassium iodide,
as shown in Figure 3.3. In response to this reaction, the potassium iodide solution changes from
an amber color, which indicates a negative result (–), to a dark blue or black color, which
indicates a positive (+) result.

Figure 3.2: Starch alpha 1, 4 linkages.

Figure 3.3: Starch wrapping around the potassium iodide molecules.

The Biuret test for detecting proteins relies on the ability of Cu2+ to react with the
nitrogen in the peptide bonds of proteins, as shown in Figure 3.4. This can only occur under
alkaline conditions. Biuret solution starts out a light blue and becomes purple in the presence of
protein, meaning that purple indicates a positive test. You will work with two solutions of
protein in order to be able to distinguish between dilute and concentrated levels of protein.

44
 Figure 3.4: Chemistry of Biuret test.

There are several simple tests for detecting lipids. Unfortunately, the more accurate
colorimetric tests use potentially harmful dyes that dissolve into the lipids as a way of indicating
the presence of the lipids. In this test, therefore, you will use a classical food chemistry test that
looks at the ability of lipids to make certain types of paper translucent. The best explanation of
this is that when paper is dry, it reflects and scatters most of the light that falls upon it. Very
little of the light passes through the paper, making it opaque. When lipids come in contact with
paper, they adhere to the cellulose fibers and also fill in the spaces between the fibers. The
lipids then act like a lens that lets most of the light pass through the paper rather than being
reflected and scattered. Water and other liquids can produce a similar effect. However, it is not
as intense and long-lasting as the translucent effect created by lipids. In this experiment you will
look for the presence of lipids in pure vegetable oil and in a mixture of vegetable oil and water,
which will serve as the positive control standards.

45
Instructor Introduction Notes
The following are recommendations for introducing this lab’s background content to your
students:
● Review the structures and functions of the four classes of macromolecules.
● Ask students for examples of simple sugars. Most will mention hexoses such as glucose
and fructose, but not other simple sugars such as glycerol, deoxyribose and ribose.
Remind students that trioses and pentoses are also simple sugars.
● Ask students for examples of polysaccharides. Starch may be the obvious answer.
Remind students that cellulose is one of the most abundant macromolecules in nature.
Explain the role of the chitin in fungi and arthropods.
● Write on the board (or display taped on a slide) a table which lists the macromolecules
and their building blocks.
● Be prepared to review the numbering of the carbon atoms in a simple sugar molecule
because students will be confused by the mention of the alpha 1,4 bond linkage. Explain
what an alpha carbon is.
● Review the formation of the peptide bond between amino acids.
● Explain why the fourth class, the nucleic acids, is not included in this exercise, because
students will be puzzled by its absence. Here is a sample:
o The main examples of nucleic acids are DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid). Single nucleotides serve as important energy carriers in the
cell, the most common one being ATP. Nucleic acids are not large contributors of
nutrients in food. The Dische's diphenylamine test can be used to detect DNA in
a mixture. In this test, a blue complex forms when diphenylamine binds to the
sugar deoxyribose under acidic conditions. RNA does not yield a colored
complex. The acidic Dische phenylamine solution is toxic and should be handled
with extreme care.
● Be prepared to discuss the misconceptions that students have about the nutritional role
of carbohydrates and fats which are considered “food villains” depending on the fad diet
of choice. Remind students about the role of carbohydrates and fats in living organisms.
● Remind students that edibles used in the lab are not to be eaten no matter how
tempting.

In this lab, you will learn

● to detect the presence of macromolecules based on their chemical structures;
● how the macromolecules found in a particular food provide evidence about the
structure of the food;
● how changing the structure of macromolecules affects their functionality.

46
Activity 1: Pre-Assessment
Answer the following question in your notebook:
1. A chemical indicator called Benedict’s solution turns from blue to brick red in the
presence of certain monosaccharides. How would Benedict's test react in the
presence of polysaccharides?
2. A chemical mixture containing copper sulfate undergoes a color change when it
binds to the nitrogen atoms in certain macromolecules. What types of
macromolecules can be detected using this solution?
3. Discuss the answers to questions 1 and 2 with your class.

Activity 1: Detecting Reducing Sugars, Starch, Proteins, and Lipids

The three major macromolecules that make up a bulk of our foods are carbohydrates,
lipids, and proteins. Carbohydrates and proteins often consist of many smaller structural units,
called monomers, linked together into long chains, called polymers. Before investigating the
presence of these macromolecules in food, it is important to first perform what is called a
positive control and negative control standards test for each macromolecule. Your positive
control samples will contain the macromolecules glucose, starch, egg white protein, and
vegetable oil. These samples will provide you with a reference for identifying each of the
representative macromolecules in your food samples. Your negative control will be distilled
water, which should provide results for the absence of the macromolecule in your food sample.

To begin your investigation, you will conduct a sequence of four tests for investigating
carbohydrates. In test 1, you will use a procedure called the Benedict’s test to confirm the
presence of glucose, which is a monosaccharide found in many types of food. For test 2, you will
use a solution of iodine to determine whether the polysaccharide starch is present in a solution.
For test 3, you will use the Biuret test to identify if a solution contains protein. Then, in test 4,
lipids will be detected using a very simple test called the paper spot test.

For this activity, you will work in pairs.

To expedite the lab activity, one team member should carry out the carbohydrate activities
while the other team member does the tests on the lipids and the proteins.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Ask students to prepare a flow of work of the lab before coming to class in order to use
their lab time efficiently.
● Review safety precautions stressing that goggles must be worn at all times, chemicals
must be disposed of in the manner prescribed by the lab guide.
● Remind students that they are supposed to check their procedure with the instructor,
who will circulate in the classroom to check each group.

47
 ● Demonstrate how to mark and read a tube at eye level to avoid parallax errors.
● For Benedict's test, remind students to label the upper part of the tube that is not
immersed in water; otherwise, the writing will come off in the water bath.
● Point out that whenever the instructions mention add, the reagent should be added
drop by drop along the side of the test tube to control the amount of solution added and
to avoid splashes.
● Have students start their experiment by heating water for the Benedict’s test and
periodically measure the temperature. Students should record the temperature at which
Benedict's test is performed.
● Remind students to 1) use a test tube holder when removing test tubes from the hot
water bath, and 2) allow hot plates to cool down after use before moving or touching
them. Hot plates should be turned off when not in use.
● If more than one group shares the beaker of hot water, remind students to number and
add one lab partner’s initials to the test tubes they will add to the water bath.
● Remind students that the Benedict’s test is the only test that requires heat. Students
may be confused and heat all the samples.
● Tell students to handle 10 percent NaOH with extreme care making sure that the Biuret
test reagents are added in the correct order. In case of a spill, they should extensively
wash their hands and alert the instructor. Any spill should be wiped carefully.
● If enough test tubes are available, save the tubes from Activity 1 as positive controls for
Activity 2.
The following are recommendations for the Guided Inquiry for Activity 1:
● Have students write individual lab reports (papers) or detailed notes and conclusions in
their lab notebooks. This is to ensure that each student contributes equally to the
project, especially if students continued with the Guided Inquiry project in pairs. It will
also encourage good lab practices while performing the actual experimental work.
● Remind students that they should submit their choices of food and procedure for
approval before they proceed with research to ensure that they are testable given the
available equipment and time. Some foods contain pigments that may give misleading
results. For example, green pigment may yield a false positive in Benedict's reaction.
● Students often will not include all vital components of a research paper or presentation
due to their inexperience. Provide a grading rubric for papers or presentations to help
guide students to include the proper analyses and critical thinking in their
papers/presentation.
● Provide an experimental set up to extract macromolecules from the foods analyzed. A
pestle and mortar works well to mash most foods. Make sure that students choose food
items that can be easily crushed.
● Ask students about which solvent they will use to extract macromolecules. Distilled
water may be the obvious choice. It will be effective for proteins and carbohydrates.
● Ask students if distilled water will extract lipids. Students will have to crush food samples
directly onto the paper.

48
 ● Remind students that no experiment is valid without a positive and negative control. Ask
them what they will use as controls. The answer should be the tests from the structured
inquiry.
● Ask students to reflect and discuss the qualitative versus quantitative aspect of their
experiments. They should discuss the limitation of their experimental method. Discuss
with the class the possibility of false positive and false negative results. For example, the
green pigment in peas may give a false positive. An incomplete extraction may give a
false negative.
● Discuss with students which components of a balanced diet were not included in their
experiments. Students should mention vitamins, fibers, and minerals.
Safety Precautions
● Safety goggles must be used throughout the duration of the laboratory activity.
● It is highly recommended that you wear rubber gloves and a lab apron when handling
any of the chemicals.
● Immediately inform your teacher of spills containing the test solutions. The acids and
bases in the solutions can harm your skin.
● Immediately inform your teacher of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent other people from slipping.
● Immediately wash your hands if they directly come in contact with the test solutions. It
is important to wash your hands after the laboratory activity to avoid any possible
contamination of other surfaces with chemical residues that might be on your hands.
For this activity, you will need the following:
● Eight test tubes (one test tube per sample being tested)
● 10 ml graduated cylinder
● Grease pencil or washable marker
● One test tube rack
● One test tube holder
● Eight sample droppers or pipettes (do not mix pipettes from one solution to another)
● One thermometer
● Hot plate or microwave
● 250 ml beaker
● Brown paper bag
● Scissors
● Small ruler
● Hair dryer or lamp

49
Chemicals
● Distilled water
● Benedict’s solution
● 1 percent copper sulfate solution or Biuret reagent
● 10 percent sodium hydroxide
● Lugol’s solution or iodine-potassium iodide solution
● 5 percent glucose solution
● 2 percent starch solution
● 1 percent albumin solution or raw liquid egg white
● Vegetable oil

Structured Inquiry
Calibrating the test tubes:
Before beginning this activity, it is important to calibrate your test tubes to ensure
accurate and consistent test results. To do this, you will need the test tube rack, seven test
tubes, the 10 ml graduated cylinder, and a marker or grease pencil. For this laboratory activity, it
is best to calibrate your test tubes in 1 ml increments. The maximum volume you will measure is
5 ml.
1. Collect eight test tubes, a test tube rack, the grease pencil or marker, and a 10 ml
graduated cylinder.
2. Write out a procedure for calibrating your eight test tubes.
3. Get your procedure approved by your teacher before you continue with the calibration.
4. Use the grease pencil or marker provided by your teacher to draw the calibration lines.
5. Explain how estimating the quantities of a solution in an uncalibrated test tube could
affect the results of an experiment. Explain if your study would have been more accurate
if you calibrated your test tube using microliter units to mark your test tube. Record your
answers in your laboratory notebook.
Carbohydrates:
As mentioned earlier, you will test for glucose and starch. In this investigation, explain
which samples are the negative controls and which are the positive controls.
Use the following steps to test for the presence of glucose and starch.
Step 1: Place four clean test tubes in the test tube rack and use the grease pencil or marker to
number each test tube as shown here.

50
Table 3.1
Test Tube Number 1 2 3 4
Distilled Glucose test Distilled Starch test
Test solution
water solution water solution

Step 2: Hypothesis/Prediction. Predict which of the tubes will show a positive result for the
Benedict’s test and which will show a negative result. Write your prediction in a table for
recording the data from the tests.
Step 3: Which tubes represent the positive and negative controls for this experiment? Explain
why in your notebook.
Step 4: Add approximately 150 ml of water into the 250 ml beaker. Use the hot plate or
microwave to heat the water to about 80oC.
Step 5: Add 1 ml of distilled water to test tube 1. Then add 1 ml of the 5 percent glucose
solution to test tube 2.
Step 6: Observe the appearance of each solution and record your observations in your table.
Step 7: Add 2 ml of the Benedict’s solution to test tube 1 and to test tube 2. Observe the color
of each solution and record your observations in your table.
Step 8: Place test tubes 1 and 2 in the beaker of heated water for 5 minutes. If using a hot plate,
turn off the heat after removing the test tubes.
Step 9: Use a test tube holder to remove test tubes 1 and 2 and allow them to cool in the test
tube rack. Observe the color of each solution and record your observations in your data table.
Step 10: Add 1 ml of distilled water to test tube 3. This will be your negative control standard for
the starch test.
Step 11: Add 1 ml of the 2 percent starch solution to test tube 4. This will be your positive
control standard.
Step 12: Observe the appearance of each solution and record your observations in your data
table.
Step 13: Add 8 drops of the Lugol’s solution or potassium iodide solution to test tube 3 and to
test tube 4. Observe the color of each solution and record your observations in your data table.
It may be useful to take photographs of your results.

51
Step 14: Cleanup:
● Empty test tubes 1 and 2 into a container designated for copper wastes or
hazardous wastes.
● Empty test tubes 3 and 4 into a container designated for iodine wastes or
hazardous wastes.
● Rinse out all of the test tubes with soap and water. Make sure to rinse out all of
the soap from the test tubes.
● Dispose of the water from the 250 ml beaker into a sink only after it has cooled.
● Place the test tubes upside down in a test tube rack to dry.
Step 15: Critical Analysis: Discuss with your partner if your results match the expected results
shown in Figure 3.5 and Figure 3.6. What could be some reasons for your results not matching
the expected results in the figures? Explain the role of starch for plants and for the organisms
that consume plants. Why can starchy foods be a problem for people with diabetes?

Figure 3.5: Benedict’s test results.

Figure 3.6: Iodine test results.

52
Proteins:
Step 1: Place three clean test tubes in the test tube rack and use the grease pencil or marker to
number each test tube as shown here.
Table 3.2
Test Tube Number 1 2 3
Concentrated
Distilled Dilute protein
Test solution protein
water solution
solution

Step 2: Hypothesis/Predict: Predict which of the tubes will show a positive result for the Biuret
test and which will show a negative result. Write your prediction in a table for recording the
data from the tests. Which tubes represent the positive and negative controls for this
experiment? Explain why in your notebook.
Step 3: Add 2 ml of distilled water to test tube 1. Then add 1 ml of distilled water and 1 ml of
the 1 percent albumin solution or raw egg white to test tube 2.
Step 4: Add 2 ml of 1 percent albumin solution or raw egg white to test tube 3.
Step 5: Observe the appearance of each solution and record your observations in your data
table.
Step 6: Add 2 ml of the 10 percent sodium hydroxide to each test tube and carefully swirl the
tubes to mix the solution.
Step 7: Add five drops of the 1 percent copper sulfate solution or Biuret reagent to each test
tube. Carefully swirl the tubes to mix the solution.
Step 8: Observe the color of each solution and record your observations in your data table. It
may be useful to take photographs of your results.
Step 9: Cleanup:
● Empty all test tubes into a container designated for copper wastes or hazardous
wastes.
● Rinse out all of the test tubes with soap and water. Make sure to rinse out all of
the soap from the test tubes.
● Place the test tubes upside down in a test tube rack to dry.
Step 10: Discuss with your partner whether your results match the expected results shown in
Figure 3.7. What could be some reasons for your results not matching the expected results?
Explain why the concentration of protein in a food is important in understanding a food’s
nutritional value for animals. Record your answers in your laboratory notebook.

53
 Figure 3.7: Biuret results.

Lipids:
As discussed in the introduction, you will use a strip of brown paper to indicate the
presence of lipids in a solution. In this investigation, explain which samples are the negative
controls and which are the positive controls. Why is it important to perform a negative control
and a positive control before using these tests to analyze foods for lipids? Explain why it is
important to test a lipid sample that is mixed with water. Write your answers in your laboratory
notebook.

Use the following steps to test for the presence of lipids.

Step 1: Design a table for recording the data from the lipid test. You should show your table to
the teacher before proceeding with the activity.
Step 2: Place three clean test tubes in the test tube rack and use the grease pencil or marker to
number each test tube as shown here.
Table 3.3
Test Tube Number 1 2 3
Water mixed
Distilled
Test solution with vegetable Vegetable oil
water
oil

Step 3: Use the scissors to cut a piece of the brown paper bag into a 7 cm × 15 cm rectangle. At
each 5 cm interval along the long side of this rectangle, draw a line using the grease pencil so
that you have three equal boxes. Label the cut sections of the bag according to Figure 3.9.

54
 Figure 3.9: Labeled section of brown paper bag for lipid test.

Step 4: Add 1 ml of distilled water to test tube 1.

Step 5: Add 1 ml of distilled water and 1 ml of vegetable oil to test tube 2.
Step 6: Add 1 ml of vegetable oil to test tube 3.
Step 7: Use a pipette to collect the distilled water from test tube 1. Transfer the liquid to the
brown paper bag. Add enough of the liquid to make a spot 1 cm in diameter on the section of
the brown paper bag labeled Water.
Step 8: Gently swirl test tube number two until the solution is uniform. Quickly, use a pipette to
collect the solution.
Step 9: Add enough of the liquid to make a spot 1 cm in diameter on the section of the brown
paper bag labeled Water mixed with vegetable oil.
Step 10: Use a pipette to collect the vegetable oil from test tube 3. Transfer the liquid to the
brown paper bag. Add enough of the liquid to make a spot 1 cm in diameter on the section of
the brown paper bag labeled Vegetable oil.
Step 11: Record your results in your table. It may be useful to take photographs of your results.
Step 12: Critical Analysis: Discuss with your partner whether your results match the expected
results shown below in Figure 3.10. What could be some reasons for your results not matching
what was determined in the examples given in the figure? Explain the roles of fats in animals
and plants. Why is knowing the lipid content of a food essential for human health? Record your
answers in your laboratory notebook.

55
 Figure 3.10: Paper spot test for lipids.

Step 13: Cleanup:

● Empty all of the test tubes into a laboratory sink.
● Rinse out all of the test tubes with soap and water. Make sure to rinse out all of
the soap from the test tubes.
● Place the test tubes upside down in a test tube rack to dry.
● Dispose of the paper sheet in a trash can.
Guided Inquiry
Step 1: Hypothesize/Predict. Explain the types of nutrient macromolecules you would expect to
find in food grouping 1, which includes potato chips, breakfast cereal, and popcorn. Do the
same assessment for food grouping 2, which includes peanuts, beans, and peas. Make a
hypothesis for which of these food groupings would be more appropriate for a balanced diet.
Feel free to use a food pyramid and other resources to help with your assessment. Write your
ideas in your laboratory notebook.
Step 2: Student-Led Planning. Write out the steps you will need to analyze the macromolecule
content of the potato chips and the peanuts. Select only one food from each grouping.
Remember to keep in mind how you will work safely in the lab and how you will dispose of the
lab wastes. Submit your experimental procedure to your teacher. Once your teacher approves
of your procedure, perform your experiment and record your data in an appropriate format.
Write your results in your laboratory notebook.
Step 3: Critical Analysis. Which of the foods had the best balance of nutrients according to the
United States Department of Agriculture? What about your procedure would you have to
modify to test for the total amount of glucose available in both food groupings once these foods
are processed by a person’s digestive system?

56
Assessments
1. A student needs to calibrate an unmarked beaker in order to mix a food sample into 100
ml of distilled water and 50 ml of dilute acid. The student is provided with a 1 ml pipette,
a 5 ml pipette, a 50 ml graduated cylinder, and a 100 ml flask. The student selects the 5
ml pipette to use in making markings at 50 ml increments on the beaker.
a. Did the student carry out the most accurate way for calibrating the beaker? Why
or why not? [SP 3, Questions and Methods]
b. Which would be the correct apparatus for calibrating a test tube in two 1 ml
increments? [SP 3, Questions and Methods]
2. Starch and glycogen are both composed of 1,4 bonded glucose polymers. Is it possible to
use potassium iodide to detect the presence of glycogen in a food sample? Explain why
or why not. [SP 1, Concept Explanation]
3. Glycoproteins are protein macromolecules that have carbohydrate groups attached to a
polypeptide chain. Which of the tests that you used would likely be for detecting
glycoproteins? Explain why. [SP 1, Concept Explanation; SP 3, Questions and Methods]
4. You need a definitive test to determine if an unknown food substance comes from an
animal or a plant. Which of the four chemical tests would be the best test to distinguish
animal from plant cells? Explain why. [SP 1, Concept Explanation; SP 3, Questions and
Methods]
5. You are doing a study on crop nutrition. The study is investigating the effects of nitrogen
fertilizer concentration on the protein content of plants. Design a simple experiment for
carrying out this investigation. [SP 3, Questions and Methods]
6. Use the image of Benedict's test (Figure 3.11) to help you design an experiment to
compare the glucose levels of different foods. [SP 3, Questions and Methods; SP 2,
Visual Representations]

Figure 3.11: Benedict’s test results for glucose concentration.

57
[Answer Key]
1.
a. No, the student should have selected the 50 ml graduated cylinder and marked
off the beaker in 50 ml increments.
b. The student should select the 1 ml pipette to calibrate the test tube into 1 ml
increments.
2. The specific arrangement of glucose molecules that characterizes starch causes the
starch to wrap tightly around the potassium iodide. This tight wrapping causes the
potassium iodide to turn blue or black. Glycogen may have enough similarity to starch
that it may partly wrap around potassium iodide. In turn, glycogen may show a partially
positive test.
3. It is more probable that the protein component of the glycoprotein has the same types
of peptide bonds as other proteins. The carbohydrate component may or may not have
the 1,4 bonds needed for the potassium iodide test to work. Therefore, is it more likely
that the Biuret test would be the best test to use to detect the glycoproteins.
4. Unlike animals, plants contain starch, which is exclusively detectable with the potassium
iodide test.
5. The Biuret test should be used to detect the protein in the plant samples, which were
given different levels of nitrogen. In order to get accurate results, you would have to
standardize the Biuret test so that low levels and high levels of protein appear in the
test.
6. Students have to make sure the mass of each food is equal and the volumes of the
reagents are each equivalent to ensure that the glucose per gram measurements of the
foods are consistent.

58
 Lab 4: Prokaryotic and Eukaryotic Cells
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED.
REQUIRED COURSE CONTENT
Topic Enduring Understanding Learning Objective Essential Knowledge
SYI-1.D.1 Ribosomes
synthesize proteins
according to mRNA
sequence.
SYI-1.D.2 Ribosomes are
found in all cells,
reflecting common
ancestry of all known life.
SYI-1.D.3 Rough
endoplasmic reticulum
compartmentalizes the
cell and is associated with
SYI-1.D Describe the
SYI-1 Living systems are membrane-bound
structure and/or function
2.1 Cell Structure: organized in a hierarchy ribosomes in eukaryotic
of subcellular
Subcellular Components of structural levels that cells, whereas
components and
interact. detoxification and lipid
organelles.
synthesis occurs in
smooth ER
SYI-1.D.4 Modification
and packaging of newly
synthesized proteins
occur in the flattened
membranous sacs of the
Golgi. Other organelles
include mitochondria,
lysosomes, vacuoles, and
chloroplasts.

SYI-1.E.1 Organelles that

interact to support cell
SYI-1.E Explain how function include ER,
mitochondria, lysosomes,
subcellular components
and vacuoles.
and organelles contribute SYI-1.F.1 The folding of the
SYI-1 Living systems are to the function of the cell. inner membrane increases
2.2 Cell Structure and organized in a hierarchy the surface area, which
Function of structural levels that SYI-1.F Describe the allows for more ATP to be
interact. structure features of a cell synthesized.
that allow organisms to SYI-1.F.3 to SYI-1.F.9 The
processes of photosynthesis
capture, store, and use
and cellular respiration occur
energy in the chloroplasts and
mitochondria, respectively.

59
 ENE-1 The highly ENE-1.B Explain the
complex organization effect of surface
of living systems area-to-volume ratios
2.3 Cell Size requires constant input on the exchange of
of energy and the materials between cells
exchange of or organisms and the
macromolecules. environment.
EVO-1.A.1
Membrane-bound
organelles evolved
from once free-living
prokaryotic cells via
EVO-1 Evolution is EVO-1.A Describe endosymbiosis.
characterized by a similarities and/or EVO-1.A.2 Prokaryotes
change in the genetic differences in generally lack internal
2.11 Origins of Cell
makeup of a population compartmentalization membrane-bound
Compartmentalization
over time and is between prokaryotic organelles but have
supported by multiple and eukaryotic cells. internal regions with
lines of evidence. specialized structures
and functions.
EVO-1.A.3 Eukaryotic
cells maintain internal
membranes that
partition the cell into
specialized regions.
EVO2-2.B.1 Many
fundamental molecular
and cellular features
EVO-2.B Describe the
and processes are
fundamental molecular
EVO-2 Organisms are conserved across
and cellular features
7.6 Evidence of linked by lines of organisms.
shared across all
Evolution descent from common EVO2-2.B.2 Structural
domains of life, which
ancestry. and functional
provide evidence for
evidence supports the
common ancestry.
relatedness of
organisms in all
domains.

60
 SCIENCE PRACTICES
Science Practice Skills
1.A Describe biological concepts and/or
SP 1 Concept Explanation processes.
1.B Explain biological concepts and/or
Explain biological concepts, processes, and processes.
models presented in written format. 1.C Explain biological concepts, processes,
and/or models in applied contexts.
2.A Describe characteristics of a biological
concept, process, or model represented
visually.
SP 2 Visual Representations
2.B Explain relationship between different
characteristics of biological concepts,
Analyze visual representations of biological
processes, or models represented visually in
concepts and processes.
applied contexts.
2.D Represent relationships within biological
models, including diagrams.
SP 3 Questions and Methods 3.D Make observations, or collect data from
representations of laboratory setups or
Determine scientific questions and methods. results.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, such
Perform statistical tests and mathematical as ratios.
calculations to analyze and interpret data.
6.A Make a scientific claim.
SP 6 Argumentation 6.B Support a claim with evidence from
biological principles, concepts, processes,
Develop and justify scientific arguments using and/or data.
evidence. 6.C Provide reasoning to justify a claim by
connecting evidence to biological theories.

Cell theory states that all cells come from preexisting cells and that cells are the basic
structure of living organisms. The subcellular structures within cells provide all the necessary
machinery for life. An organism can be single-celled or multicellular. The human body contains
trillions of cells. The latest research shows that you have as many bacteria in you as your own
human cells. You have probably spent time in the past making lists of the parts of cells, but what
will help you identify which type of cell you are looking at under a microscope?
All cells contain DNA, RNA, proteins, and a plasma membrane. There are two basic types
of cells: prokaryotic and eukaryotic. Prokaryotic cells are simpler, do not contain internal
membranes, and tend to be small (1 to 10 µm). Generally, prokaryotes are single-celled
organisms, although recent studies suggest that prokaryotes can be multicellular (see
References). There are two domains within prokaryotes: archaea and bacteria. Bacteria have

61
cell walls, which include the molecule peptidoglycan. Eukaryotic cells are more complex. DNA is
enclosed in a membrane-bound structure called a nucleus. Eukaryotic cells also contain
organelles (membrane-bound compartments), and tend to be larger (generally, 10 to 100 µm,
with some egg cells large enough to be visible to the human eye). See Figure 4.1 for an
illustration of the relative sizes of cells.

Figure 4.1: This figure shows relative sizes of microbes on a logarithmic scale. Recall that each unit of increase in
the logarithmic scale represents a 10-fold increase in the quantity being measured.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Show diagrams of bacterial, plant, and animal cells so that students can compare and
contrast each cell structure.
● Provide a scale diagram that compares the size of a typical bacterial cell to the size of a
typical eukaryotic cell to help students visualize differences in cell size.

62
 ● Show micrographs of different cells so that students can see how cell structure varies.
Bacterial micrographs could include coccus (spherical or round), bacillus (rod-shaped),
and spiral (corkscrew-shaped) bacteria. A plant micrograph could be a cross section of a
leaf. Point out that kidney-shaped guard cells, which differ in structure from other cells
of the epidermis, allow air to travel in and out of the leaf. Animal cell micrographs could
include nerve cells. Point out that the long extensions of the nerve cells allow
communications to travel from one part of the body to another.
● Ask students to think about how they could determine if an unknown cell is a bacterial
cell, plant cell, or animal cell. Then show the students one example of each cell type and
ask them to identify what type it is.

In this lab, you will learn

● how to make wet mounts of bacteria, plant and animal cells and view them under the
microscope
● how to observe and identify differences between cells and cell structures under low and
high magnification and record observations
● how and why microscope stains are used when viewing cells under the microscope

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: What is the difference between the
cells of a bacterium and the cells of your own body?
2. Answer the following question in your notebook: Compare and contrast the structures
of prokaryotic and eukaryotic cells.
3. Discuss the answers to questions 1 and 2 with a partner and then the class.

Activity 1: Observation of Anabaena or Nostoc

Prokaryotes, unicellular organisms lacking a nucleus, include cyanobacteria (formerly
blue-green algae; this name is now considered inaccurate because algae are eukaryotes).
Cyanobacteria, like those shown in Figure 4.2, are photoautotrophs—organisms that carry out
photosynthesis by using light energy, water, and carbon dioxide from the air and converting to
sugars, and providing oxygen to the atmosphere as a waste product. Cyanobacteria contain
pigments capable of capturing light energy but do not contain chloroplasts. Cyanobacteria are
single-celled organisms, but some can form colonies with differentiated cell types. For example,
some species can form specialized cells called heterocysts—structures containing enzymes
which can take atmospheric nitrogen (nitrogen fixation) from the air and convert into usable
molecules for DNA, RNA, and protein synthesis. Nitrogen fixation involves converting nitrogen
gas (N2) from the atmosphere to ammonia (NH3). Ammonia is a form of nitrogen that can be
used to build other molecules, including DNA, RNA, and proteins. Oxygen, a waste product of

63
photosynthesis, interferes with a key enzyme in nitrogen fixation. Thus, only a few cells in a
colony (about 1 in 10) become heterocysts. Resources, such as ammonia and sugars from
photosynthesis, are then shared between cells. These cells range from 1–40 micrometers in size.
Not all bacteria can carry out photosynthesis; there are many species of heterotrophic bacteria
living virtually everywhere on Earth. These cells are much smaller ranging from 0.5–8
micrometers. Eukaryotic cells have many more features and organelles and range in size from
10–500 micrometers.

Figure 4.2: Cyanobacteria Nostoc with larger heterocysts.

Safety Precautions
● Be careful when handling glass slides, as the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Wash your hands with soap and water after handling live organisms.
For this activity, you will need the following:
● Light compound microscope (10x, 40x, 100x)
● Lens paper
● Prepared slide of Anabaena or Nostoc or images of Anabaena or Nostoc
● Special slide with micron ruler or clear millimeter ruler (you can photocopy ruler on
overhead transparency, then cut and tape to microscope slide)
For this activity, students will work in pairs.

64
Activity 1 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 1:
● Explain that slides should be handled at the edge only so they do not get smudged with
fingerprints. Remind students to be careful when handling glass slides and to inform you
immediately if glass breaks. Be sure that students know how to dispose of broken glass.
● Explain that a microscope is delicate and must be handled with care. Tell students to use
two hands to carry the microscope and to use only lens paper to clean the lenses.
● Identify the parts of the microscope, including the stage, coarse focus knob, fine focus
knob, nosepiece, objective lenses and ocular lens. Show students how to adjust the
amount of light that enters the microscope and how to change the objective lens. Tell
students to watch carefully as they adjust the magnification so they do not strike the
lens against the slide.
● Explain that magnification can be calculated by multiplying the magnification of the
ocular lens by the magnification of the objective lens. Have students calculate all
possible magnifications for the scopes they are using.
● Explain how to focus the microscope. Initially, adjust the focus using the coarse focus
knob with the low power (10x) objective lens in place. Once the object is visible, use the
fine adjustment knob to sharpen the focus. To increase magnification, switch to the
medium power lens and use the fine focus knob to adjust the focus. Repeat this process
to switch to the high-power lens. Make students aware that they must never use the
coarse adjustment knob when focusing at medium or high magnification. If students
cannot see the object they are trying to focus at high magnification, they will need to
switch back to the low power lens and start over.
● Show students what an air bubble or a piece of lint looks like as students can mistake
these for cells. Have students tell you when they think they have the cells in focus so you
can confirm that they are actually looking at cells. Make sure students know how to
calculate the magnification and to estimate the cell size.

Structured Inquiry
Step 1: Estimate the size of the field of view at all the magnifications of your microscope by
placing a clear millimeter ruler on the stage of the microscope. This will allow you to estimate
the sizes of cells. Convert your millimeter estimates to micrometers for this activity.

65
 Figure 4.3: Example of a millimeter ruler taped to a microscope slide.

Step 2: Hypothesize/Predict. In your notebook, predict (draw) what you would expect to see
through the microscope. How big do you predict the cells will be? What features do you expect
to see? Do you expect to see organelles or a cell wall?
Step 3: In your notebook, create a detailed drawing, with a sharp pencil, of the structure of the
cyanobacterium. An example of a detailed drawing is seen in Figure 4.4. Record the estimated
size of the cells at the magnification used. Use color in your drawings if appropriate. Identify the
colors used and label any obvious structures. Note the shapes or organization of the cells.

Figure 4.4: Example of a student drawing including labels and magnification.

Step 4: Critical Analysis: Think about the cell types you observed. Do your observations match
your expectations? For example, given that the cyanobacteria is photosynthetic, was it the color
you expected? Why? Did you expect the cells to have organelles? Did they? Did they have a cell
wall? Did you find heterocysts? Discuss with your partner and write your answers in your
notebook.

Assessments
1. Create a diagram of a general prokaryotic cell and a general eukaryotic cell. Label the cell
structures that differ between the two cell types. [SP 2, Visual Representations]

66
 2. How would internal membrane-bound structures, such as chloroplasts and
mitochondria, allow chemical reactions to occur more efficiently in cells? [SP 1, Concept
Explanation]
[Answer Key]
1. The main differences between the two diagrams should be that the eukaryotic cell
should contain membrane-bound organelles, such as chloroplasts, mitochondria, etc., as
well as a nucleus. The prokaryotic cell should not contain any of these organelles.
2. Internal membrane-bound structures can create isolated compartments for chemical
reactions. Within these compartments, the cells can create ideal conditions for the
chemical reactions, allowing them to proceed at a faster rate.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: What new structures would you
observe in Elodea cells which are not present in a cyanobacterium cell?
2. Answer the following question in your notebook: Which of those structures would you
expect to observe in an onion skin cell? Can you explain why some structures will be
present in an Elodea cell but not in an onion epidermal cell?
3. Discuss the answers to questions 1 and 2 with a partner and with the class.

67
Activity 2: Comparing Plant Cells
Plant cells are eukaryotic; they have subcellular organelles. Like the bacteria, they have a
cell wall to help keep the cell rigid—in plants, the cell wall is composed of a complex
carbohydrate called cellulose. Plant cells, like that shown in Figure 4.5, also have a nucleus with
DNA and a large central vacuole full of water and other important substances for maintaining
life (such as carbohydrates, non-nutrients, and wastes) and cell pressure.

Figure 4.5: Structures found in a typical plant cell.

Safety Precautions
● Be careful when handling glass slides, the edges may be sharp.
● Dispose of used cover slips in a glass disposal box.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Wash your hands with soap and water after handling live organisms.
For this activity, you will need the following:
● Elodea anacharis, moss or Spirogyra green algae
● Light compound microscope
● Clean microscope slides and cover slips
● Lens paper

68
 ● Water and dropper
● Yellow onion
● Forceps
● Iodine solution (optional)
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Notes

The following are recommendations for the Guided Inquiry for Activity 2:
● Explain that leaf sections must be very thin or not enough light will pass through for
students to see the cells. Explain that to prepare the slide, students should lay the leaf
flat and add a drop of water. Then lay the cover slip over the liquid. Air bubbles can be
removed by carefully pressing down on the edge of the cover slip. Explain that even in a
very thinly sliced leaf, multiple cell layers will be present. The lens can be adjusted to
focus on different layers.
● Ask students to note the color of the unstained cells and to think about why the color of
Elodea leaves is different from that of onion leaves (Elodea leaves are photosynthetic
and contain green pigment.)
● Explain that a drop of dye can be added at the edge of the cover slip while the slide is
still on the stage. Ask students to think about why the dye is used, and to predict what
structures will be visible in the absence and presence of dye.
● Students that have difficulty preparing a good slide can be directed to look at a better
slide prepared by a different pair of students, or consult another group to determine
how to re-prepare the slide.

Guided Inquiry
Step 1: Hypothesize/Predict. What features do you expect to see in the plant cell Elodea and
the onion skin under low and high magnification? Draw and label your prediction in your
notebook.
Step 2: Student-led Planning. Prepare wet mounts of Elodea and the onion skin. View with
water only, and again in iodine solution (biological stain). Record your observations as drawings.
Use color if present, label the magnification, and estimate the size of the cells in your notebook.
Each partner is expected to prepare one sample. Each person should view, draw, state the size
and magnification, and label each sample.
Step 3: Critical Analysis. What differences do you expect to see between Elodea and the onion?
What similarities? If you used iodine as a stain, did that reveal any other differences in either

69
plant? Why would these organisms have anything in common based on your prediction in Step
1?

Assessments
1. Were the features you predicted to see in Elodea cells and the onion cell visible at low and
high magnification? Which structures in Figure 4.5 can you identify in the Elodea cells? What
about the onion cells? Why do you think this is the case? [SP 2, Visual Representations; SP 6,
Argumentation]
2. What are the similarities and differences between cyanobacterium and a plant cell? [SP 1,
Concept Explanation]
[Answer Key]
1. In the Elodea, you can clearly see the cell walls, and probably see cytoplasmic streaming of
the chloroplasts around the central vacuole. The nucleus is not obvious. In the onion cell,
the cell walls and central vacuole are visible, but there are no chloroplasts. This part of the
onion is not exposed to sunlight and does not have active chloroplasts, so it is not green.
Sometimes you can see the nucleus as a circular object. The nucleus is much more visible
when an iodine solution is added to the onion.
2. Prokaryotic cells, like those of cyanobacteria, are smaller than eukaryotic cells, like those
of plants. In Elodea, the obvious chloroplasts are membrane-bound organelles, which
prokaryotic cells lack. In addition, the nucleus visible in the onion cells is not present in
prokaryotic cells like the cyanobacteria.

Activity 3: Pre-Assessment
1. Answer the following question in your notebook: How do plant cells and animal cells
differ? Why would these differences likely evolve in plant and animal cells?
2. Answer the following question in your notebook: What microscope techniques could
help us see more structures within cells?
3. Discuss the answers to questions 1 and 2 with a partner and then the class.

70
Activity 3: Observe Animal Cells and Identify their Components
Animal cells are eukaryotic and possess subcellular components in common with the
plant cells you observed in Activity 2. Organelles that plant and animal cells share in common
include the nucleus, Golgi apparatus, mitochondria, ribosomes, and the endoplasmic
reticulum. These are all participants in protein synthesis. An illustration of an animal cell is
shown in Figure 4.6. There are some exceptions to these general components. For example,
mature red blood cells (RBC) which have ejected their nuclei to have more room for
hemoglobin, the protein that carries oxygen around the body. One of the easiest eukaryotic
cells to obtain in the lab is the squamous epithelial cell (your cheek cells). These cells are
arranged in a flat layer and are easy to remove and observe.

Figure 4.6: Diagram of the parts of an animal cell.

Safety Precautions
● Be careful when handling glass slides, as the edges may be sharp.
● Dispose of used cover slips in a glass disposal box.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.

71
 ● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Used cotton swabs are considered biohazard; dispose of swabs in the biohazard trash
container as soon as you have used them.
● Methylene blue is a dye; be cautious not to ingest methylene blue.
● Wash your hands with soap and water after handling live organisms.
For this activity, you will need the following:
● Prepared slide of red blood cells
● Light compound microscope
● Clean microscope slide, cover slip
● Clean cotton swab
● 0.5–1 percent methylene blue solution
● Dropper or pipette
● Small squares of paper towels
For this activity, you will work in pairs.

Activity 3 Instructor Preparation and Teaching Notes

The following are recommendations for the Guided Inquiry for Activity 3:
● Remind students to handle the dye with care and to be careful with broken glass.
● Tell students to carefully wash their hands after preparing the cheek cell slides;
otherwise they could spread germs.
● Ask students to think about how the color and shape of animal cells differs from that of
bacterial cells and plant cells.
● Remind students that only lens paper can be used to clean the lenses and to use two
hands to put the scope away.

Guided Inquiry
Step 1: Hypothesize/Predict. Predict the different features you expect to see in the animal cell
versus the plant cell. Predict the differences you will see between animal cells and prokaryotic
cells under low and high magnification. Include in your prediction the size differences between
a cyanobacterium, plant cells, and animal cells. Create a table in your notebook to draw and
label your predictions in your notebook.

72
Step 2: Student-led Planning. Observe the red blood cell prepared slide. Record your
observations (draw and label any visible parts, use color if visible, include magnification and
size of cells) in your notebook. Both partners should view, draw, state the size and
magnification, and label each sample.
Step 3: Prepare your cheek cell slides as shown in Figure 4.7 and Figure 4.8 and outlined
below.
a. Take a clean cotton swab and gently scrape the inside of your mouth.
b. Smear the cotton swab on the center of the microscope slide for 2 to 3 seconds.
c. Add a drop of methylene blue solution (a dye) and place a coverslip on top.
d. Remove any excess solution by allowing a paper towel to touch one side of the
coverslip.
e. View the slide at all magnifications.
f. Record your observations as drawings. Use color if present, label the magnification,
and estimate the size of the cells in your notebook. Record your observations
(drawings, color if present, labels, magnification, and size of cell) in your notebook.

Figure 4.7: How to prepare a cheek swab.

Figure 4.8: How to stain cheek cells with methylene blue dye.

Step 4: Critical Analysis. Were differences observed between the RBC and the cheek cell? What
does the methylene blue stain reveal in the cheek cell? There should be small blue dots visible
on the cheek cells much smaller than the nuclei. Hypothesize what those blue dots might be?

73
How does the animal cell compare to the plant cells in Activity 2 and the cyanobacteria in
Activity 1? Record the answers to these questions in your notebook.

Figure 4.9: Cheek cells stained with methylene blue dye.

Assessments
1. Based on the staining technique you performed in this activity, how could you
distinguish stained prokaryotic cells from stained eukaryotic cells? [SP 1, Concept
Explanation; SP 2, Visual Representation; SP 3, Questions and Methods]
2. What do all cells have in common, whether prokaryotic or eukaryotic? What major
differences would you expect to find? [SP 1, Concept Explanation]
3. Identify whether the following images show an animal cell, a plant cell, or a prokaryote
cell. Explain how you know the difference. [SP 1, Concept Explanation; SP 2, Visual
Representations]

74
[Answer Key]
1. The stained prokaryotic cells should be much smaller than the eukaryotic cells and
would likely be located on the outside of the eukaryotic cells. In addition, the eukaryotic
cell would contain a stained nucleus while the prokaryotic cells would not contain the
nucleus.
2. In common: Living cells must have nucleic acid (DNA) whether present in an organelle in
eukaryotes or in the nucleoid in prokaryotes. They must have a boundary membrane to
separate their metabolic processes from the environment. Differences: Eukaryotes
would be larger and have a visible nucleus.
3. Cell (a): Plant cell, cell wall, geometric shape, visible chloroplasts, central vacuole
Cell (b): Prokaryote, very small at high magnification, no obvious subcellular features.
Cell (c): Animal cell, no cell wall, obvious nucleus, visible subcellular structures, no
chloroplasts or large central vacuole.

75
 Lab 5: Subcellular Structures
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT:
Enduring
Topic Learning Objective Essential Knowledge
Understanding
SYI-1.D.1 Ribosomes
comprise rRNA and protein
and are the sites of protein
synthesis.
SYI-1.D.2 Ribosomes are
found in all cells, reflecting
common ancestry of all
known life.
SYI-1.D.3 Endoplasmic
reticulum occurs in two
forms—smooth and rough.
SYI-1.D Describe the
SYI-1 Living systems Rough ER is associated with
2.1 Cell Structure: structure and/or
are organized in a membrane-bound
Subcellular function of subcellular
hierarchy of structural ribosomes and also
Components components and
levels that interact. compartmentalizes the cell.
organelles.
Smooth ER functions include
detoxification and lipid
synthesis.
SYI-1.D.4 Folding and
chemical modification of
newly synthesized proteins
occur in the flattened sacs of
the Golgi complex. Other
organelles include
mitochondria, chloroplasts,
lysosomes, and vacuoles.
SYI-1.E Explain how
SYI-1.E.1 Organelles and
subcellular components
subcellular
and organelles
components—including
contribute to the
mitochondria, ER,
SYI-1 Living systems function of the cell.
ribosomes, lysosomes, and
2.2 Cell Structure are organized in a SYI-1.F Describe the
vacuoles—interact to
and Function hierarchy of structural structural features of a
support cellular function.
levels that interact. cell that allow
SYI-1.F.1 The folding of inner
organisms to capture,
membranes increases
store, and use energy.
surface area, which allows
for more synthesis of ATP.

76
 SYI-1.F.2 to SYI-1.F.7 The
light-dependent reactions of
photosynthesis occur in the
grana of the chloroplasts,
whereas the carbon-fixing
reactions occur in the
stroma.
EVO-1.A Describe
similarities and/or
differences in EVO-1.A.1 & EVO-1.B.1
compartmentalization Membrane-bound
EVO-1 Evolution if
between prokaryotic organelles evolved from
characterized by a
and eukaryotic cells. once free-living prokaryotic
2.11 Origins of Cell change in the genetic
cells via endosymbiosis.
Compartmentaliza makeup over time and
EVO-1.B Describe the EVO.1.A.3 Eukaryotic cells
tion is supported by
relationship between maintain internal
multiple lines of
the functions of membranes that partition
evidence.
endosymbiotic the cell into specialized
organelles and their regions.
free-living ancestral
counterparts.
ENE-1.I Describe the ENE-1.I.1 Photosynthesis
photosynthetic first evolved in prokaryotic
ENE-1The highly processes that allow organisms, resulting in an
complex organization organisms to capture oxygenated atmosphere.
of living systems and store energy. These pathways were the
3.5 Photosynthesis requires constant ENE-1.J Explain how foundation of eukaryotic
input of energy and cells capture energy photosynthesis.
the exchange of from light and transfer ENE-1.J.2 Photosystems I
macromolecules. it to biological and II are embedded in the
molecules for storage internal membranes of
and use. chloroplasts.
EVO-2.B.1 Many
EVO-2.B Describe the
fundamental molecular and
fundamental molecular
cellular features and
EVO-2 Organisms are and cellular features
processes are conserved
7.6 Evidence of linked by lines of shared across all
across organisms.
Evolution descent from common domains of life, which
EVO-2.B.2 Structural and
ancestry. provide evidence of
functional evidence supports
common ancestry.
the relatedness of organisms
in all domains.

77
 SCIENCE PRACTICES:

Science Practice Skill

SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.A Describe characteristics of a biological

SP 2 Visual Representations concept, processes, or model represented
visually.
Analyze visual representations of biological 2.B Explain relationships between characteristics
concepts and processes. of biological concepts, processes, or models
represented visually in applied contexts.
3.B State and null and/or alternative hypotheses,
SP 3 Questions and Methods
or predict the results of an experiment.
3.D. Make observations, or collect data from
Determine scientific questions and methods.
representations of laboratory setups or results.
6.A. Make a scientific claim.
SP 6 Argumentation
6.B Support a claim with evidence from biological
principles, concepts, processes, and/or data.
Develop and justify scientific arguments using
6.E Predict the cause or effects of a change to one
evidence.
or more components in a biological system based
on data.

Eukaryotic cells are recognizable by the presence of subcellular structures like

organelles. Organelles like mitochondria for cellular respiration, lysosomes, endoplasmic
reticulum, and chloroplasts that capture light energy and manufacture energy rich compounds.
Visualizing these structures under the light microscope can be difficult since the organelles
appear transparent without the use of biological stains. However, some organelles are much
easier to visualize due to the presence of organic molecules such as chlorophyll and other
pigments.
Plastids are double-membraned subcellular structures found in algae and plants. The
theory of endosymbiosis, illustrated in Figure 5.1, is supported by the findings of Lynn Margulis
at the University of Massachusetts in the 1960s. Evidence that a cyanobacterium was engulfed
and took up residence inside a eukaryotic ancestor is strengthened by the presence of circular
DNA and bacterial sized ribosomes inside the plastids. Plastid DNA was transferred to the host
DNA, leaving only the partial circular genomes of their free-living cousins.

78
 Figure 5.1: Endosymbiosis is the theory that eukaryotes gained membrane-bound organelles by evolving a
symbiotic relationship with prokaryotic cells.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Make sure students understand the definition of symbiosis, and explain that a symbiotic
relationship can be obligatory, meaning that one organism cannot survive without the
other.
● Explain that mitochondria and chloroplasts are believed to have arisen through
endosymbiosis, a process in which an ancestral eukaryotic cell engulfed a smaller
cyanobacterium. The two cells formed an obligatory symbiotic relationship in which the
cyanobacterium provides energy to the larger cell, and the larger cell protects the
cyanobacterium.
● Show students diagrams of a chloroplast and mitochondrion. Ask them to identify
evidence of endosymbiosis in these organelles, including the presence of inner and
outer membranes, DNA, and ribosomes.
● Ask students to describe a common function of chloroplasts and mitochondria.
Response should indicate that both of these organelles provide energy for the cell.
Chloroplasts capture energy from sunlight, and mitochondria extract energy from food.
● Explain that a chloroplast is a type of plastid. Other types of plastids include
chromoplasts, which contain colored pigments such as those found in flowers, and
amyloplasts, which store starch.

In this lab, you will learn

● to prepare wet mounts of carrot slices and observe chromoplasts;
● to prepare wet mounts of potato slices and observe before and after staining with
iodine;

79
 ● to make wet mounts of red onion skin;
● the effect of pH on red onion skin;
● to observe Nissl bodies in a neuron as an example of endoplasmic reticulum;
● to observe striated muscle as an example of cytoskeleton thin filaments;
● to visualize mitochondria using a biological stain.

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: Why would pigments make visualizing
subcellular structures easier under the light microscope?
2. Answer the following question in your notebook. How could you tell the difference
between a lipid-soluble pigment and a water-soluble pigment under a light microscope?
3. Discuss the answers to questions 1 and 2 with a partner (think, pair, and share) and
then the class.

Activity 1: Chromoplasts and Amyloplasts

All plastids originate from proplastids that can differentiate into several mature types of
plastids, as shown in Figure 5.2, similar to the idea of a stem cell. Chloroplasts (“green bodies”)
are an example of a mature plastid that contain pigments, water insoluble lipid molecules that
can be found in membranes and are capable of absorbing light energy. These start the process
of photosynthesis, as shown in Figure 5.3. You will examine a few different types of plastids in
this lab. The two plastids that are examined in this activity are the chromoplasts and the
amyloplasts, shown in Figure 5.4 and Figure 5.5, respectively. Chromoplasts, or colored bodies,
are mature plastids that are used primarily for the synthesis of pigments and their storage.
Chromoplast pigments are found in many plant parts such as flowers, roots, and aging leaves.
The purpose of these plastid pigments is to attract pollinators to the plant and to advertise to
different animals that eat the fruit (such as mammals, birds, insects, and reptiles) and then
disperse the seeds, generally with a bit of fertilizer. These pigments are also found in root
vegetables such as carrots and yams. Amyloplasts, meaning starch bodies, are a type of
leucoplast, or white body, that are colorless; these plastids synthesize glucose into starch, store
starch, and help the plant detect gravity using statoliths.

80
Figure 5.2: Protoplastids are the stem for several other plastids.

Figure 5.3: A micrograph of chloroplasts within plant cells.

Figure 5.4: A micrograph of chromoplasts within plant cells

81
 Figure 5.5: A micrograph of amyloplasts within plant cells.

Safety Precautions
● Be careful handling glass slides, the edges may be sharp.
● Cut away from your body. Sectioning of potato and carrots involves using a single-edged
razor blade. Use care to avoid cuts.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Dispose of biological stains according to teacher instructions and local regulations.
● Wash your hands with soap and water after handling biological stains.

For this activity, you will need the following:

● Clean microscope slides and coverslips
● Single-edge razor blade (only for preparation purposes, not for students to use)
● Carrot and potato
● Iodine stain (or Lugol’s stain) in dropper bottle
● Paper towels
For this activity, you will work in pairs.

82
Activity 1 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 1:
● Let students know that slides should be handled at the edge only so they do not get
smudged with fingerprints. This activity recommends you, as the instructor, use the
razor blade or knife to pre-cut the carrot and potato slices. Remind students to be
careful handling glass slides and to inform you immediately if glass breaks. Be sure
students know how to dispose of broken glass.
● Explain that a microscope is delicate and must be handled with care. Tell students to use
two hands to carry the microscope and to use only lens paper to clean the lenses.
● Identify the parts of the microscope, including the stage, coarse focus knob, fine focus
knob, nosepiece, objective lenses, and ocular lens. Show students how to adjust the
amount of light that enters the scope, and how to change the objective lens. Tell
students to watch carefully as they adjust the magnification so they do not strike the
lens against the slide.
● Explain that magnification can be calculated by multiplying the magnification of the
ocular lens by the magnification of the objective lens. Have students calculate all
possible magnifications for the scopes they are using.
● Explain how to focus the microscope. Initially, adjust the focus using the coarse focus
knob with the low power (10x) objective lens in place. Once the object is visible, use the
fine adjustment knob to sharpen the focus. To increase magnification, switch to the
medium power lens and use the fine focus knob to adjust the focus. Repeat this process
to switch to the high-power lens. Make students aware that they must never use the
coarse adjustment knob when focusing at medium or high magnification. If students
cannot see the object they are trying to focus at high magnification, they will need to
switch back to the low power lens and start over.

Structured Inquiry
Step 1: Make extremely thin slices of the carrot. The slice should be as transparent as possible.
Place the carrot slice on a clean microscope slide with a coverslip. If the coverslip does not lie
flat, the slice is too thick. Observe under middle (100x) and high power (400x) and draw and
label what you observe in your notebook, and record the magnification. Note: you do not need
to add a drop of water before placing the coverslip.
Step 2: Hypothesize/Predict. In your notebook predict (draw) what you expect to see in the
microscope with a potato slice. Will the addition of a biological stain (a dye that stains cellular
structures) help visualize subcellular structures? Which structures do you think will be more
obvious with the stain? Write your predictions in your notebook.

83
Step 3: Repeat Step 1 with the potato slice. Do not add iodine. Observe under high power
(100x) and then draw and label what you observe in your notebook, recording the
magnification.
Step 4: Add a drop of iodine stain to the edge of the coverslip. Grasp a small piece of paper
towel and dab it to the opposite side of the coverslip to draw the iodine through the potato
slice, as illustrated in Figure 5.6. Observe the slice under high power and draw and label what
you observe in your notebook, recording the magnification.

Figure 5.6: Process for making a wet mount cell of potato tissue.

Step 5: Critical Analysis: Are the predictions you made in Step 2 supported by your data and
observations? Why or why not? Are there any methods you could use to improve your results?
Discuss with your partner and then write your answers in your notebook.

Assessments
1. How do your drawings of the microscope slides compare to the internet images in Figure
5.3, Figure 5.4, and Figure 5.5? How are you convinced that the chromoplast pigments
were lipids? [SP 3, Visual Representations; SP 3, Questions and Methods]
2. Differentiate between the pigment functions in chromoplasts and in chloroplasts. [SP 1,
Concept Explanation]
3. Iodine (or Lugol’s solution) is not a very water-soluble stain. What type of molecules do
you think the iodine solution is staining in the potato slice? [SP Concept Explanation; SP
3 Questions and Methods]
[Answer Key]
1. Depending on the student’s microscope skills, the internet images may be higher
magnification. Students should note that the plastids are colored in the carrot (carrots
have carotenoids—lipid soluble pigments) that range from yellow to red and are visible
to the human eye but hard to see in the potato without the iodine stain. (Iodine stain is
not very water soluble, since starch is non-polar the iodine does stain the starch as well
as other lipids in the cell membrane and other subcellular membranes).

84
 2. Chloroplasts are a specialized chromoplast that contain the pigment chlorophyll, which
captures light energy for use in photosynthesis. Other chromoplasts have lipid soluble
pigments, such as carotenoids that are also used in photosynthesis and provide orange
color to flowers and leaves. Chloroplasts can change into chromoplasts when fruit
ripens.
3. Iodine (or Lugol’s solution) is not very water soluble and primarily used to detect
non-polar starches and lipids. Therefore, iodine can also stain cell membranes and other
membrane-bound organelles.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: How do plant cells and animal cells
capture energy that they use to survive?
2. Answer the following question in your notebook. How do the different ways that plant
and animal cells acquire energy reflect the different ways in which they live?
3. Discuss the answers to questions 1 and 2 with a partner and then the class.

85
Activity 2: Central Vacuole and Anthocyanins
Plant cells are eukaryotic; they have subcellular organelles. Like bacteria they have a cell
wall to help keep the cell rigid—although in plants the cell wall is composed of a complex
carbohydrate called cellulose. Plant cells also have a nucleus with DNA and a large central
vacuole (see Figure 5.7) full of water and other important substances (such as carbohydrates,
non-nutrients, wastes) for maintaining life and cell pressure.

Figure 5.7: Structures of a generalized plant cell.

The second major type of pigment found in plastids is water soluble. These pigment
molecules, called flavonoids, are stored in the central vacuole of plants. The specific pigment
found in the central vacuole that is examined in this activity is called anthocyanin (meaning
flower blue) the addition of a sugar group to its structure makes it water soluble. If cations
(positively charged ions or polyatomic ions) are added or removed from the anthocyanin
structure the pigment color will change, showing the anthocyanins are sensitive to pH changes.
Anthocyanins are found in most plant tissues as well as algae and some bacteria. Anthocyanins
are hypothesized to protect plant tissues from harmful UV radiation and are also used as
camouflage from herbivores.
Safety Precautions
● Be careful handling glass slides, the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Wear goggles when handling pH solutions.

86
 ● If any pH solutions get on your hands, flush with water to remove.
● Dispose of pH solutions according to teacher instructions and local regulations.
● Wash your hands with soap and water after handling pH solutions.
For this activity, you will need the following:
● Red onion
● Clean microscope slides and cover slips
● Water and dropper
● Forceps
● pH solutions in dropper bottles (pH 3.0, pH 7.0, and pH 8.5)
● Notebook to observe and draw features of plant cells.
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● Show a diagram of a plant cell and to point out the nucleus, central vacuole,
chloroplasts, and cell wall. Ask students to describe the function of each of these
structures.
● Show a picture of flavonoid-containing fruits and vegetables, including strawberries,
blueberries, cherries, and tomatoes.
● Show the structure of anthocyanin. Explain that the structure of anthocyanin changes
with pH, which causes a change in color.
● Demonstrate that anthocyanin can be used as a pH indicator. To prepare a
demonstration, boil red cabbage in water. Dilute the cabbage juice enough to see
through and add the diluted juice to a series of test tubes. Add substances that differ in
pH to each test tube (it helps if the substances are colorless) and ask students to note
the color. Substances that can be used include white vinegar; lemon-lime flavored soft
drinks, tap water, baking soda, and ammonia.
● Remind students to handle the microscope with care, and to use care with glass slides
and coverslips.

Structured Inquiry
Step 1: Hypothesize/Predict: What subcellular features do you expect to see in red onion cells?
Will the central vacuole, nucleus, and chromoplasts be obvious? Will any features differ

87
between red and white onion cells? If not, hypothesize how you would be able to see them?
What do you hypothesize will happen to the red onion cells in various pH solutions? Record
your ideas and hypothesis in your notebook.
Step 2: Student-led planning: Prepare your plant cell slides as shown in Figure 5.8. Each partner
is expected to prepare one sample. Each of you should view, draw, state the size and
magnification, and label each sample. To prepare the onion skin, cut the onion in quarters, take
the outer red peel of an onion section, and place it on the microscope slide. Experiment with a
few different cuts to determine the best technique to get the thinnest possible slice. Spread the
onion skin on the microscope slide, put one small drop of water and top with a coverslip. Record
your observations (drawings, color if present, labels, magnification, and size of cell) in your
notebook, keeping in mind your hypotheses from above.

Figure 5.8: Process for preparing red onion wet mounts.

Step 3: Critical Analysis: Were the predictions you made supported by your data
(observations)? Why or why not? Are there any methods you could have used that would
improve your results? Discuss with your partner and then write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: A change in pH can often alter the function of macromolecules
and cell structures. How do you think a change in pH will affect the pigment of the red onion

88
cells? Write one hypothesis each describing how an increase and a decrease in pH would affect
the onion cell pigment.
Step 2: Student-led planning. Your teacher has provided you with solutions of different pH.
Discuss, with your partner, how you will examine each onion skin under the different pH
solutions (e.g., How long should you observe each slide? How many times should you repeat
your observations?). Record your plan in your notebook and create date tables in which to
record your results.
Step 3: Carry out the experiment you designed in Step 2, preparing your slides using the
technique in Figure 5.8. Create drawings in your notebook to record all of your observations.
Step 4: Critical analysis: Did the results you predicted match what you observed as the pH
changed? In a paragraph, describe how increasing and decreasing pH affected the red onion
cells. Include an explanation of why you think pH affected the cells in the way you observed.
Discuss your analysis with your partner and write it in your notebook.

Assessments
1. How are you able to find the vacuole of the red onion cells? What should you be looking
for in the cell? [SP 3, Questions and Methods]
2. Red wines range in color from pink, red, and even violet-blue. If red grapes are used to
make red wine, what part of the grape would explain that coloration? Predict the pH of
wine based on the color. [SP 1, Concept Explanation]
[Answer Key]
1. Anthocyanins are contained in the central vacuole. Anthocyanins are water-soluble and
the central vacuole solvent is water. The vacuole is full of anthocyanins and the outline
of the vacuole between the cell wall/cell membrane and the clear cytoplasm should
hopefully stand out. Note this picture shows the vacuole in a hypertonic solution.

2. Students may or may not know that the skin of the grape provides color. If the grapes
are crushed with their skin, then the solution will contain anthocyanins that were
located in the vacuoles of the skin cells. The activity above had students explore the

89
 relationship between pH and the color of the red onion cells. The wine coloration is
linked to the pH of the wine. Red would indicate a lower pH than violet.

Activity 3: Pre-Assessment
1. Answer the following question in your notebook. What structures would you expect to
find in plant cells but not animal cells?
2. Answer the following question in your notebook. Do you expect that the subcellular
structures will be easy to see? Why or why not?
3. Discuss the answers to questions 1 and 2 with a partner (think, pair, and share) and
then the class.

90
Activity 3: Observation of Subcellular Structures in Animal Tissues
Animal cells (Figure 5.9) are eukaryotic and possess subcellular components in common
with the plant cells you observed earlier. Some example structures include, nuclei (contains
DNA, controls cell function), Golgi apparatuses (sorting, packing, and modification of proteins,
mitochondria (energy production from organic molecules such as glucose), ribosomes
(translation of messenger RNA into proteins), and the endoplasmic reticulum (folding of
proteins and manufacture of lipids). Animal cells are predominantly colorless. Exceptions
include the red of hemoglobin in red blood cells, red myoglobin in striated muscle, and melanin
in skin cells. Thus; SPecific biological stains are required to make visible cellular features under a
light microscope.

Figure 5.9: Structures of a generalized animal cell.

Nissl bodies, shown in Figure 5.10, present in neurons can be stained and allow you to
see the rough endoplasmic reticulum and rosettes of free ribosomes. The cytoskeleton includes
thin and thick filaments which are involved in cell shape, movement, and muscle contractions.

91
 Figure 5.10: In this cross section from a rodent brain cells containing Nissl bodies are stained purple.

Safety Precautions
● Be careful handling glass slides, the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Wear goggles when using staining solutions.
● Dispose of staining solutions according to teacher instructions and local regulations.
● Wash your hands with soap and water after handling live organisms and biological
stains.
For this activity, you will need the following:
● Prepared slides of Nissl bodies and striated muscle
● Elodea anacharis
● Clean microscope slides, cover slips
● Janus Green B stain
● Dropper
● Small squares of paper towels
● Notebook to observe and record data.
For this activity, you will work in pairs.

92
Activity 3 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 3:
● Show a diagram of an animal cell and to point out the nucleus, mitochondria, and
endoplasmic reticulum (ER). Ask students to describe the function of each of these
structures. Response should indicate that the nucleus carries genetic information,
mitochondria provide energy to the cell by breaking down food, and the rough ER makes
fats, steroids, membrane proteins, and secretory proteins.
● Show students a micrograph of an aniline-stained neuron. Ask students to identify the
function of neurons and to think about how the long, thin axons and dendrites might
help the cell accomplish this function. Response should indicate that neurons are
involved in cell-cell communication, and that the long extensions allow the neuron to
form connections with other cells. Explain that Nissl bodies, which are stained purple by
aniline stain, are rough ER.
● Show students a diagram of a striated muscle cell. Explain that these cells are found in
skeletal muscle, and that bands called sarcomeres give the cells a striated appearance.
Ask students to think about why the cells form long thin bands. Response should
indicate that the bands form muscle fiber, and all the cells act together to cause the
muscle to contract.
● Ask students to think about how animal cells differ from plant cells. Response should
indicate that plant cells have a cell wall, plastids, and a central vacuole and animal cells
do not.

Structured Inquiry
Step 1: Hypothesize/Predict: Predict how big the structures of the Nissl bodies, thin and thick
filaments, and mitochondria will appear when you look at them under the microscope. Which
structures do you think you can see without staining?
Step 2: Observe the prepared slides and record (draw, label any visible parts, use color if
visible, magnification, and size of cells) in your notebook.
Step 3: Critical analysis: How visible were the Nissl bodies, thin and thick filaments, and
mitochondria before and after staining? Write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: Given the function of mitochondria, predict how a biological
stain would help you visualize mitochondria. Record your prediction in your notebook.

93
Step 2: Prepare a wet mount microscope slide with Elodea anacharis (each partner is
expected to prepare one sample and each of you should view, draw, state the size and
magnification, and label each sample). Experiment with a few different cuts to determine the
best technique to get the thinnest possible slice. Observe your prepared slide, record (draw,
label any visible parts, use color if visible, magnification, and size of cells) in your notebook.
Step 3: Prepare a second wet mount, this time with Janus Green B stain (Figure 5.12). No
Figure 12 is provided. Observe over a period of time. Record your observations, drawings,
label any visible parts, use color if applicable, and magnification in your notebook.
Step 4: Critical Analysis: Were the structures smaller or larger than you thought? How do the
structures compare to the general size of prokaryotes? There should be small blue dots visible
on the Elodea cells—much smaller than the chloroplasts. Hypothesize what those blue dots
might be. Why are some of the dots turning pink over time? Come up with an experimental
design that would allow you to test your idea. Record the answers to these questions in your
notebook.

Assessments
1. Explain how your experimental design allowed you to identify a mitochondrion versus a
nucleus in terms of function in the Elodea cell slide. [SP 1, Concept Explanation]
2. Describe some of the functions of thin and thick filaments in the muscle cells, based on
your observations of their structure. [SP 1, Concept Explanation]
[Answer Key]
1. Methylene blue stains nucleic acid, and will therefore stain the nucleus, while Janus Green
B stains the components of mitochondria. Therefore, the nucleus and chloroplasts will
appear different in color due to the staining. The nucleus is also much larger than a single
mitochondrion.
2. In muscle cells, thin and thick filaments combine their efforts to pull on the edges of the
muscle cells, allow them to contract and relax. This allows muscles to move various parts
of the body.

94
 Lab 6: Diffusion and Osmosis
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring Essential Knowledge
Topic Learning Objective
Understanding
ENE-1.B.1 Surface
area-to-volume ratios affect
the ability of a biological
ENE-1.B Explain the
system, including cells, to
effect of surface
exchange nutrients, wastes,
area-to-volume ratios on
and energy with the
ENE-1 The highly the exchange of
environment.
complex organization materials between cells
ENE 1.B.2 The surface area of
of living systems and the environment.
the plasma membranes must
2.3 Cell Size requires constant
be large enough to adequately
input of energy and ENE-1.C Explain how
exchange materials; smaller
the exchange of specialized structures
cells typically have a larger
macromolecules. and strategies are used
SA:V ratio.
for the efficient
ENE-1.C.1 Organisms have
exchange of molecules to
evolved efficient strategies to
the environment.
obtain nutrients and eliminate
wastes, using specialized
exchange surfaces.
ENE-2.A.1 Cell membranes
consist of a phospholipid
bilayer with hydrophilic and
hydrophobic regions, with
ENE-2.A Describe the fatty acid regions facing each
ENE-2 Cells have roles of each of the other within the interior of
membranes that allow components of the cell the membrane.
them to establish and membrane in ENE-2.A.2 Proteins embedded
2.4 Plasma maintain internal maintaining the internal in the bilayer can be
Membranes environments that are environment of the cell. hydrophilic or hydrophobic.
different from their ENE-2.B.1 Cell membranes
external ENE-2-B Describe the consist of a structural
environments. Fluid Mosaic Model of framework (bilayer) of
cell membranes. phospholipid molecules with
embedded proteins, steroids
such as cholesterol in
eukaryotes, glycoproteins, and
glycolipids.

95
 ENE-2.C.1 The structure of cell
membranes results in selective
permeability.
ENE-2.C.2 Cell membranes
separate the internal
environment of the cell from the
external environment.
ENE-2 Cells have
ENE-2.C.3 Selective permeability
membranes that allow
ENE-2.C Explain how the is a direct consequence of
them to establish and
2.5 Membrane structure of biological membrane structure.
maintain internal
Permeability membranes influences ENE-2.C.4 Small, nonpolar
environments that are
selective permeability. molecules freely pass across the
different from their
membrane, whereas larger polar
external environments.
molecules and ions move across
through the embedded channel
and transport proteins.
ENE-2.C.5 Polar uncharged
molecules, including water, pass
through the membrane in small
amounts.
ENE-2.E.1 Passive transport is the
ENE-2 Cells have net movement of molecules from
ENE-2.E Describe the
membranes that allow high concentration to low
mechanisms that organisms
them to establish and concentration without the direct
2.6 Membrane use to maintain solute and
maintain internal input of metabolic energy.
Transport water balance.
environments that are ENE-2.E.2 Passive transport plays
different from their a primary role in the import of
external environments. materials and the export of
wastes.
ENE-2 Cells have
ENE-2.G.1 Membrane proteins
membranes that allow ENE-2.G Explain how the
are required for facilitated
them to establish and structure of a molecule
2.7 Facilitated diffusion of charged and large
maintain internal affects its ability to pass
Diffusion polar molecules. Large quantities
environments that are through the plasma
of water pass through
different from their membrane.
aquaporins.
external environments.
ENE-2.H.1 External environments
can be hypotonic, hypertonic, or
isotonic to international
environments of cells, with water
ENE-2.H Explain how moving by osmosis from areas of
concentration gradients high water potential/low solute
ENE-2 Cells have
affect the movement of concentration to areas of low
membranes that allow
molecules across water potential/high solute
them to establish and
2.8 Tonicity and membranes. concentrations.
maintain internal
Osmoregulation ENE-2.I.1 Growth and homeostasis
environments that are
ENE-2.I Explain how are maintained by the constant
different from their
osmoregulatory mechanisms movement of molecules across
external environments.
contribute to the health and membranes.
survival of organisms. ENE-2.I.2 Osmoregulation
maintains water balance and
allows organisms to control their
internal solute composition/water
potential.

96
 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation 1.A Describe biological concepts and/or
processes.
Explain biological concepts, processes, and 1.B Explain biological concepts and/or processes.
models presented in written format. 1.C Explain biological concepts, processes, and/or
models in applied contexts.
2.B Explain relationship between difference
SP 2 Visual Representations characteristics of biological concepts, processes,
or models represented visually in applied
Analyze visual representations of biological contexts.
concepts and processes. 2.D Representation relationships within biological
models, including mathematical models and
diagrams.
3.A Identify or pose a testable question based on
an observation, data, or a model.
3.C Identify experimental procedures that are
SP 3 Questions and Methods aligned to the questions, including variables and
controls.
Determine scientific questions and methods. 3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E Propose a new/next investigation based on a
previous experiment.
SP 4 Representing and Describing Data
4.B Describe data from a table or graph,
describing patterns and/or relationships.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, including
equations in the curriculum, rates, and
Perform statistical tests and mathematical
percentages.
calculations to analyze and interpret data.
SP 6.B Support a claim with evidence from
SP 6 Argumentation
biological principles, concepts, processes, and/or
data.
Develop and justify scientific arguments using
SP 6.C Provide reasoning to justify a claim by
evidence.
connecting evidence to biological theories.

Cells are like miniature factories, using materials and energy to break down and
manufacture substances that are important to their survival. The movement of materials into
and out of the cell is important for proper cell functioning. This movement is regulated by the
plasma membrane, which determines what goes in and what comes out of the cell. The plasma
membrane is said to be selectively permeable which means that it only allows certain
molecules to cross the membrane while others cannot (Figure 6.1). This is largely because the
membrane’s phospholipid bilayer has a hydrophobic (water-fearing) core, so hydrophilic
(water-loving) substances and large, polar molecules need assistance to cross the membrane.
Conversely, molecules that are small, nonpolar and hydrophobic can cross the membrane easily

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without using energy through a method called passive transport. Passive transport involves the
diffusion of molecules down a concentration gradient. Recall that diffusion is the natural
tendency of particles (e.g., atoms, molecules, ions) to move from an area of high concentration
to an area of low concentration. Osmosis is a special type of diffusion that involves the
movement of water across a selectively permeable membrane. In this lab, you study both
diffusion and osmosis.
The selective permeability of the cell membrane allows the cell to control what
substances enter or leave. Polar molecules, such as water, must enter the cell through
membrane proteins that facilitate their diffusion across the membrane. This is called facilitated
diffusion. Other molecules, such as those that are very large or are already in a high
concentration within the cell, must be pumped across the membrane with the assistance of a
membrane protein that uses energy (ATP). Membrane transport that requires energy is called
active transport.

Figure 6.1: The plasma membrane is mainly made up of phospholipids which have hydrophobic and nonpolar tails
in the interior and polar and hydrophilic heads toward the exterior.

Instructor Introduction Notes

The following are recommendations for introducing the lab’s background content to your
students.
● Show students a labelled diagram of a cell while introducing the lab.
● Review the terms polar, nonpolar, hydrophilic, and hydrophobic by showing a diagram
that distinguishes the properties.
● Reinforce the distinctions between passive transport, active transport, and osmosis
with short videos or animations from the internet or YouTube.

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In this lab, you will learn
● how simple diffusion works;
● how various factors affect the rate of diffusion;
● to calculate diffusion rates of substances based on molecular weight;
● how different media affect the rate of diffusion;
● how surface area-to-volume ratio influences the effectiveness of diffusion;
● how the process of osmosis works.

Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: What happens when an air
freshener is sprayed in a corner? What is the name of the process that causes the
molecules to move?
2. Answer the following questions in your notebook: Do you think that the rate of the air
freshener molecules moving would change if the room temperature was warmer or
colder? Why or why not?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 1: Diffusion

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry Activity 1:
● Tell students that certain dyes will not completely wash out of their clothing.
● Ask the students to hypothesize temperatures for what they think very cold or very hot
water is.
● Tell students that they should not stir the solution or move the setup while diffusion is
taking place.
● Instruct students to avoid touching the surface of agar with their fingers because it could
affect diffusion.

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The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 1:
● Have students identify the skills learned in the structured inquiry, such as quantifying
the rate of diffusion. They will need to carry out the guided inquiry.
● Place students in work groups to expedite the activity. Encourage each group to assign
specific tasks to group members.
● Ask students to write their own paper written in their words. Check for plagiarism by
comparing the answers between group members.
Diffusion is the movement of molecules from an area where the molecule is highly
concentrated to an area of low concentration, as illustrated in Figure 6.2. The rate of diffusion is
dependent upon the temperature of a system, molecular size, and the medium through which
diffusion is occurring (i.e., semi-solid, liquid, air). In this activity, we will be observing the
diffusion of a dye through a beaker of water and through agar (a gelatinous substance),
diffusion as a function of temperature, and diffusion as a function of molecular weight.

Figure 6.2: In diffusion, molecules move from areas of high concentration to areas of low concentration.

Safety Precautions
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent other people from slipping.
● Be careful with the dye as it can stain your clothes, and it should not be ingested.
● Wash your hands with soap and water after completion of the activity.

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For this activity, you will need the following:
● Three 250 mL beakers
● Water
● Food coloring
● Agar plates
● Potassium permanganate
● Congo red
● Methylene blue
● Thermometer
● Incubator
● Refrigerator
● Clock or timer
For this activity, you will work in groups of four.

Structured Inquiry
Step 1: Measure 200 mL of room temperature water in a beaker. Put three drops of food
coloring into the water. Time how long it takes for the dye to completely diffuse throughout the
water. Record the time and describe in your notebook what you observe. Create a data table for
your observations.
Step 2: Hypothesize/Predict: Predict what would happen to the rate of diffusion if you had
beakers with both very hot and very cold water in them. Add your predictions to the data table
you created in Step 1.
Step 3: Student-led Planning: Determine how diffusion of the food color would be affected
when the water is either very hot or very cold. Use a thermometer and record the temperature
for each. Use a timer to measure how long it takes for complete diffusion to occur in all
scenarios.
Step 4: Critical Analysis: Create a graph that shows how the diffusion rate is affected because of
temperature change. Are the predictions you made in Step 2 supported by your data? Why or
why not? What methods could you use to improve your results? Discuss with your group and
then write your answers in your notebook.

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Guided Inquiry
Step 1: Gather four agar plates and the three dyes, provided by your instructor, that differ in
molecular size: Congo red (mol. wt. 696.66 g/mol), methylene blue (319.85 g/mol), and
potassium permanganate (mol. wt. 158.03 g/mol).
Step 2: Hypothesize/Predict: How would the rate of diffusion of a molecule through a gel
compare to its rate of diffusion through water? How would the rate of diffusion differ between
molecules of different molecular sizes? Write your ideas in your notebook.
Step 3: Student-led planning: Use 1 plate for determining how molecular size affects diffusion
using the 3 dyes. Determine how best to measure movement of the dye in an agar plate. Be
sure to keep the dyes far enough apart so that they do not touch once they start diffusing. Get
your instructor’s approval before proceeding with the experiment. Measure the distance that
the dye spreads in 20-minute intervals for 1 hour.
Step 4: Examine the effect of temperature on the rate of diffusion for 1 dye of your choosing.
With your group, determine 3 temperatures that would be appropriate. Measure the diameter
of the dye spread for each. Write the results in your notebook.
Step 5: Critical Analysis: Rank all 3 dyes in terms of diffusion rate. What was the relationship
between diffusion rate and molecular size? What is the relationship between temperature and
diffusion rate? Discuss your answers with your group and write them in your notebook.

Assessments
1. In a system, there is a concentration of molecules. However, on the outside, there is
little to no concentration of this particular molecule. In which direction would the
molecules be moving more so than the other direction? [SP 1, Concept Explanation]
2. Diffusion is affected by what factors? [SP 1, Concept Explanation]
3. Dye tends to move faster in warmer temperatures. Why is this? [SP 1, Concept
Explanation]
[Answer Key]
1. The molecules would move from the area of higher concentration to lower. So, the
movement would be from inside to outside.
2. Temperature, molecular size, and the type of medium all affect the rate of diffusion.
3. Increased temperature means increased molecular motion.

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Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: What do you think happens to a
plant when it is placed in salt water? Why might this occur?
2. Discuss the answers to question 1 with the class.

Activity 2: Measuring Osmosis

Osmosis is the diffusion of water across a selectively permeable membrane (i.e., cell
membrane). Because of osmosis, water will move where water is less concentrated from an
area of low solute (high water content) to an area with high solute concentration (low water
content) (Figure 6.3). Dialysis tubing is used to model selectively permeable membranes
because it will prohibit large molecules from crossing the membrane but will allow small
molecules to cross. For example, water and glucose are small molecules that can easily cross the
membrane. However, starch (a polymer of glucose), cannot cross due to its large molecular size.
Additionally, cell surface area and volume can affect the rate of diffusion across a membrane.
The surface area-to-volume ratio describes the relationship between the area outside the cell
to the volume inside the cell (Figure 6.4). In this lab, we first will use dialysis tubing to model
how the membrane selectively allows certain molecules to cross the membrane. Then, we will
compare the diffusion rate of dialysis tubing with a small surface area-to-volume ratio to one
that has a large surface area-to-volume ratio.

Figure 6.3: Osmosis is the diffusion of water across a semipermeable membrane. Water moves from an area of
high water and low solute concentration to an area of low water and high solute concentration. Note that the
solute does not move.

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 Figure 6.4: Smaller cells have higher surface areas compared to larger cells.

Safety Precautions
● Safety goggles/glasses should be worn when chemicals or solutions are heated.
● Handle all chemicals safely.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent other people from slipping.
● Dispose of all chemicals per local regulations.
● Use caution when performing the Benedict’s reaction which involves the use of a hot
water bath.
For this activity, you will need the following:
● Dialysis tubing, two pieces, about 15 cm each long and narrow (pre-soaked)
● Dialysis tubing, one piece, short and wide (pre-soaked)
● Six Dialysis tubing clamps (optional)
● Three 500 mL beaker
● Water
● 5 percent Glucose solution, 10 mL

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 ● Starch suspension, 10 mL
● Iodine
● Benedict’s reagent
● Graduated cylinder
● Test tube
● Hot water bath
For this activity, you will work in groups of four.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry Activity 2:
● Remind students that the minimum molecular weight of starch in solution is likely
50,000 MW. The best dialysis tubing for retaining starch has a molecular weight cutoff of
12,000 to 14,000 MW.
● Demonstrate how to properly clamp the dialysis tubing. You can have an image of the
setup or a model setup where the students can view it.
● Discuss with students that the dyes used in class may stain the skin for a few hours to a
couple of days.
The following are recommendations for the Guided Inquiry for Activity 2:
● Have students identify the skills learned in the structured inquiry, such as detecting the
presence of glucose, they will need to carry out the guided inquiry.
● Place students in work groups. Encourage each group to assign specific tasks to group
members.
● Ask students to write their own paper written in their words. Make sure to check for
plagiarism by comparing answers between group members.

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 Figure 6.5: Directions for how to seal a dialysis tube.

Structured Inquiry
Step 1: Use a graduated cylinder to measure 250 mL of room temperature water in a beaker. Set
aside. Obtain 1 piece of long and narrow dialysis tubing. Seal 1 end of the tube by tying a secure
knot or attaching a dialysis tubing clamp to the end of the tubing. Put 10 mL of starch
suspension and 10 mL of glucose solution in the tube. Then tie or clamp the other end of the
tube. Place the sealed tube into the beaker (Figure 6.6). Develop and implement the use of an
appropriate controlled experiment to go alongside your experiment. Allow both to sit for 20–30
minutes.

Figure 6.6: Your dialysis tube should be placed into the beaker and allowed to sit for the time specified.

Step 2: Hypothesize/Predict: Consider the molecular sizes of glucose and starch. What do you
think will happen with the starch and the glucose inside of the dialysis tube? Predict whether
starch and/or glucose will remain inside of the tube or if they will move out.

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Step 3: Student-led planning: Briefly remove the dialysis bag from the beaker and set aside.
Pour 2 mL of the water from the beaker into a test tube. Put 10 drops of Benedict’s reagent into
the tube (note the initial color of the solution) and place into the hot water bath for 5 minutes.
Note whether there is a color change. Place the bag back into the beaker. Put 20 drops of iodine
into the water in the beaker. Allow this to sit for about 10 minutes. Determine whether starch is
present in the bag and/or the beaker. Describe in your notebook what you observe. Create a
data table for your observations that shows whether starch and glucose were inside and/or
outside of the dialysis tubing.
Step 4: Critical Analysis: Are the predictions you made in Step 2 supported by your data? Why
or why not? What methods could you use to improve your results? Discuss with your group and
then write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: Predict what would happen to the rate of osmosis if the surface
area-to-volume ratio was large (as it would be with small cells compared to larger ones). Record
your prediction.
Step 2: Student-led planning: Determine how the rate of osmosis of water using a dialysis bag
with starch solution is affected if surface area-to-volume ratio is altered. Use dialysis tubing of 2
different lengths and widths to determine this. Record weights of each bag at different time
intervals. Discuss with your group the time points at which you will check the bags for
differences in weight over a 20-minute period. Record your data for each. Discuss with your
group how best to set up these experiments.
Step 3: Critical Analysis: Create a graph that shows how osmosis rate is affected over time
because of alterations in surface area-to-volume ratios. Are the predictions you made in Step 2
supported by your data? Why or why not? What methods could you use to improve your
results? Discuss with your group and then write your answers in your notebook.

Assessments
1. Would a large protein have difficulty crossing the membrane of a dialysis tube? Why or
why not? [SP 1, Concept Explanation; SP 3, Questions and Methods]
2. Why do smaller cells have more efficient diffusion compared to larger ones? [SP 1,
Concept Explanation; SP 5, Statistical Tests and Data Analysis]
[Answer Key]
1. Yes. Large molecules cannot cross the membrane.
2. Smaller cells have a larger surface area-to-volume ratio compared to larger cells.

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Activity 3: Pre-Assessment
1. Answer the following question in your notebook: Saline solutions are given to patients
in an IV. Why might the salt concentration in the solution need to be the same as that in
the blood cells?
2. Discuss the answer to question 1 with your group.

Activity 3: Osmosis in Living Cells

The inside and the outside of a cell is mainly composed of water with dissolved solutes.
The differences in solute concentration direct the movement of water across the plasma
membrane of the cell. This difference between the two solutions (i.e., the cytoplasm and the
extracellular fluid) is known as tonicity. Solutions are said to be isotonic if they both have equal
concentrations of solute. The extracellular fluid is hypertonic to the intracellular fluid if it has a
higher solute concentration. On the other hand, a lower solute concentration in the
extracellular fluid compared to the inside of the cell would mean that the outside of the cell is
hypotonic to the inside.
We can observe tonicity in Elodea leaves by placing them into different solutions with
various solute concentrations.
Safety Precautions
● Be careful handling glass slides; the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent other people from slipping.
● Handle all chemicals safely.
For this activity, you will need the following:
● Two glass slides
● Elodea leaves
● Two coverslips
● Distilled water
● 10 percent NaCl solution
● 30 percent NaCl solution

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 ● Microscope
● Potato cubes
● Electronic balance
● Weigh boat
● Three 50 mL beakers
For this activity, you will work in groups of four.

Activity 3 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry Activity 3:
● Tell students to handle the slides and coverslips carefully to prevent breaking slides and
cutting their fingers.
● Project or make available an image of Elodea cells to help students understand what
they are viewing under the microscope.
● Reinforce that students must be consistently measuring the actual weight of the potato
cubes during their experiment.
● Use freshly cut potato cells that do not show brown discoloration. The cut surfaces of
potatoes oxidize and dehydrate which affects diffusion and osmosis results.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 3:
● Have the students identify the skills, such as identifying how to measure changes in the
Elodea leaf, they will need to carry out the guided inquiry. These skills were learned in
the structured inquiry.
● Place students in work groups. Encourage each group to assign specific tasks to group
members.
● Ask students to write their own paper written in their words. Make sure to check for
plagiarism by comparing answers between group members.

Structured Inquiry
Step 1: Obtain 2 glass slides and 2 cover slips. On 1 slide, place an Elodea leaf and put 1 drop of
distilled water. Make a second slide with an Elodea leaf but put a drop of 10% NaCl. Allow these
to sit for 2–3 minutes.
Step 2: Hypothesize/Predict: Predict what will happen with the leaf that has been placed into
NaCl. What do you think will happen to the leaf in water? Record your predictions.

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Step 3: Student-led planning: Determine what happens to the Elodea leaf cells in both solutions
by observing them under the microscope. Record your observations for each. Draw pictures
that demonstrate what you observe.
Step 4: Critical Analysis: Are the predictions you made in Step 2 supported by your data? Why
or why not? What methods could you use to improve your results? Discuss with your group and
then write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: What happens to a cell in an isotonic solution? Hypertonic?
Hypotonic? Predict what might happen to a potato in these solutions. Will it gain, lose, or
maintain its weight? Write your ideas in your notebook.
Step 2: Student-led planning: Design a study that would test your hypothesis in Step 1. Plan
which solutions you will use and how to determine if osmosis happened in each. Show your
design to a teacher for approval then create data tables that you would need to test your
hypothesis. Write your results in your notebook and create a graph or table to summarize your
results.
Step 3: Critical Analysis: Discuss your results with your group. Are your results what you
expected? How can you improve your experiment? Write your ideas in your notebook.

Assessments
1. Have you ever seen a slug hanging out on the steps? Oftentimes, people use salt to get
rid of them. What do you think this does to the slug in terms of osmosis? [SP 1, Concept
Explanation]
2. How can you predict whether osmosis will occur into or out of a cell? Explain in terms of
water potential. [SP 5, Statistical Tests and Data Analysis; SP 6, Argumentation]
3. Explain what is meant when we say that a cell membrane is selectively permeable. What
types of molecules might have a difficult time crossing the membrane? [SP 1, Concept
Explanation]
[Answer Key]
1. Putting salt on a slug is like placing a cell into a solution with a higher salt concentration.
This causes water to leave out of the cell to balance the environment and equalize the
concentrations of the two environments.
2. You can determine the direction of osmosis by knowing the solute concentration inside
relative to the outside of the cell. Water potential is a measure of potential energy in a
sample of water, and describes the tendency of water to move from one place to
another, from high water potential to low water potential. Water potential, which can be
mathematically calculated, is influenced by solute concentration and pressure.

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3. The cell membrane is selectively permeable which means that it determines what goes
in and what comes out of the cell. Basically, it selects what it will allow to cross the
membrane. Large molecules, as well as polar molecules, have a difficult time crossing
the membrane due to the makeup of the membrane (i.e., phospholipids).

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Lab 7: Factors Affecting the Enzymatic Activity of Lactase
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Topics Enduring Understanding Learning Objective Essential Knowledge
ENE-1.D.1 The structure of
enzymes includes the active
site that specifically
ENE-1 The highly complex interacts with substrate
organization of living molecules.
systems requires constant ENE-1.D Describe the ENE-1.D.2 For an
3.1 Enzyme Structure
input of energy and the properties of enzymes. enzyme-mediated chemical
exchange of reaction to occur, the shape
macromolecules. and charge of the substrate
must be compatible with
the active site of the
enzyme.
ENE-1.E.1 The structure and
ENE-1 The highly complex function of enzymes
organization of living contribute to the regulation
ENE-1.E Explain how
systems requires constant of biological processes by
3.2 Enzyme Catalysis enzymes affect the rate
input of energy and the acting as catalyzes and
of biological reactions.
exchange of lowering the activation
macromolecules. energy of a chemical
reaction.
ENE-1.F.1 Change to the
molecular structure of a
component in an enzymatic
ENE-1.F Explain how system resulting from
changes to the structure denaturation, or less than
ENE-1 The highly complex
of an enzyme may optimal temperature
organization of living
3.3 Environmental affect its function. and/0r pH, may result in a
systems requires constant
Impacts on Enzyme change of the function or
input of energy and the
Function ENE-1.G Explain how efficiency of the system.
exchange of
the cellular ENE-1.G.1 – 1.G.4 Factors
macromolecules.
environment affects that affect enzyme activity
enzyme activity. include pH, temperature,
concentration of substrates,
and competitive and
noncompetitive inhibitors.

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 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.A Describe characteristics of a biological

concept, process, or model represented visually.
2.B Explain relationships between different
SP 2 Visual Representations
characteristics of biological concepts, processes,
or models represented visually in applied
Analyze visual representations of biological
contexts.
concepts and processes.
2.D Represent relationships within biological
models, including mathematical models and
diagrams.
3.A Identify or pose a testable question based on
an observation, data, or a model.
3.B State the null and alternative hypotheses, or
predict the results of an experiment.
SP 3 Questions and Methods
3.C Identify experimental procedures that are
aligned to the questions, including variables and
Determine scientific questions and methods.
controls.
3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E Propose a new/next investigation.
SP 4 Representing and Describing Data
4.A Construct a graph, plot, or chart.
4.B Describe data from a table or graph.
Represent and Describe Data.

SP 5 Statistical Tests and Data Analysis

5.A Perform mathematical calculations, including
Perform statistical tests and mathematical equations in the curriculum (pH) and rates.
calculations to analyze and interpret data. 5.B Use data to evaluate a hypothesis/prediction.

6.B Support a claim with evidence from biological

SP 6 Argumentation
principles, concepts, processes, and/or data.
6.E Predict the causes or effects of a change in
Develop and justify scientific arguments using
one or more components in a biological system
evidence.
based on data.

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 Enzymes are biological catalysts, substances that serve to increase the rate of chemical
reactions. Most enzymes are proteins, but some RNA molecules have also been shown to
demonstrate catalytic activity. Enzymes typically work by binding to starting chemicals, called
substrates, and holding them in a way that will break chemical bonds and form new bonds
more readily. In doing so, enzymes lower the activation energy, the energy needed to form or
break chemical bonds (Figure 7.1) and convert the substrate(s) to product(s). These are the
molecules that result from such chemical reactions (Figure 7.2). After facilitating the transition
from substrate(s) to product(s), an enzyme releases the product(s) and becomes available for
reuse.

Figure 7.1: Enzymes lower the activation energy of chemical reactions. Here, we can see that the activation energy
of a chemical reaction in the presence of an enzyme (red) is lower than the same reaction without an enzyme
(purple).

Figure 7.2: Over the course of a chemical reaction, the substrate concentration (purple) will decrease as the
product concentration (green) increases.

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 Because enzymes are normally proteins, they are composed of amino acids, each
containing a side chain or R group with distinct chemical properties. Side chains may be
hydrophobic or hydrophilic (polar, acidic, or basic). The combination of side chains within an
enzyme and the chemical interactions between them contribute to the enzyme’s unique
three-dimensional structure. This structure also determines the shape of its active site, the
structural portion of the enzyme that binds to the substrate(s). There are a variety of factors
that may influence enzymatic activity; including temperature, pH, and substrate concentration.

In this lab, you will learn

● how to measure enzymatic activity of the enzyme lactase over time and represent it
graphically
● how to monitor the effects of environmental conditions on enzymatic activity
● how to determine the specificity of the enzyme lactase

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students.
● Show a brief animation or video on enzyme action as a review of enzyme function.
● Project and draw a diagram showing the primary through quaternary structures of
proteins as a review of protection structure.
● Do a quick inquiry to see what information students have retained about the terms
hydrophobic and hydrophilic.
● Ask students what it means to be lactose intolerant. Do not define it immediately
because lactose intolerance is briefly described in Activity 1.

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: How would the concentration of an
enzyme, its substrate(s), and its product(s) change over time as an enzymatic reaction
takes place?
2. Answer the following questions in your notebook: What molecule(s) would you
measure to monitor the progress of an enzymatic reaction? How might you observe the
relative concentrations of these molecules over the course of the enzymatic reaction?
3. Discuss the answers to questions 1 and 2 with the class.

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Activity 1: Measuring the Enzymatic Activity of Lactase
How would we measure the activity of a specific enzyme? One easy way to measure the
concentration of a chemical is by using a colored indicator. As an enzymatic reaction proceeds,
the color of the solution changes, and can be monitored visually.
In this lab, you will measure the enzymatic activity of lactase, an enzyme that breaks
down lactose, a disaccharide found in milk and other dairy products, into the monosaccharides
galactose and glucose (Figure 7.3). While human infants naturally produce lactase, most adults
do not, making them lactose intolerant, or unable to produce enough lactase to digest ingested
lactose. Interestingly, in the past 10,000 years, several populations of humans have developed
lifelong lactase activity. These human populations are often people who raise livestock and
drink animal milk. For most individuals who are lactose intolerant, lactase may be purchased in
tablet form from pharmacies.

Figure 7.3: The enzyme lactase degrades the disaccharide lactose into the monosaccharides galactose (1) and
glucose (2).

Glucose, one of the products of lactose degradation, can be detected visually using
glucose detection strips, which can also be purchased from pharmacies. One type of glucose
detection strip contains the dye toluidine blue and the enzyme glucose oxidase (Figure 7.4).
Glucose oxidase within the strip converts the glucose to gluconic acid and hydrogen peroxide.
Hydrogen peroxide then interacts with the toluidine blue within the strip, bringing about a color
change from blue to green to yellow to brown.

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 Figure 7.4: Diastix glucose detection strips turn from blue to green to yellow to brown with increasing
concentrations of glucose.

Safety Precautions

● Measure fluids carefully using graduated cylinders to avoid breakage and spillage.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
For this activity, you will need the following:
● Graduated cylinder
● Beaker
● Water
● Milk
● Lactase (obtained from laboratory supply company)
● Stirring rod
● Test tubes
● Glucose (powdered)
● Balance
● Labeling pencil
● Glucose test strips

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 ● Graph paper
● Timer
For this activity you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry 1:
● Reduce the number of glucose test strips used in this activity by carefully slitting them in
half lengthwise. Make sure that the indicator patch remains intact on each side.
● Store lactase solution in a refrigerator to reduce a loss of activity. The solution should be
used at room temperature.
● Tell students not to touch the indicator patch on the glucose test strips. Oils, sweat, and
particles on their fingertips can contaminate the strip and give false results.
● You could use a Benedict’s test in place of the glucose test strips in this activity to
measure glucose, if desired.
● Confirm that test strips are actually measuring glucose by comparing a test strip result to
a Benedict’s test result on the glucose solution. They should be more familiar with
Benedict's test.
● Project an unlabeled image of lactose and have the class identify the two
subcomponents.
● Look for glucose powder in grocery stores or health food shops.
● Purchase lactase in grocery stores, health food shops, and pharmacies. Perform a test
run of this activity before using it in class.
The following are recommendations for the Guided Inquiry for Activity 1:
● Have the students identify the skills, such as determining the breakdown of lactose, they
will need to carry out the guided inquiry. These skills were learned in the structured
inquiry. Place students in work groups. Encourage each group to assign specific tasks to
group members.
● Ask students to write their own paper written in their words.

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Structured Inquiry
Step 1: Make 100 mL of a 20 mg/mL glucose solution or obtain this from your instructor. Set up
tubes as follows:
Negative control: 3 mL of water
Positive control: 3 mL of 20 mg/mL glucose solution
Insert a glucose test strip into each tube, and compare the color you observe to the color chart
that comes with the test strips. What is the concentration of glucose in each tube? Are your
results what you expected?
Step 2: Hypothesize/Predict. Based upon your knowledge of enzymes and lactase function,
predict whether glucose will be detected in milk in the presence of lactase enzyme compared to
milk lacking the lactase enzyme. Predict how the glucose concentration of milk will change over
time under each of these two conditions. Write your predictions in your lab notebook.
Step 3: With your partner, prepare two tubes each containing 2 mL of milk. Additionally, make
your lactase enzyme solution per your teacher’s instructions.
Step 4: Student-led Planning. Your experiment should last for 15 minutes. Create a data table to
record glucose concentrations in each of the two tubes of milk at 3-minute intervals. To start
the experiment, add 1 mL of water to one of the tubes of milk and 1 mL of the lactase solution
to the other. Then, every 3 minutes for 15 minutes, record the concentrations of glucose in each
tube using the color chart that came with the test strips and record this in your data table.
Step 5: Critical Analysis. Using graph paper, graph your data of glucose concentration versus
time. Which is the independent variable? Which is the dependent variable? Calculate the rates
of glucose production for each tube. Are the predictions you made in Step 2 supported by your
data? Explain how you know in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict. How do you think increasing the amount of lactase will affect
glucose formation? How do you think decreasing the amount of lactase will affect glucose
formation? Formulate a hypothesis involving the effect of the lactase amount on glucose
formation.
How do you think increasing the amount of milk will affect glucose formation? What would
happen if you decrease the amount of milk? Formulate a hypothesis involving the effect of the
amount of milk on glucose formation.
Write your hypotheses and predictions in your notebook.

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Step 2: Student-led Planning. Select one of the hypotheses above to discuss with your lab
partner. Design an experiment to address this hypothesis and submit it to your teacher for
approval. Once your teacher approves, create a table to record your data, prepare your test
tubes per your design, and record data on glucose production every 3 minutes for 15 minutes.
Graph your data and calculate the rate of glucose production for each tube.
Step 3: Critical Analysis. Are the predictions you made in Step 1 supported by your data? Is
there any way you could improve your experiment? Discuss your answers with your lab partner
and write it in your notebook.

Assessments
1. In this experiment, assume that all the lactose provided is broken down to glucose and
galactose. Based upon this assumption, if one diluted the amount of milk by half in the
tube containing milk and the lactase enzyme, what would happen to the amount of
glucose produced? Explain why in terms of molecular interactions of the substrate with
the enzyme and enzyme function. [SP 1, Concept Explanation; SP 3, Questions and
Methods; SP 6, Argumentation]
2. In this experiment, describe the role of each of the following: [SP 3, Questions and
Methods]
a. The water-only tube
b. The tube of 20 mg/mL glucose solution
[Answer Key]
1. One would expect the amount of glucose produced to be halved. This is because the
number of interactions between substrate and enzyme would be cut in half if the
amount of substrate (milk) is diluted by half. Thus, the rate of the enzymatic reaction
would be cut in half.
2.
a. The water-only tube serves as the negative control for the glucose test strips.
b. The tube of 1 percent glucose solution serves as the positive control for the
glucose test strips.

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Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: Which environmental conditions
could alter the rate at which an enzymatic reaction takes place? Why would this occur?
2. Answer the following questions in your notebook: Which environmental conditions
could affect an enzyme’s active site? Why would this occur?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 2: The Effects of Temperature and pH on Enzymatic Activity

What types of environmental factors may affect enzymatic activity? Why? Several factors
known to affect enzymatic activity are temperature, pH, and substrate concentration. In a
typical chemical reaction, increasing temperature causes the substrates to become more
energetic and hence more likely to bump into each other in solution. However, changes in
temperature can cause an enzyme to denature, which changes the three-dimensional structure
of the enzyme molecule. In addition, cellular enzymes each work within a certain pH range
because the side chains within their active sites are optimized for efficient catalysis and are thus
quite sensitive to changes in pH. Different enzymes may have different pH ranges and pH
optima, conditions under which they work maximally; while many enzymes work best around a
neutral pH, some are adapted to an acidic pH, while others are adapted to a basic pH.
Safety Precautions
● Measure fluids carefully using graduated cylinders to avoid breakage and spillage.
● Be careful not to touch solutions of concentrated acids and bases directly.
● Take precautions when using a hot plate and touching hot glassware.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
For this activity, you will need the following:
● Graduated cylinder
● Beaker
● Water
● Buffer solutions of pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, and pH 9.0
● Milk
● Lactase (obtained from laboratory supply company)
● Test tubes

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 ● Stirring rod
● Labeling pencil
● Hot plate
● Ice bath
● Refrigerator set to approximately 4 °C
● Incubator set to 37 °C
● Thermometers
● Glucose test strips
● Timer
● Graph paper
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● Reduce the number of glucose test strips used in this activity by carefully slitting them in
half lengthwise. Make sure that the indicator patch remains intact on each side.
● You could use a Benedict’s test in place of the glucose test strips in this activity to
measure glucose, if desired.
● Store lactase solution in a refrigerator to reduce a loss of activity. The solution should be
used at room temperature.
● Perform a quick demonstration of protein denaturation can be shown by placing a drop
of 1N HCl on liquid egg white in a flat container. Have the students explain what
happened to the egg white solution.
● Reinforce student safety when they are handling the different pH solutions and working
with the hot plates.

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The following are recommendations for the Guided Inquiry for Activity 2:
● Have the students identify the skills, such as determining the rate of glucose production
from lactose, they will need to carry out the guided inquiry. These skills were learned in
the structured inquiry.
● Place students in work groups. Encourage each group to assign specific tasks to group
members.
● Ask students to write their own paper written in their words.

Structured Inquiry: Temperature

Step 1: Prepare a large beaker of boiling tap water on a hot plate. Prepare five identical test
tubes, each containing 2 mL of milk. Label five test tubes accordingly with each of the following
temperatures: 0°C (ice bath), 4°C (refrigerator), room temperature, 37°C, and 100°C (boiling
temperature). Place one tube of milk at each of the five temperatures. Create a data table to
enter your results for each of these test tubes over time. Measure the room temperature using
a thermometer.
Step 2: Hypothesize/Predict. Based upon your knowledge of enzymes and the effects of
temperature on their activity, rank the tubes from fastest (1) to slowest (5) glucose production
predicted over time after the addition of lactase. Add your predictions to the data table you
created in Step 1.
Step 3: Student-led Planning. Discuss with your partner how you could use the data you collect
to calculate a rate of lactase activity for each temperature.
Step 4: Make your lactase enzyme solution per your teacher’s instructions. Add 1 mL of the
lactase enzyme solution to each of the five tubes listed above and immediately start timing.
Immediately after adding the lactase enzyme solution, determine the glucose concentration in
each tube using glucose test strips and the color chart that came with the test strips. Record this
in your data table.
Monitor the temperatures of each of these locations, both before and after the experiment
using thermometers.
Step 5: Every 3 minutes for 15 minutes, record the concentrations of glucose in each tube using
the color chart that came with the test strips and record in your data table.
Step 6: Critical Analysis. Calculate the rate of enzymatic activity for lactase at each temperature
using the method you devised in Step 3. Using graph paper, graph your data of rates of lactase
activity versus temperature. Which is the independent variable? Which is the dependent
variable? Are the predictions you made in Step 2 supported by your data? Explain how you
know in your notebook.

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Guided Inquiry: Temperature
Step 1: Hypothesize/Predict. Based upon the data already collected, predict a temperature
range that includes the optimal temperature for lactase activity. How do you think you could
more finely pinpoint the optimal temperature for lactase activity? Write your ideas in your
notebook.
Step 2: Student-led Planning. Determine how you could change the set-up of your test tubes to
determine the optimal temperature for lactase activity. Once your teacher approves, create a
table to record your data, prepare your test tubes per your design, and record data on glucose
production every 3 minutes for 15 minutes. Determine the rates of lactase enzyme activity
under each of your chosen conditions as you did in the Structured Inquiry. Graph the rates of
lactase enzyme activity versus temperature and estimate the optimal temperature.
Step 3: Critical Analysis. Are the predictions you made in Step 1 supported by your data? Is
there any way you can improve your experiment? Discuss your answer with your lab partner
and write it in your notebook.

Structured Inquiry: pH
Step 1: Prepare three test tubes, each containing 2 mL of milk and label the three tubes as
follows: 4.0 (acidic), 7.0 (neutral), and 9.0 (basic). To the first test tube, add 1 mL of pH 4.0
buffer. To the second test tube, add 1 mL of pH 7.0 buffer. To the third test tube, add 1 mL of pH
9.0 buffer. Create a data table to enter your results for each of these test tubes over time.
Step 2: Hypothesize/Predict. Based upon your knowledge of enzymes and the effects of pH on
their activity, order the tubes from highest (1) to lowest (3) glucose production predicted over
time. Add your predictions to the data table you created in Step 1.
Step 3: Student-led Planning. Discuss with your partner how to calculate a rate of lactase
activity for each pH.
Step 4: Make your lactase enzyme solution per your teacher’s instructions. Add 1 mL of the
lactase enzyme solution to each of the three tubes listed above and immediately start timing.
Immediately after adding the lactase enzyme solution, determine the glucose concentration in
each tube using glucose test strips and the color chart that came with the test strips. Record this
in your data table.
Step 5: Every 3 minutes for 15 minutes, record the concentrations of glucose in each tube using
the color chart that came with the test strips and record in your data table.
Step 6: Critical Analysis. Calculate the rate of enzymatic activity for lactase at each pH. Using
graph paper, graph your data of rates of lactase activity versus pH. Which is the independent
variable? Which is the dependent variable? Are the predictions you made in Step 2 supported
by your data? Explain how you know in your notebook.

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Guided Inquiry: pH
Step 1: Hypothesize/Predict. Based upon the data already collected, predict a pH range that
includes the optimal pH for lactase activity. How do you think you could more finely pinpoint
the optimal pH for lactase activity? Write your ideas in your notebook.
Step 2: Student-led Planning. Determine how you would change the set-up of your test tubes to
determine the optimal pH for lactase activity. Once your teacher approves, create a table to
record your data, prepare your test tubes per your design, and record data on glucose
production every 3 minutes for 15 minutes. Determine the rates of lactase enzyme activity
under each of your chosen conditions. Graph the rates of lactase enzyme activity versus pH and
estimate the optimal pH.
Step 3: Critical Analysis. Are the predictions you made in Step 1 supported by your data? Is
there any way you could improve your experiment? Discuss your answer with your lab partner
and write it in your notebook.

Assessments
1. If an enzyme has an optimal activity at 25°C, what do you think will happen to the
enzyme’s activity if the temperature is raised to 37°C? Why? [SP 1, Concept Explanation;
SP 6, Argumentation]
2. If an enzyme has a largely acidic active site, what do you think will happen to the
enzyme’s activity if the pH is made basic? Why? [SP 1, Concept Explanation; SP 6,
Argumentation]
[Answer Key]
1. Since 37°C is well above the optimum, one would expect a decrease in enzyme activity,
possibly due to denaturation.
2. A basic solution would neutralize the active site, likely reducing the activity of the
enzyme.

Activity 3: Pre-Assessment
1. Answer the following question in your notebook: How could we determine
experimentally the specificity of an enzyme to its substrate?
2. Discuss the answer to question 1 with the class.

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Activity 3: Substrate Specificity of the Enzyme Lactase
An enzyme’s active site contains side chains of the amino acids that can only bind to
certain molecules. For example, if the active site is largely positively charged, then negatively
charged substrates will be attracted. Additionally, the three-dimensional shape of the active site
within the enzyme is key in determining which substrates will fit into the active site (Figure 7.5).
What does this suggest about how specific enzymes are to their substrates?

Figure 7.5: The three-dimensional shape of the active site is also important in determining which substrates will
bind to the active site.

Safety Precautions
● Measure fluids carefully using graduated cylinders to avoid breakage and spillage.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
For this activity, you will need the following:
● Graduated cylinder
● Beaker
● Water
● 20 mg/mL lactose solution
● 20 mg/mL sucrose solution
● Various foods for testing for presence of lactose, including both dairy products and those
marketed as lactose-free
● Lactase (obtained from laboratory supply company)
● Test tubes
● Stirring rod

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 ● Timer
● Labeling pencil
● Glucose test strips
● Graph paper
For this activity, you will work in pairs.

Activity 3 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 3:
● Provide students with images of lactose and sucrose. Have the students compare and
contrast the structure of the two molecules.
● A short video or animation of enzyme-substrate interactions provides a good review for
students.
● You can reduce the number of glucose test strips used in this activity by carefully slitting
them in half lengthwise. Make sure that the indicator patch remains intact on each side.
● A Benedict’s test can be used in place of the glucose test strips in this activity to measure
glucose, if desired.
● Lactase solution must be stored in a refrigerator to reduce a loss of activity. The solution
should be used at room temperature.
● Take precautions to prevent redundancy if you are requesting students to bring in food
for this activity. Assign students to particular food groups and have them at first
hypothesize about the foods they are selected as containing or not containing lactose.
The following are recommendations for the Guided Inquiry for Activity 3:
● Have the students identify the skills, such as determining the relative concentration of
lactose, they will need to carry out the guided inquiry. These skills were learned in the
structured inquiry.
● To expedite the activity, it is best to place the students in work groups. Encourage each
group to assign specific tasks to group members.
● To facilitate individual assessments, students should be asked to write their own paper
written in their words.

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Structured Inquiry
Step 1: Prepare two test tubes, one with 2 mL of 20 mg/mL lactose and the other with 2 mL of
20 mg/mL sucrose. Label one tube as lactose-containing and the other as sucrose-containing.
Create a data table to enter your results for each of these test tubes over time.
Step 2: Hypothesize/Predict. Based upon your knowledge of enzymes and their specificity,
predict whether you will observe enzyme activity for each of the two tubes indicated above.
Add your predictions to the data table you created in Step 1.
Step 3: Student-led Planning. Discuss with your partner how to calculate a rate of lactase
activity for each substrate.
Step 4: Make your lactase enzyme solution per your teacher’s instructions. Add 1 mL of the
lactase enzyme solution to each of the two tubes listed above and immediately start timing.
Immediately after adding the lactase enzyme solution, determine the glucose concentration in
each tube using glucose test strips and the color chart that came with the test strips. Record this
in your data table.
Step 5: Every 3 minutes for 15 minutes, record the concentrations of glucose in each tube using
the color chart that came with the test strips and record in your data table.
Step 6: Critical Analysis. Calculate the rate of enzymatic activity for lactase in each substrate.
Using graph paper, make a bar graph showing the rates of lactase activity in each of the two
substrates. Are the predictions you made in Step 2 supported by your data? Explain in your
notebook how you know.

Guided Inquiry
Step 1: Hypothesize/Predict. Based upon what you have learned, how could we use this assay
for lactase activity to determine the relative amounts of lactose in various food products,
including typical dairy products as well as those that are advertised as lactose-free? Write your
ideas in your notebook.
Step 2: Student-led Planning. With your lab partner, choose two food products to test—one
that you suspect contains lactose and one that is supposed to be lactose-free. Determine how
you would change the setup of your test tubes above to assay for lactase activity in food
products. Once your teacher approves your experimental design, create a table to record your
data, prepare your test tubes per your design, and record data on glucose production every 3
minutes for 15 minutes. Determine the rates of lactase enzyme activity for each food product.
Make a bar graph showing the lactase activity for each of the two foods chosen.
Step 3: Critical Analysis. Are the predictions you made in Step 1 supported by your data? Is
there any way you could improve your experiment? Discuss your answers with your lab partner
and write it in your notebook.

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Assessments
1. How can substrate specificity of an enzyme be used to determine the presence of its
substrate in a sample? Explain in terms of the active site. [SP 1, Concept Explanation]
2. What might you conclude if you observed glucose in a lactose-free food at the start of
the experiment? [SP 3, Questions and Methods]
3. How could enzyme specificity be used to determine the concentration of a substrate in a
food product? [SP 3, Questions and Methods]
[Answer Key]
1. Because enzymes are specific, an enzyme should only exhibit activity if its specific
substrate is present within the sample. Due to the three-dimensional shape of the active
site, the active site will only fit its specific substrate. Other substrates will not be of the
appropriate shape to fit into the active site, so they will be unable to interact with the
enzyme’s active site and the enzyme will not function with incorrect substrates.
2. One would conclude that there was glucose present in the food product prior to lactase
addition.
3. One could determine the rate of enzyme activity in the presence of a fixed amount of
enzyme but different known concentrations of substrate to develop a standard curve of
enzyme activity versus substrate concentration. Because a higher substrate
concentration would lead to more interactions of the substrate with the enzyme, one
would expect higher rates of enzyme activity at higher substrate concentrations. Then,
one could add the fixed amount of enzyme to a food product, monitor the
concentrations of product made over time, determine the rate of enzyme activity, and
then compare that rate of enzyme activity to the standard curve to determine the initial
amount of substrate in the food product.

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 Lab 8: Cellular Respiration
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
SYI-1.D.4 Mitochondria
SYI-1.D Describe the
SYI-1 Living systems are have a double membrane.
2.1 Cell Structure: structure and/or
organized in a The outer membrane is
Subcellular function of subcellular
hierarchy of structural smooth, but the inner
Components components and
levels that interact. membrane is highly
organelles.
convoluted, forming folds.
SYI-1.E.1 Mitochondrial
double membrane
provides compartments
for different metabolic
reactions.
SYI-1.E Explain how
SYI-1.F.1 The folding of
subcellular components
the inner membrane
and organelles
increases the surface
contribute to the
SYI-1 Living systems are area, which allows for
function of the cell.
2.2 Cell Structure organized in a more ATP synthesis.
and Function hierarchy of structural SYI-1.F. 8 The Krebs cycle
SYI-1.F Describe the
levels that interact. (citric acid cycle)
structural features of a
reactions occur in the
cell that allow
matrix of the
organisms to capture,
mitochondria.
store, and use energy.
SYI-1.F.9 Electron
transport and ATP
synthesis occur on the
inner mitochondrial
membrane.
ENE-1 The highly ENE-1.E.1 Enzymes are
complex organization biological catalysts that
of living systems ENE-1.E Explain how facilitate chemical
3.2 Enzyme Catalysis requires constant input enzymes affect the rate reactions in cells and
of energy and the of biological reactions. contribute to the
exchange of regulation of biological
macromolecules. processes.
ENE-1 The highly
ENE-1.H Describe the ENE-1.H.1 All living
complex organization
role of energy in living systems require constant
3.4 Cellular Energy of living systems
organisms. input of energy.
requires constant input

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 of energy and the ENE-1.H.2 Life requires a
exchange of highly ordered system
macromolecules. and does not violate the
second law of
thermodynamics.
ENE-1.H.3 Energy-related
pathways in biological
systems are sequential to
allow for a more
controlled and efficient
transfer of energy.
ENE-1.K.1 Fermentation
and cellular respiration,
characteristics of all forms
of life, use energy from
biological molecules to
produce ATP.
ENE-1.K.2 Cellular
respiration in eukaryotes
involves a series of
enzyme-catalyzed
reactions.
ENE-1.K.3 The electron
ENE-1.K Describe the transport chain transfers
processes that allow energy from the electron
ENE-1 The highly organisms to use energy in a series of coupled
complex organization stored in biological reactions that establish
of living systems macromolecules. an electrochemical
3.6 Cellular
requires constant input gradient across
Respiration
of energy and the ENE-1.L Explain how membranes.
exchange of cells obtain energy from ENE-1.L.1 Glycolysis
macromolecules. biological molecules in releases energy in glucose
order to power cellular to form ATP, NADH, and
functions. pyruvate.
ENE-1.L.2 Pyruvate is
transported from the
cytosol to the
mitochondrion, where
further oxidation occurs.
ENE-1.L.3 In the Krebs
cycle, CO2 is released, ATP
is synthesized, and
electrons are transferred
to NADH and FADH2.
ENE-1.L.4 Electrons are
transferred by NADH and

131
 FADH2 to the electron
transport chain.
ENE-1.L.5 As electrons
pass through the ETC, a
proton gradient is
established across the
inner mitochondrial
membrane.
ENE-1.L.6 In
fermentation, glycolysis
occurs in the absence of
oxygen.
ENE-1.L.7 The conversion
of ATP to ADP releases
energy necessary to
power metabolic
processes.

SCIENCE PRACTICES
Science Practices Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.A Describe characteristics of a biological

concept, processes, or model represented
SP 2 Visual Representations visually.
2.B Explain relationships between different
Analyze visual representations of biological characteristics of biological concepts, processes,
concepts and processes. or models represented visually in applied
contexts.
2.D Represent relationships within biological
models, including diagrams and flowcharts.
3.A Identify or pose a testable question based on
an observation, data, or model.
3.B State the null and alternative hypotheses, or
predict the results of an experiment.
SP 3 Questions and Methods
3.C Identify experimental procedures aligned to
the question, including variables and controls.
Determine scientific questions and models.
3.D. Make observations and collect data.
3.E Propose a new/investigation based on
evaluation of evidence from a previous
experiment.
SP 4 Representing and Describing Data 4.A Construct a graph, plot or chart.
4.B Describe data from a table or graph.

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 Represent and describe data.
5.A Perform mathematical calculations, including
SP 5 Statistical Tests and Data Analysis equations from the curriculum and rates.
5.B Use confidence level intervals and/or error
Perform statistical tests and mathematical bars to determine whether sample means are
calculations to analyze and interpret data. statistically different.
5.C Use data to evaluate a hypothesis.
6.A. Make a scientific claim.
6.B Support a claim with evidence.
6.C. Provide reasoning to justify a claim by
SP 6 Argumentation connecting evidence to biological theories.
6.D Explain the relationship between
Develop and justify scientific arguments using experimental results and larger biological
evidence. concepts, processes, or theories.
6.E Predict the causes of effects of a change in
one or more components in a biological system
based on data.

Living organisms require an external source of energy to sustain growth, metabolism,

and reproduction. Where does this energy come from? Phototrophs can tap solar energy and
convert it into chemical energy through the process of photosynthesis. Chemotrophs obtain
chemical energy from food molecules and store it in the small molecules ATP and NADH. Cells
release the energy of glucose and other food molecules through stepwise redox reactions in
which electrons and energy are transferred from one compound to another. Oxidation
describes the loss of electrons by a molecule referred to as an electron donor. Where do these
electrons go? The accompanying reaction, reduction, is the gain of electrons by another
molecule which serves as an electron acceptor. The overall chemical reaction of aerobic
respiration is summarized below:

.
In the process of aerobic respiration, electrons are oxidized from glucose molecules
(C6H12O2) to create ATP (adenosine triphosphate), the energy molecule of the cell. The
electrons oxidized from glucose undergo a series of redox reactions, each of which releases
energy that drives the formation of ATP. Some of these redox reactions occur during glycolysis
and the Krebs cycle, but most occur during the electron transport chain of aerobic respiration.
Eventually, the electrons are accepted by oxygen atoms and removed from the system. This
allows additional electrons from other glucose molecules to enter the chain of redox reactions,
synthesizing more ATP. During the multi-step oxidation of glucose, carbon dioxide is formed and
released as a waste product. Another waste product of aerobic respiration is heat, as the redox
reactions are not 100% efficient at transferring energy to ATP. Fortunately, this heat is used to
maintain our body temperature, explaining why exercise increases heat production in our
bodies.

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 Aerobic respiration can be divided in three main phases which are localized in different
compartments of the eukaryotic cell, as shown in Figure 8.1.

Figure 8.1: The three phases of cellular respiration are localized in different compartments of the eukaryotic cell.

Glycolysis, the partial breakdown of glucose to pyruvate with production of NADH and
ATP, takes place in the cytoplasm. The intermediate step is the oxidation of pyruvate into acetyl
CoA followed by the citric acid cycle in the matrix of the mitochondrion. In the citric acid cycle,
glucose is completely oxidized to CO2. Electrons are transferred to small electron carrier
molecules, NADH and FADH2.
The final step, oxidative phosphorylation takes place in the inner membrane of the
mitochondrion, as shown in Figure 8.2. Oxidative phosphorylation is the process by which
reduced electron carriers generated in glycolysis and the citric acid cycle are re-oxidized by an
electron transport chain embedded in a membrane. The energy released by the oxidation of the
electron carriers fuels the production of ATP by chemiosmosis across the membrane (Figure
8.2).

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 Figure 8.2: Electron transport chain embedded in mitochondrial membrane. Oxygen acts as the final electron
acceptor.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Begin the first lab by discussing the background of this topic by including a brief
PowerPoint lecture on the topic or even an animation of the various chemical reactions
of cellular respiration. This will be especially helpful if your students have not had a
general chemistry lab class in a long time or if this is their first time being in a biology
lab.
● Use a single-edge razor blade or knife with caution to prepare the specimen slices. Do
not leave blades exposed on the bench.
● Emphasize the importance of cellular respiration, the locations of where the reactions
occur in cells, and the end results (products) that are produced by these chemical
reactions. A circle of life type of illustration to show the interconnectedness of what
happens in plants and animals will be useful for further guidance (i.e., discuss or include
an illustration of how carbon dioxide released from animals is used by plants for carrying
out photosynthesis and likewise discuss how Oxygen produced by plants is used by
animals for the cellular respiration). This information would be helpful to assist students
in seeing the entire bigger picture of why these processes are important.
● Remind students of lab safety guidelines and proper disposal of sharps items such as
razors, slides, and coverslips.

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 ● Remind students that human biological samples (i.e., cheek cells) may be used for part
of this experiment. Highlight the proper disposal of this potentially hazardous waste (i.e.,
bleach or biohazard container). Be sure to have one of these items on hand as part of
the preparation for the lab setup.
● Supply students with either lab coats or aprons to protect their clothing and/or skin
from exposure. It will be useful to also require the students to wear gloves/goggles
when handling the dyes.

In this lab, you will learn

● how to determine the site of respiration in the cell;
● how to measure the consumption of oxygen during respiration;
● how to measure the effect of environmental conditions on respiration in pea seeds.

Activity 1: Pre-Assessment:
1. Answer the following questions in your notebook: What function do mitochondria
fulfill in the cell? What kind of staining would allow visualization of mitochondrial
activity?
2. Answer the following questions in your notebook: Which plant tissue would you
choose to stain mitochondria? Explain your choice.
3. Discuss the answers to questions 1 and 2 with the class. Activity 1: Staining Mitochondria
with Janus Green B

You will investigate the site of oxidative phosphorylation and the effect of environmental
conditions on the mitochondria. Janus Green B is a stain that appears blue-green when oxidized
(that is, when it loses electrons) and is colorless or light pink when reduced (when it gains
electrons).
Safety Precautions
● Dispose of coverslips in a broken glass container.
● Be careful handling glass slides, the edges may be sharp.
● If using cheek cells, dispose of flat toothpicks and slides in a jar containing 10 percent
bleach.
● Handle dyes with care and be careful not to ingest.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.

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 ● Wash your hands with soap and water after completion of the activity.
For this activity, you will need the following:
● Onion; alternatively slices of celery branch or cheek epithelial cells may be used
● Toothpicks and 10 percent bleach container if using epithelial cells
● Forceps
● Water and dropper
● 0.001 percent solution of Janus Green B
● Absorbent paper such as a paper towel or filter paper
● Microscope
● Slides and coverslips
● Mounting fluid to be chosen by students (7 percent sucrose, 0.9 percent salt, vinegar,
ammonia)
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Assist students in performing a wet mount by doing a proper demonstration in front of
the entire class prior to letting them commence with this activity (i.e., demonstrate how
to safely use the razor to slice the onion).
● Remind students of the potential changes in color they will see upon adding the Janus
Green B dye indicator. Remind them to write their hypotheses first prior to commencing
the experiment. Additionally, discuss the various mounting solutions that are available
that they will have to choose from.
● Having a brief discussion of the proper use and handling of the microscope may also be
useful to discuss prior to letting the students begin their experiments. Tips to consider
discussing include instructions on how to properly carry the microscope, the locations
and uses of the various parts (i.e., oculars, condenser, fine and coarse adjustment knob,
stage, etc.), and how to store the microscopes will be very helpful in assisting your
students in conducting this part of their lab exercises. Keep in mind that if they are
beginners, you may have to instruct them on how to focus their specimen as well as on
how to correctly record the total magnification of the specimen they are viewing (i.e.,
Total Magnification = Magnification of Ocular lens x Magnification of Objective lens).

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The following are recommendations for the Guided Inquiry for Activity 1:
● Students could remain in groups of two while doing the Guided Inquiry project, but you
may wish to have each student write their own hypotheses to ensure that they are
submitting their own original work.
● Although an illustration has been provided in Figure 8.3, you may wish to demonstrate
how to perform the wicking method prior to letting the students perform this part of the
experiment. Be sure to provide the students with a timer so that they can accurately
check their results at the designated times.
● If time allows, you may want to instruct the students to try redoing their experiment by
changing the mounting solutions they are using for their wet mounts and have them
compare the results yielded and/or observations made when utilizing the different types
of mounting solutions. Remind the students that both should be observing their results
microscopically and writing their observations in their respective notebooks to ensure
that everyone is doing their fair/equal share of the work.
● In Step 4, clarify to students how to analyze their results. Providing a grading rubric for
this activity with criteria describing each of the mandatory parts of this activity can help
guide students to include the proper components in their experiment for this activity.

Structured Inquiry
Step 1: Preparation and observation of wet mount:
1. Grab the edge of the layer with the forceps peeling back a thin transparent layer of
epidermal tissue. The thickness of the layer is one or a few cells which will allow you to
visualize clearly the inside of the cells.
2. Add a drop of water and place a cover slip over the onion slice; do not remove air
bubbles if they form.
Step 2: Hypothesize/Predict: Janus Green B is an indicator of the redox state. Knowing that it
appears blue/green in its oxidized state, and loses its color when it is reduced, allows you to
predict which organelle will show a progressive change in color because it is the active site of
oxidation-reduction. Discuss with your lab partner how you would expect Janus Green B color to
reflect active respiration. Which experimental conditions would you choose to investigate with
your current setup? Record your prediction in your notebook.
Step 3: Student-Led Planning: Decide which mounting solution(s) you will choose to observe
changes in respiration in the epidermal layer. View the sample first under low magnification to
focus on the cells. Proceed to the highest magnification available to you (highest dry objective
or oil immersion) and observe internal structures.

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Stain with Janus Green B by using the wicking method as shown in Figure 8.3. Place a piece of
filter paper or tissue on one side of the cover slip. Add one or two drops of Janus Green on the
opposite site of the coverslip close to the edge. The stain solution will flow in by capillary action.

Figure 8.3: Staining a wet mount by capillary action.

Monitor the changes in stain appearance for five to 10 minutes, taking turns observing the slide.
Record your observation in your lab notebook.
Step 4: Critical Analysis: Draw and label all the structures that you can identify. Do not forget to
add a title and the final magnification to all the drawings. Draw only what you observe. Do not
copy from existing micrographs from published or online work. Compare the effect of different
mounting solutions to distilled water. Did you observe a change in the color of mitochondria?
Did it change over time? If there was an effect, how can you explain your observations? Can you
think of other experiments that would support your conclusions?

Guided Inquiry
Step 1: Hypothesize/Predict: Janus Green B is a vital stain and an indicator of redox state.
Knowing that it appears blue/green in its oxidized state and loses its color when it is reduced,
allows you to predict which organelle will show a progressive change in color because it is the
active site of oxidation-reduction and which organelle will appear colorless. What
environmental conditions would be essential to observe a blue color of stain?
Step 2: Student-led planning: Prepare a wet mount of tissue under the microscope. Stain with
Janus Green B, using the wicking method described earlier, while observing under the
microscope. Which organelles can you distinguish? Are there any changes with time? Record
your observations in your notebook. Repeat your experiment with a different mounting
medium, staining with Janus Green B according to the wicking method. Record your
observations in your notebook.

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Step 3: Critical analysis: How did the various mounting fluids you used influence the response
of the mitochondria to Janus Green B? How can you explain the effect that you observed?
Compare the effect of different mounting solutions to distilled water. Was there an effect? If
there was an effect, how can you explain your observations? Can you think of other
experiments that would support your conclusions? Write your ideas in your notebook.

Assessments
1. What do you predict would be observed if the epidermal layer of an onion is incubated
in a solution of rotenone, an inhibitor of respiration? [SP 3, Questions and Methods; SP
6, Argumentation]
2. A student carefully mounts a specimen of onion epidermal layer, pushing out all the air
bubbles. She is very disappointed that she does not observe a change in the color of
Janus Green B. Can you explain her observation? [SP 1, Concept Explanation; SP 3,
Questions and Methods]
3. Cyanide is a known metabolic poison that acts mainly by blocking cytochrome oxidase,
an enzyme embedded in the inner membrane of mitochondria, and preventing the
reduction of oxygen. If cyanide were added to an onion layer stained with Janus Green B,
what you would observe and why? [SP 1, Concept Explanation; SP 3, Questions, and
Methods; SP 6, Argumentation]
[Answer Key]
1. Janus Green B loses its stain only if active mitochondria are present and use up oxygen.
In an oxidizing atmosphere, where oxygen is present. The mitochondria would look
green.
2. The student created an anaerobic environment. The stain cannot be oxidized by the
mitochondria because there is no oxygen.
3. The mitochondria would stay green because respiration is inhibited.

Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: Students stain corn seeds over a period
of several days after the seeds are soaked with water to promote germination with iodine.
Iodine stains starch blue. The students observe that the amount of starch decreases during
germination. Can you explain this observation? Which metabolic process uses up starch?
2. Answer the following questions in your notebook: What kind of biological catalysts are
involved in the reactions of respiration? If the rate of a chemical reaction doubles with the
temperature, would you expect that rate of respiration to increase continuously with
temperature?
3. Discuss the answers to questions 1 and 2 with the class.

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Activity 2: Measurement of Respiration and Effect of Temperature on
Respiration Rate
Imagine that you plan to monitor respiration in a whole organism, such as a small
invertebrate or a seedling. You may decide to follow the disappearance of the reactants, either
glucose or oxygen. Your second choice is to measure the formation of the products, either water
or carbon dioxide. In this laboratory, you will design experiments to assess the effect of
environmental conditions on the process of cellular respiration.
In respiration, oxygen is consumed and CO2 is released. In this experiment, we will
measure the disappearance of oxygen. A respirometer consists of an enclosed chamber in
which the studied organism is placed and a graduated pipette with which we measure changes
in the gas volumes. The CO2 gas which forms will be removed by adding Ca(OH)2 that reacts with
carbon dioxide to form the insoluble salt CaCO3, calcium carbonate.
While measuring the changes in the amount of gas produced, you will consider the ideal gas law
equation which can be stated as

.
P represents the atmospheric pressure in mmHg, V is the volume of the gas in liters, n is the
number of moles of gas, R is the ideal gas constant, and T is the temperature in degrees Kelvin.
In the respirometer, pressure remains constant as the gas produced displaces water in the tube.
We will set up the respirometers in a water bath to minimize fluctuations in temperature.
In this experiment, you will use pea seeds. In a seed, like the yellow peas shown in Figure 8.4, a
tough coat protects the plant embryo. Nutrients in the form of starch and lipids surround the
embryo and support its germination, or growth from seed, until the appearance of
photosynthetic structures. Seeds are normally dormant, that is metabolically inactive, until the
environmental conditions helpful for growth are available. In order to bring the seeds to an
active state, (out of dormancy), the seeds you will use were soaked in water via a process called
imbibition, for 6 to 8 days.

Figure 8.4: Dried yellow peas are the seeds of a variety of the plant Pisum sativum and are considered a staple
food. They contain yellow cotyledons and a large reserve of starch.

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Cellular respiration involves three major sequential stages: glycolysis, the citric acid cycle, and
oxidative phosphorylation. Oxygen serves as a terminal electron acceptor. Glycolysis takes place
in the cytoplasm whereas mitochondria are the site of the citric acid cycle and the electron
transport chain.
All the steps of respiration are mediated by enzymes, biological catalysts—mainly
proteins—that lower the activation energy, the energy required to be available in a system
before a chemical reaction can take place. Enzymes are not used up by the reactions they
catalyzed. The process of respiration responds to the same environmental factors that affect the
activity of enzymes. In this activity, you will measure the effect of temperature on respiration
rates.
Safety Precautions
● Handle test tubes or glass containers with care; insert the plug by holding the
container in a paper towel.
● Use plastic pipettes rather than glass pipettes.
● Wear goggles or safety glasses.
● Wear gloves when working with KOH or lime [Ca(OH)2] that are corrosive chemical
compounds.
● Use care while handling hot water. Wear mitts and do not leave boiling water or a
hot plate unattended.
● Protect your clothes with an apron.
● Inform your teacher immediately of any broken glassware as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
● Wash your hands with soap and water after completion of the activity.
For this activity, you will need the following:
● Dried yellow peas
● Glass beads
● Balance and weigh boats
● Paper towels to imbibe seeds
● KOH or lime water
● Food coloring
● Absorbent and nonabsorbent cotton

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 ● Drilled rubber stoppers which fit the opening of the test tubes or bottles
● 1-ml plastic pipettes
● Top loading balance
● Thermometers
● Water baths
● Weights such as clamps or hex keys
● Wide glass test tubes or bottles
● Stirring rod
● Ice
● Hot plate to boil water
● Mitts
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● Remind students of the importance of lab safety and that they should wear appropriate
clothing as well as shoes to the lab for each period. Additionally, since a hot plate is
being used for this activity, it is important to also underscore the importance of the
proper use of this piece of equipment to avoid burn injuries and possible fire hazards.
● Provide the students with instructions on the popular handling of mercury
thermometers as well as what to do in the event that one breaks during lab.
● Be sure to have any extra materials available while prepping for this experiment.
Students working in pairs may require a bucket or other carrying container for their
materials (i.e., pipettes, seeds, ice, etc.)
● Since the first part of this activity requires students to utilize a respirometer, it will be
useful to demonstrate the proper use and setup of this equipment to the students prior
to letting them begin their experiments.
● Be sure to have three water baths set up ahead of time at the following temperatures:
10°C, room temperature (see Structured Inquiry), and 50°C. You will want to check the
water baths periodically to ensure that the temperatures remain stable.

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The following are recommendations for the Structured Inquiry for Activity 2:
● Students could remain in groups of two while doing the Guided Inquiry project, but you
may wish to have each student write their own redesign of the experiment. This is to
ensure that each student contributes equally to a group project.
● You will want to check the student’s hypothesis and initial experimental design during
the Structured Inquiry before allowing them to proceed with performing their
experiment. This will help to ensure that they are performing an experiment that can
actually be conducted in your lab, given the available equipment and time you have
allotted for this activity.
● Students often will not include all vital components of an experiment due to their
inexperience. Therefore, they will also likely need advice on how to determine what
controls to use for their experiment as well as identifying their independent and
dependent variables.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 4, clarify to students how to analyze their results. Often students don’t
understand the importance of why they must perform experiments multiple times or
have multiple variables, controls, etc., therefore remind them of the importance of the
reproducibility of data when trying to analyze results for scientific significance. Instruct
them on how to determine if any human error could have been introduced into the
experiment that could have skewed the results.
● Providing a grading rubric for this activity with criteria describing each of the mandatory
parts of this activity can help guide students to include the proper components in their
experiment for this activity.
The following are recommendations for the Guided Inquiry for Activity 2:
● You will want to check the water baths periodically to ensure that the temperatures
remain stable.

To ensure that the respirometers are working properly, you may want to instruct the
students on how to test for leaks by immersing the respirometer with the plug and
pipette before filling it with reagents and cotton.
● Students often will not include all vital components of an experiment due to their
inexperience. Therefore, they will also likely need advice on how to determine what
controls to use for their experiment as well as identifying their independent and
dependent variables during the guided portion of the exercise.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.

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 ● In Step 4, clarify to students how to analyze their results. Often students don’t
understand the importance of why they must perform experiments multiple times or
have multiple variables, controls, etc., therefore remind them of the importance of the
reproducibility of data when trying to analyze results for scientific significance. Instruct
them on how to graph the changes in gas volumes from the data in your table for all
three temperatures for the experimental and control set-up.
● Providing a grading rubric for this activity with criteria describing each of the mandatory
parts of this activity can help guide students to include the proper components in their
experiment for this activity.

Structured Inquiry
Step 1: Obtain 25-30 germinating peas, dry peas and glass beads to start your experiment. Place
the germinating peas in a weigh boat and measure their weight. Record the weight in your
notebook and then repeat for the dried peas and glass beads.
Step 2: In this activity, you will indirectly measure the rate of respiration of the peas by
monitoring the decrease in gas when the peas are placed in the respirometer chamber. What
gas will decrease in the chamber as the peas undergo respiration? Hypothesize how much the
gas levels will likely change for the germinating seeds, dry seeds, and glass beads. Record your
hypotheses and predictions in your notebook.
Step 3: Student-Led Planning: Which of your treatments serve as a control? Is this a positive or
negative control? How will this control reveal whether or not the experiment is functioning
properly? Write your answers in your notebook.
Step 4: Assemble a respirometer using Figure 8.5 as a guide and following the steps below.

145
 Figure 8.5: Assembled respirometer.

a. In a wide test tube (or bottle), drop a pad of absorbent cotton. Pack down the
cotton with a stirring rod. Add lime water Ca(OH)2, being careful not to
oversaturate the pad or drip the lime water on the side of the tube.
b. Insert a thin layer of nonabsorbent cotton, pushing down with the glass rod. The
cotton protects the seeds from lime water; however, if it is too thick, it will
interfere with the diffusion of CO2.
c. Plug the test tube with a bored rubber stopper. Add a drop of colored water in a
1-ml graduated pipette and insert the pipette in the hole of the stopper. Adjust
the position of the drop by inserting a syringe in the stopper until you can easily
read the position of the dye. (The syringe is not shown in Figure 8.5.) Rub some
petroleum jelly where the pipette comes into contact with the rubber stopper.
The respirometer must be water tight to yield reliable results. It is also possible
to wrap the openings with stretchable plastic film.
d. You may want to test for leaks by immersing the respirometer with the plug and
pipette before filling it with reagents and cotton.
Step 5: Assemble the respirometer containing the control sample in the same manner.

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Step 6: Immerse the respirometers with the experimental sample and the control in the water
bath. Lining the water bath with a white paper towel will make it easier to read the markings on
the pipettes. Make sure that the pipettes are resting across a piece of ribbon or string that
spans the width of the water bath, as illustrated in Figure 8.6. The goal is to keep the pipettes
out of the water while the test tubes remain submerged.

Figure 8.6: Respirometers in water bath.

Step 7: Let the respirometers equilibrate for 5–10 minutes.

Step 8: Read the starting volume on the pipette. This is time 0 min. Record the displacement of
the colored bead for all samples every 2 minutes for 20 minutes and enter data in a table of
measurements.
Step 9: Critical Analysis: Calculate the changes in volume where the reading at time 0 is
subtracted from every subsequent reading. Subtract the rate of volume change measured in the
control samples to obtain a corrected rate of respiration. Graph the changes in volume in
respirometers as a function of time and calculate the rate of change from the slopes of the line
plots. Calculate the rate of change per gram of seed. This will allow you to compare values
obtained from different samples. Draw a plot of changes in gas volumes from the data in your
table. What measurements will you enter on the x-axis? What measurements will you enter in
the y-axis? Determine the rate of respiration in your experiment. How did you use the data from
your control or controls? Did volumes change during the experiment? Which gas caused the
change in volume? Do the results support your hypothesis? Can you explain unexpected
results? Were the respirometers water-tight at all times? How could you modify the experiment
in the future? Write your answers in your notebook.

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Guided Inquiry
Step 1: Repeat the steps to set up the respirometers described in the Activity 2 Structured
Inquiry. Use three water baths at the following temperatures: 10°C, room temperature (see
Structured Inquiry), and 50°C.
Step 2: Hypothesize/Predict: Discuss with your partner what kind of influence temperature
might have on metabolic processes. How would respiration rate measured at 10°C compare to
the rate measured at room temperature? Will the rate of respiration be higher at 30°C than
room temperature? Do you predict that the rate of respiration will be higher at 50°C than at
room temperature or 30°C? Enter your hypotheses in your notebook.
Step 3: Student-Led Planning: You will now measure the rate of respiration at three different
temperatures. Discuss with your partner if you need to run the experiment at room
temperature again. Decide which control you will set up for this experiment. Make a note of all
the steps you will perform, as you did in Activity 2, and create tables for your observations in
your lab notebook. You will take readings of the colored water bubble at 2-minute intervals for
20 minutes. Have your teacher approve your experimental procedure before proceeding.
Step 4: Once approved, carry out your experimental procedure, closely monitoring the
temperature as you take measurements.
Step 5: Critical Analysis: Graph the changes in gas volumes from the data in your table for all three
temperatures for the experimental and control set-up, as you did for the Structured Inquiry.
Determine the rate of respiration for each temperature. Because the gas law shows that differences
in temperature affect volumes, you must correct for any changes in volume that are a consequence
of temperature variations rather than respiration. To do this, subtract changes in volumes measured
in the respirometer containing glass beads from the changes in volume measured in the tubes
containing germinating seeds held at the same temperature. Do the results support your
hypothesis? Explain whether your results support or refute your hypothesis. How could you modify
the experiment in the future? Write your ideas in your notebook.

Assessments
1. Students record changes in gas released from respirometers containing germinating
seeds and dry seeds. They set up their tubes in air rather than in a water bath. A
thermometer probe is inserted in each respirometer. The tube that contains germinating
seeds shows an increase in temperature. No such increase is recorded in a respirometer
that contains dry seeds. What is the reason for the difference in temperature? [SP 1,
Concept Explanation; SP 3, Questions and Methods; SP 6, Argumentation]
2. The ideal gas law shows that volume depends on temperature as well as pressure. Why
do you set your respirometers in a water bath? [SP 1, Concept Explanation; SP 3,
Questions and Methods]

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 3. A classmate insists that there are no mitochondria in leaves because chloroplasts
produce ATP through photosynthesis. How would you experimentally disprove this
claim? [SP 3, Questions and Methods; SP 6, Argumentation]
[Answer Key]
1. Respiration is an exergonic reaction. The metabolically active seeds release heat as well
as produce ATP.
2. Water has a high heat capacity. By immersing the respirometer in a comparatively large
volume of water, we buffered the change in temperature.
3. Set up a respirometer with leaves in the chamber. Tightly wrap the respirometer in
aluminum foil to block all light. Measure the changes in gas over time. The changes are
due to oxygen consumption because there is no photosynthesis. You may also use Janus
Green B to make visible mitochondria which are noticeably smaller than chloroplasts.

149
 Lab 9: Fermentation in Yeast
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
ENE-1 The highly ENE-1.E.1 Enzymes are
complex organization biological catalysts that
of living systems ENE-1.E Explain how facilitate chemical
3.2 Enzyme Catalysis requires constant input enzymes affect the rate reactions in cells and
of energy and the of biological reactions. contribute to the
exchange of regulation of biological
macromolecules. processes.
ENE-1.H.1 All living
systems require
constant input of
energy.
ENE-1.H.2 Life requires
ENE-1 The highly
a highly ordered system
complex organization
and does not violate
of living systems ENE-1.H Describe the
the second law of
3.4 Cellular Energy requires constant input role of energy in living
thermodynamics;
of energy and the organisms.
energy input must
exchange of
exceed energy loss.
macromolecules.
ENE-1.H.3 Energy
pathways are
sequential for a more
efficient transfer of
energy.
ENE-1.K.1
ENE-1.K Describe the Fermentation and
processes that allow cellular respiration,
organisms to use characteristic of all life,
ENE-1 The highly energy stored in use energy from
complex organization biological biological
of living systems macromolecules. macromolecules to
3.6 Cellular Respiration requires constant input produce ATP.
of energy and the ENE-1.L Explain how ENE-1.L.6 Fermentation
exchange of cells obtain energy allows glycolysis to
macromolecules. from biological proceed in the absence
macromolecules in of oxygen and produces
order to power cellular organic molecules,
functions. including alcohol and
lactic acid.

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 SCIENCE PRACTICES
Science Practices Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and models in applied contexts.
models presented in written format.
2.A Describe characteristics of a biological
concept, processes, or model represented
SP 2 Visual Representations visually.
2.B Explain relationships between different
Analyze visual representations of biological characteristics of biological concepts, processes,
concepts and processes. or models represented visually in applied
contexts.
2.D Represent relationships within biological
models, including diagrams and flowcharts.
3.A Identify or pose a testable question based on
an observation, data, or model.
3.B State the null and alternative hypotheses, or
predict the results of an experiment.
SP 3 Questions and Methods
3.C Identify experimental procedures aligned to
the question, including variables and controls.
Determine scientific questions and models.
3.D. Make observations and collect data.
3.E Propose a new/investigation based on
evaluation of evidence from a previous
experiment.
SP 4 Representing and Describing Data
4.A Construct a graph, plot or chart.
4.B Describe data from a table or graph.
Represent and describe data.
5.A Perform mathematical calculations, including
SP 5 Statistical Tests and Data Analysis equations from the curriculum and rates.
5.B Use confidence level intervals and/or error
Perform statistical tests and mathematical bars to determine whether sample means are
calculations to analyze and interpret data. statistically different.
5.C Use data to evaluate a hypothesis.
6.A. Make a scientific claim.
6.B Support a claim with evidence.
6.C. Provide reasoning to justify a claim by
SP 6 Argumentation connecting evidence to biological theories.
6.D Explain the relationship between
Develop and justify scientific arguments using experimental results and larger biological
evidence. concepts, processes, or theories.
6.E Predict the causes of effects of a change in
one or more components in a biological system
based on data.

151
 Non-photosynthetic organisms extract and store energy from organic molecules, such as
glucose. The chemical energy in these molecules is used to make ATP, the energy source that
allows cells to function. The transfer of the chemical energy in food to ATP defines the process
of cellular respiration. Two types of cellular respiration are aerobic respiration and
fermentation. Aerobic respiration depends on the presence of oxygen as a final electron
acceptor and yields large quantities of ATP from each organic molecule. Due to its dependence
on oxygen, it is called an aerobic mechanism. Fermentation also yields ATP from organic
molecules but occurs without the presence of oxygen. Fermentation is therefore called an
anaerobic mechanism. In the presence of increased oxygen, cells of organisms capable of both
processes will transition from fermentation to cellular respiration, because it is a more efficient
way to obtain larger quantities of energy. The scientist, Louis Pasteur, was the first to observe
this pattern, and therefore, it is called the Pasteur Effect.
Some organisms, such as yeast, can utilize both processes but it is more efficient for
them to perform aerobic respiration as opposed to fermentation, as aerobic respiration
produces more ATP per organic molecule. However, when oxygen levels are low or inadequate,
these cells can switch to fermentation in order to keep producing ATP. In addition to yeast, some
human cells, such as our muscle cells, can switch from aerobic respiration to fermentation for
brief periods of time. You may be familiar with sore muscles or cramps from exercise, which are
caused by the lactic acid produced by fermentation in our muscle cells. In this lab you will
explore fermentation in yeast cells.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Begin the lab by giving the students a brief overview on the topic of the two types of
cellular respiration, along with examples of each. You can utilize real life examples such
as on the ones mentioned in the lab’s introduction to make it more understandable to
the students. For instance, you can have the students do a quick one-minute workout in
class (i.e., jumping jacks) that may exert them to the point of producing lactic acid to
illustrate your point further.
● Underscore the importance of proper lab safety for your students since they will be
working with a living microorganism (dry yeast).
● Instruct students on the proper way to dispose of their yeast samples, Petri dishes (if
disposable) into a biohazard container, if you have one present in the lab.

In this lab, you will learn

● how different carbon compounds affect the rate of fermentation;
● how temperature affects the rate of fermentation.

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Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: What is the difference between real
sugar and a sugar substitute? Could either substance be used during cellular respiration?
2. Answer the following questions in your notebook: How could you tell that an organism,
such as yeast, has switched from aerobic respiration to fermentation?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 1: Comparing Rates of Fermentation in Yeast

Yeast carry out fermentation as a means to access the chemical energy from their food,
which, in this case, will be sugars such as glucose. In this activity, you will be comparing
fermentation between these food sources and a control group of water. Yeast can exist in a state
of dormancy, where they are alive, but their physical activity has temporarily stopped to
minimize energy use. This adaptation allows these organisms to survive extended periods of
drought and other harsh environmental conditions. As you add water to the dry yeast, you will
be activating the cells out of dormancy, and they will resume physical activity and begin eating.
Once the yeast absorbs the food molecule, it will first break the molecule down in a process
called glycolysis. The word glycolysis literally translates into the breaking apart of glucose and
this is the first step of the metabolism of sugars (Figure 9.1).
Shortly after mixing the activated yeast with the sugar molecules, fermentation will
begin, and you will be able to observe gas bubbles being produced. This gas is carbon dioxide,
one of the products of fermentation.

Figure 9.1: Glycolysis is the first step of both aerobic respiration and fermentation. Aerobic respiration only
produces carbon dioxide as a toxic byproduct, but fermentation also produces alcohol or lactate.

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Safety Precautions
● Use care when using glassware.
● Be careful when inverting the graduated cylinder into a Petri dish.
● Inform your teacher immediately of any broken or cracked glassware, as it could cause
injuries.
● Clean up any spilled liquids to prevent people from slipping.
For this activity, you will need the following:
● Graduated cylinder (50 ml)
● Petri dishes
● Beaker/container for yeast solution (150–200 ml)
● One package of dry yeast
● Warm water
● Room temperature water
● Glucose
● A sugar substitute
● Pipette or droppers
● Paraffin wax paper
● Paper towels
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Demonstrate how and where to properly dispose of the reconstituted yeast and it’s
metabolic by products prior to letting students begin their experiments. Continue to
remind the students of the importance of lab safety. Since pipettes are being used,
remind them to never pipet anything by mouth.
● Since the yeast will be producing alcohol as a byproduct of fermentation, it may be
interesting to have the students smell the alcohol produced as a result of the anaerobic
respiration process. If you choose to do this, it is highly recommended to teach students
how to do the waft test in lieu of directly smelling the chemical agent. Please
demonstrate this prior to initiation of the experiments, as you may have students who
may be learning how to do this for the first time.
● Remind students of the locations of the following lab safety items in your lab in the
event they need them during this activity: first aid kit, emergency exits, shower, fire
extinguisher, eyewash, waste containers (broken-glass waste), safety glasses bin, gloves,
and any other safety equipment and supplies in the lab.

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 ● Have students create their data tables prior to beginning the lab exercise, to ensure that
they are including the appropriate criteria in their lab reports for later analysis.
● Remind students that they are to perform the tests with each of the variables (i.e., sugar
vs sugar substitute) multiple times, per the Scientific Method.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 1:
● Have each student write their own hypotheses regarding the proposed outcomes of the
experiment. This is to ensure that each student contributes equally and submit their
own original content.
● Check the students’ hypotheses and results from the initial experimental design during
the Structured Inquiry before allowing them to proceed with performing their
experiment. This will help to ensure that they are on the right track and will not waste
too many materials not needed for the activity.
● Offer students advice on how to determine what controls to use for their experiment as
well as identifying their independent and dependent variables. Students often will not
include all vital components of an experiment due to their inexperience.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 3, clarify to students how to analyze their results. Additionally, providing a
grading rubric for this activity with criteria describing each of the mandatory parts of this
activity can help guide students to include the proper components in their experiment
for this activity.

Structured Inquiry
Step 1: Add the yeast packet to the beaker and add 100 ml of warm water to activate it. Swirl
slightly until water and yeast are mixed. Allow 10 minutes for activation. During this time, create
a data table to measure the volume in milliliters of the carbon dioxide gas bubbles as a function
of time. Include space for two trials for each variable of water, glucose, and the sugar substitute,
as well as predictions of the amount of respiration that will occur for each sugar.
Step 2: Hypothesize/Predict: Based on what you know about the differences between water,
glucose, and the sugar substitute, predict how much carbon dioxide concentrations will differ in
the presence of 1) glucose, 2) the sugar substitute, and 3) water. Add your predictions to the
data table created in Step 1.

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Step 3: Student-Led Planning: You will now start with the first trial of measuring carbon dioxide
gas bubbles produced from fermentation. Choose a time interval to take down measurements.
After the first trial, adjust the time intervals if necessary for taking down measurements. These
should be in your data table. Determine and record the quantity of glucose and sugar substitute
that you will use, as you should perform more than one trial of each test.
Step 4: Pour 15 ml of yeast solution into the graduated cylinder. Then add in the water and fill
up the rest of the cylinder. For the other trials, glucose or sugar substitute will be added instead
of water. As soon as you add the water, cover the cylinder with paraffin wax paper and invert it,
to avoid spilling the fluid. One person can hold the graduated cylinder in place displacing it
carefully and completely onto the Petri dish, as shown in Figure 9.2. As fermentation takes
place, bubbles will rise into the cylinder, and the volume of the bubbles can be recorded. To do
this, you will read the measurement markings at either end of the bubbles to get at total
volume reading.
Step 5: Student-Led Planning: Discuss with your partner how the first trial worked and whether
any adjustments need to be made to the set-up to ensure more accurate results. If the original
trial did not go well due to measurement issues, repeat after adjusting the procedure. Then use
the corrected trial as the first one. All subsequent trials will then use the same exact procedure.

Figure 9.2: As fermentation takes place, carbon dioxide bubbles will rise into the cylinder. Volume measurements
can be taken at specific time increments.

Step 6: Critical Analysis: Record the amount of carbon dioxide gas produced in your data table.
Are the predictions made in Step 2 supported by the data? Why or why not? What is the control
in this experiment? Is it a positive or negative control and why? What changes to the quantities
of liquids, time period, or set up can you make to improve your results? Discuss with your
partner and write your answers in your notebook.

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Guided Inquiry
Step 1: Hypothesize/Predict: How do you think the chemical make-up of the compound added
to the yeast solution affects the rate of fermentation? How different do you think the results
will be with the glucose and sugar substitute trials? Write your hypotheses in your notebook.
Step 2: Student-Led Planning: Make any adjustments to your first trial set up and re-do if
necessary. Then, carry out the second trial for water and two additional trials each using
glucose and sugar substitute. Record your measurements in the data table.
Step 3: Critical Analysis: Which compound led to the highest rate of fermentation? Was there a
significant difference between the glucose and sugar substitute trials? Discuss your answers
with your partner and write them in your notebook.

Assessments
1. What does data from the experiment show about the energy needs of yeast? [SP 5, Data
Analysis; SP 6, Argumentation]
2. What do you think would happen to the activated yeast if no carbon-based molecules
were provided? [SP 6, Argumentation]
3. What environmental reasons would cause yeast to go dormant? [SP 1, Concept
Explanation; SP 6, Argumentation]
[Answer Key]
1. Possible answer: Yeast will absorb and process carbon-based food molecules to access
energy that they require to survive and carry out all cellular functions.
2. Possible answer: Without carbon-based molecules, the yeast could not undergo cellular
respiration. Eventually, the water would evaporate and the yeast would return to a
dormant state.
3. Yeast may go dormant if there is a lack of food molecules available, which would prevent
them from undergoing a high rate of cellular respiration.

Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: How does temperature affect the
rate of chemical reactions? Explain your answer.
2. Answer the following questions in your notebook: What would likely influence the
optimum temperature of cellular respiration for an organism? Do you think there is a
temperature that cellular respiration would cease and what would determine this upper
temperature limit for cellular respiration?
3. Discuss the answers to questions 1 and 2 with the class.

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Activity 2: Dependence of Fermentation on Temperature
All cellular processes, like fermentation, consist of an interconnected series of chemical
reactions. However, temperature can influence the rate of chemical reactions by affecting how
quickly the reactants move, and therefore, how often they collide with each other. If the
temperature gets too high, enzymes or other cellular proteins involved with cellular respiration
can break apart, or denature, rendering them inactive. Therefore, it is very important for cells
and organisms to regulate their internal temperature to ensure that cellular respiration and
other chemical reactions can continue at the proper rate.
Safety Precautions
● Use protection when handling hot glass and materials.
● Do not mix very hot water with very cold water in glass containers.
● Clean up any spilled liquids to prevent slipping.
For this activity, you will need the following:
● Graduated cylinders
● Water
● Ice
● Thermometer
● Beakers
● Sugar substitute solution
● Yeast solution (one packet of yeast with 100 ml of water; can use from Activity 1)
● Hot plate
● Droppers or pipettes
● Medium-size, un-inflated balloons
● Cloth measuring tape or pieces of string and a ruler
● Balloons

For this activity, you will work in pairs.

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Activity 2 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 2:
● Demonstrate how to properly utilize this equipment prior to letting students begin their
experiments. Continue to remind the students of the importance of lab safety and first
aid in the event of a burn injury.
● Demonstrate how to apply the balloons to the graduated syllabus. If students are not
familiar with the concept of denaturation, a brief overview on this topic should also be
given prior to the experiment, preferably during the initial lab lecture/setup discussion.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● Demonstrate how to measure the circumference of the balloons prior to beginning the
lab exercise, to ensure that students are utilizing proper technique for their data yields.
● Remind students that they are to perform the tests with each of the variables (i.e., sugar
vs. sugar substitute) multiple times, per the Scientific Method.
● Since students are recording results at different time intervals, it will be useful to have
multiple timers present for this exercise.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 2:
● Have each student write their own hypotheses regarding the proposed outcomes of the
experiment. This is to ensure that each student contributes equally and submit their
own original content.
● Advise the students to develop their proposed experimental design prior to allowing
them to proceed with performing their experiment. This will help to ensure that they are
on the right track and will not waste too many materials not needed for the activity.
● Remind students to include controls for their experiment as well to identify the
independent and dependent variables.
● Instruct students to create their data table prior to conducting their experiment so that
it can be already prepared when they need to record their results.
● Remind students to wait until the temperature measurements stabilize before placing
their beakers into the various water baths for acquisition of their results.
● In Step 3, clarify to students how to analyze their results. Additionally, providing a
grading rubric for this activity with criteria describing each of the mandatory parts of this
activity can help guide students to include the proper components in their experiment
for this activity.

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Structured Inquiry
Step 1: Hypothesize/Predict: Unlike Activity 1, you will measure the volume of carbon dioxide
gas by placing balloons over the graduated cylinders after the reactants are added. Use 10 ml of
each reactant. As the balloons fill with gas, you can measure their circumference to find the
volume. How do you think temperature will affect the rate of fermentation? How can this be
tested? Write your hypotheses in your notebook.
Step 2: Student-Led Planning: In this lab, you will measure the changes in the circumference of
a balloon. Decide, with your partner, how you will take this measurement using a practice
balloon that you inflate with your mouth. Make sure that your measurement is repeatable
regardless of the size of the balloon. Create a data table for your measurements of balloon
circumference versus solution temperature. You should record the volume of the gas in the
balloon every 2 minutes for 10-15 minutes per trial.
Step 3: Prepare the first beaker with 100 ml of water at room temperature, approximately
21 °C. Add cold or hot water to get the water as close to 21 °C as possible. Once the
temperature is stable, place the graduated cylinder containing the yeast and sugar substitute
directly into the water beaker. Be sure to record the actual temperature of your room
temperature solution in your notebook.
Step 4: Repeat Step 3 for the cold and hot water beakers one at a time. For the cold water
treatment, fill the beaker with cold water and then add ice. Wait until the temperature stabilizes
before beginning the timer. For the hot water beaker, bring the water to a boil using a hot plate.
Then, turn down the hot plate to maintain the water at a low boil. Record the stabilized
temperatures for both treatments before immersing the graduated cylinders containing the
yeast into the beakers.
Step 5: Critical Analysis: Does this set up seem to capture the carbon dioxide gas effectively?
Should you adjust the quantities of any of the liquids and/or the time intervals? Discuss with
your partner and then write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: How do you think the more extreme temperatures, such as
(~ 0–4 °C) and (55–60 °C) will affect the rate of fermentation? Are there any new safety
concerns to consider with such changes to the temperature?
Step 2: Student-Led Planning: Pick two additional temperatures to test within the range
(~ 0–4 °C) and (55–60 °C). Use the water, ice, hot plate, and thermometer to make these baths
before adding the graduated cylinders containing the yeast and sugar substitute solutions. Then
perform your trials as in the structured inquiry, measuring the volume of gas in the balloon.
Create appropriate data tables before beginning your trials.

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Step 3: Critical Analysis: How did temperature affect the rate of fermentation for this species of
yeast? Is it likely any of the proteins involved in the reaction denatured and at what
temperature did this occur? Graph the circumference of the balloon versus the temperature of
the reaction. Based on your graph, what is the best estimate of the optimal temperature for
fermentation in yeast? Discuss your answers with your partner and write them in your
notebook.

Assessments
1. What likely caused the differences in reaction rate at the three temperatures examined
in Activity 2? [SP 1, Concept Explanation; SP 6, Argumentation]
2. How could this experiment better measure the effect of temperature changes in the
natural habitat of the yeast? [SP 6, Argumentation]
3. What other biotic and abiotic factors would likely affect the rate of cellular respiration in
yeast? Explain your answers. [SP 6, Argumentation; SP 1, Concept Explanation]
4. What patterns can you identify in the data you collected between the yeast and the
abiotic factor of temperature? [SP 4, Representing and Describing Data]
[Answer Key]
1. The reaction rate was slow at the coldest temperature because the reactants were
moving slowly and had a low rate of collision. The reaction rate was fastest at room
temperature because the reactants were moving fast and the enzymes were still
functioning. At the highest temperature, no reaction occurred because the enzymes had
likely denatured.
2. Rather than exposing the yeast to a single temperature at a time, the same sample of
yeast could be exposed to a steady temperature change, during which fermentation
rates are constantly monitored.
3. Anything that affects reaction rate would affect the rate of cellular respiration, including
the concentration of the reactants and the concentration of the enzymes
4. Student answers will vary, but will most likely show an increase in fermentation rates
with warmer temperatures, to a point where protein denaturing took place.

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 Lab 10: Plant Pigments
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
SYI-1.D Describe the SYI-1.D.4 Chloroplasts are
SYI-1 Living systems
2.1 Cell Structure: structure and/or specialized organelles that are
are organized in a
Subcellular function of subcellular found in photosynthetic algae
hierarchy of structural
Components components and and plants and have a double
levels that interact.
organelles. outer membrane.
SYI-1.F.1 The folding of the
inner membrane increases the
surface area, which allows for
more ATP to be synthesized.
SYI-1.F Describe the SYI-1.F.2 Within the
SYI-1 Living systems
structural features of a chloroplasts are thylakoids and
2.2 Cell Structure are organized in a
cell that allow the stroma.
and Function hierarchy of structural
organisms to capture, SYI-1.F.3 The thylakoids are
levels that interact.
store, and use energy. organized in stacks.
SYI-1.F.4 Membranes contain
chlorophyll pigments and
electron transport proteins that
comprise the photosystems.
ENE-1 The highly
complex organization
of living systems ENE-1.H Describe the ENE-1.H.1 All living systems
3.4 Cellular
requires constant role of energy in living require constant input of
Energy
input of energy and organisms. energy.
the exchange of
macromolecules.
ENE-1.I Describe the
photosynthetic
ENE-1 The highly processes that allow
ENE-1.I.1 Photosynthesis
complex organization organisms to capture
captures energy from the sun
of living systems and store energy.
3.5 and produces sugars.
requires constant ENE-1.J Explain how
Photosynthesis ENE-1.J.1 During
input of energy and cells capture energy
photosynthesis, chlorophylls
the exchange of from light and transfer
absorb energy from light.
macromolecules. it to biological
molecules for storage
and use.

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 SCIENCE PRACTICES
Science Practices Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.A Describe characteristics of a biological

concept, processes, or model represented
SP 2 Visual Representations visually.
2.B Explain relationships between different
Analyze visual representations of biological characteristics of biological concepts, processes,
concepts and processes. or models represented visually in applied
contexts.
2.D Represent relationships within biological
models, including diagrams and flowcharts.
3.A Identify or pose a testable question based on
an observation, data, or model.
3.B State the null and alternative hypotheses, or
predict the results of an experiment.
SP 3 Questions and Methods
3.C Identify experimental procedures aligned to
the question, including variables and controls.
Determine scientific questions and models.
3.D. Make observations and collect data.
3.E Propose a new/investigation based on
evaluation of evidence from a previous
experiment.
SP 4 Representing and Describing Data
4.B Describe data from a table, diagram, or graph.
Represent and describe data.

SP 5 Statistical Tests and Data Analysis

5.A Perform mathematical calculations, including
equations from the curriculum.
Perform statistical tests and mathematical
calculations to analyze and interpret data.

6.A. Make a scientific claim.

6.B Support a claim with evidence.
SP 6 Argumentation
6.C. Provide reasoning to justify a claim by
connecting evidence to biological theories.
Develop and justify scientific arguments using
6.E Predict the causes of effects of a change in
evidence.
one or more components in a biological system
based on data.

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 Photoautotrophs such as plants, algae, and photosynthetic bacteria transform the
energy from sunlight into chemical energy that is used to power life activities. The sun emits a
wide range of electromagnetic waves. However, photosynthetic pigments only absorb within
the narrow range of visible light, from 380 nm to 700 nm, which is referred to as
photosynthetically active radiation (PAR).
Photons must be absorbed before light energy can be converted into chemical energy.
Arrays of pigments, which are brightly colored chemical compounds, absorb specific
wavelengths of photons while other wavelengths are reflected. The color we see is determined
by the wavelengths of light that are not absorbed, but reflected off the structure, as shown in
Figure 10.1. For example; Spinach leaves look green because they reflect green wavelengths of
visible light.

Figure 10.1: Absorption spectra of chlorophyll a, chlorophyll b, and beta-carotene, which are photosynthetic
pigments in land plants. Which color of light do you expect to be the most efficient for photosynthesis?

Chlorophyll a is the molecule responsible for the transformation of light energy into
chemical energy in photosynthetic plants. Most pigment molecules in photoautotrophs are not
actually involved in the photosynthesis. These accessory pigments can be compared to the
satellite dishes that collect radio waves. They capture photons from a broad range of
wavelengths and re-emit them to special chlorophyll-protein complexes called reaction centers.
The photons are eventually absorbed by chlorophyll a in the reaction center, exciting its
electrons to a higher energy state. The ensuing flow of high-energy electrons through an
electron transport chain produces the ATP and NADPH that fuels the conversion of carbon
dioxide into organic molecules.

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 As shown in Figure 10.2, many types of photosynthetic pigments are found in nature
giving plants and algae their characteristic colors. They promote photosynthesis by widening the
spectrum of visible light that can be used for photosynthesis. Chlorophylls and carotenoids are
present in all land plants, algae, and cyanobacteria. Carotenoids fulfill the dual role of accessory
and protective pigments. They prevent photo damage caused by intense light energy.

Figure 10.2: Molecular structures of plant pigments. The pigments display characteristic colors as follows: (a)
chlorophyll a is green-blue, (b) chlorophyll b is olive green, (c) carotenoids are yellow-orange, (d) and xanthophylls
are lemon yellow

What is the main environmental factor that determines which colored pigments are
present in a photosynthetic organism? In this lab, you will make predictions about the pigments
that are present in a plant leaf and study the pigments found in leaves by separating them on
paper per their solubility in a solvent system.

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Instructor Introduction Notes
The following are recommendations for introducing this lab’s background content to your
students:
● Show a diagram of a chloroplast. Explain that energy from sunlight is captured in the
light reactions, which take place in the thylakoid membrane. This energy is used to make
sugars in the dark reactions, which take place in the stroma.
● Show a diagram of the thylakoid membrane. Explain that light is captured by accessory
pigments and re-emitted to chlorophyll a in the reaction center. Chlorophyll a shuttles
electrons into the electron transport chain, generating ATP and NADPH.
● Ask students why accessory pigments are needed. Response should indicate that these
pigments can capture different wavelengths of light than chlorophyll a, increasing the
total amount of sunlight that the plant can use.
● Explain that all photosynthetic tissue contains chlorophyll a, but the amount of other
pigments present can vary. As a result, the color of photosynthetic tissue can vary.

In this lab, you will learn

● how to extract pigments from plant material;
● how to separate pigments by paper chromatography;
● how to measure Rf (retention factor) values for pigments.

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: Think about what colors you have
seen in leaves of plants. What determines the color of a leaf? What color or colors do
you expect to see if you extract pigments from leaves and separate them chemically?
2. Answer the following question in your notebook: Other structures in plants besides
leaves are brightly colored. Fruit, flowers, and some roots display colors. Are these
colors associated with photosynthesis? What are the purposes of those colors?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 1: Extracting Pigments from Plant Material

The chloroplast, as illustrated in Figure 10.3, is the photosynthetic organelle in the plant
cell. The chloroplast is surrounded by a double membrane. Within the inner membrane is a fluid
compartment called the stroma. The thylakoids form a complex network of membranes that
appear as stacked disks called grana (singular, granum). The photosystems, and photosynthetic
pigments they contain, are embedded in the thylakoid membrane. Carbon fixation takes place

166
in the stroma. What chemical properties would you expect photosynthetic pigments to display
and how can these chemical properties be used to extract the pigments from tissues? How do
the solubility properties enable scientists to separate individual pigments for analysis?

Figure 10.3: A section through a chloroplast shows the arrangement of the thylakoid membranes in stacks and
single membranes. The thylakoids are pockets enclosed by membranes.

Safety Precautions
● Wash your hands after completion of this activity.
For this activity, you will need the following:
● Plant material: intact leaves of spinach and Coleus (one leaf of each per pair of students)
● Filter or chromatography paper
● Ruler (one per group)
● Coins
● Pencils
● Colored pencils
● Forceps
● Scissors

In this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Show examples of plant tissue that is colored by different pigments. For example, the
orange color in carrots is produced by carotenoids; the red color in grapes is produced
by anthocyanins; the green color in spinach is produced by chlorophyll.

167
 ● Ask students whether two colors are visible after paint has been mixed (no). Based on
this answer and on what they know about plant pigments, ask students to predict
whether more than one pigment will be present in the leaves they will be testing, and to
predict the color of the pigments.
● Demonstrate how to rub the coin on the leaf.

Structured Inquiry
Step 1: Hypothesize/Predict: In this activity, you will extract pigments from spinach leaves.
Discuss and predict what colored pigments you will observe after extraction. Write your
hypotheses in your notebook.
Step 2: Student-led Planning: Assemble all the material needed for extracting pigments from
spinach leaves. Read the procedure in Step 3 and Figure 10.4 to extract pigments from plant
material. Throughout the procedure, you will need to avoid touching the paper with your
fingers, because oils from your skin can interfere with the chromatography. Handle the paper by
the edges. Devise a method for handling the paper with your partner. Use a few scraps of paper
to practice extracting pigments from loose leaves. If you smear pigments all over the piece of
paper while you apply pigment, the stains will also separate by chromatography and interfere
with the interpretation of the results. Throw any smeared paper away and start over.
As pigments separate along the paper, the color intensity of each pigment decreases and makes
it more difficult to identify each color. What approach will you use to obtain a dark line of
concentrated pigment on the paper?
Step 3: Complete the steps below to extract pigments from plant material, as illustrated in
Figure 10.4. Use the method you devised in Step 2 to avoid touching the paper with your
fingers.
1. Measure the height and the diameter (D) of the container with your partner to
determine the size of the piece of chromatography paper you need for the
experiment. Subtract 1 cm from the height of the container. The width of the
chromatography paper should be about 1 cm shorter than the circumference of the
container. Record your calculations and show them to your instructor before you cut
the paper.
2. With a pencil, draw a line across the entire width of filter/chromatography paper
about 1.5 cm from the bottom. This line is called the origin or start of the separation.
It is the place where the sample is applied.
3. Select 1 large spinach leaf and carefully blot it dry with paper towels. Note in your
lab notebook why it is important to blot dry the leaf.
4. Position the leaf on top of the pencil line leaving a space on each end. The leaf
should reach the edge of the paper.

168
 5. Align a plastic ruler over the leaf with the edges of the pencil line that was left
uncovered by the leaf.
6. Roll a coin pressing down firmly along the ruler edge without crushing the rest of the
leaf and smearing pigment on the surface of the filter paper. You may find it helpful
to lay the filter paper on top of the paper towels taking care not to cover the line
where the pigments are deposited.

Figure 10.4: Extract pigments by rolling a coin along a ruler. Make sure that the leaf does not cover the entire paper
and align the ruler with the starting line to avoid edge effects.

7. Let the pigment line dry for 3 to 5 minutes. Move to a different section of the leaf
and roll the coin again over the same line. Keep the band of pigments as narrow as
possible. The pigments will diffuse during separation and the bands will smear if they
are laid down too thick.
Step 4: Critical Analysis: Record the results from your experiment. Try to identify the pigment or
pigments you extracted. Can you state conclusively what types of pigments are present in your
extract on the line? How could you determine which pigments are present in the extract
without performing a chemical separation? Write your answers in your lab notebook. You may
refer to Figure 10.1 while discussing the answer.

Guided Inquiry
Step 1: Hypothesize/Predict: If pigments are colored molecules, can you decide which pigments
are present by looking at biological material? Do spinach leaves contain other pigments besides
the green chlorophylls? Do Coleus leaves contain additional pigments? Do you see a red
pigment on the line for Coleus? Write your hypotheses in your notebook.

169
Step 2: Student-led Planning: Decide with your lab partner how you will test your hypotheses
on the kinds of pigments you expect to find in your extracts of spinach and Coleus leaves. One
lab partner should use spinach and the other one should use Coleus. Decide with your lab
partner which plant material you will use to test your hypothesis. Extract pigments from leaves
by the coin method described in Step 2 of the Structured Inquiry. Record all the steps you
perform and draw the colors seen on the line in your lab notebook.
Step 3: Critical Analysis: Observe the pigment lines on the paper. Record in your lab notebook
the color or colors you detected. Note the differences, if any, between the spinach and Coleus
samples.

Assessments
1. Plants in the rainforest that live under the canopy of tall trees must adapt to low light
conditions. Can you predict how the pigments of plants that receive low light levels will
differ from the pigments of plants that receive high light levels? What would happen to
the forest if the tall trees are removed for timber? [SP 6, Argumentation]
2. You analyze a pigment extract from a mutant plant and discover that the plant is
defective in its carotenoid biosynthetic pathway. What would happen to that plant if it
is grown in bright sunlight? Where would you expect it to thrive and how would such a
plant compensate for the loss of carotenoids? [SP 6, Argumentation; SP 1, Concept
Explanation]
3. The Rhodophyta are algae that can grow deeper in the ocean than other plants. Based
on the information shown below in Figure 10.5, what color of the visible spectrum
would you expect to be absorbed by an accessory pigment found in Rhodophyta? Why
do you think plants have reddish colors? [SP 6, Argumentation; SP 1, Concept
Explanation]

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[Answer Key]
1. Leaves have wide surfaces and contain large amounts of light-harvesting pigments;
however, the pigments are the same as for other land plants. The adaptive strategy is to
increase the amount of light collected by leaves. If tall trees are removed, the
ecosystem will be damaged, because the lower plants are adapted to low light intensity
and will be bleached by sunlight.
2. The plant would likely survive in the shade, but undergo photobleaching, loss of color
due to light, under intense sunlight. Carotenoids are mostly protective pigments. Plants
will increase the synthesis of chlorophyll a and b and associated light-harvesting
complex proteins.
3. The pigment would absorb in the blue and green region. The pigment itself would be
red. Having red pigments would allow plants to have access to a larger region of the
visible spectrum on land.

Activity 2: Pre-Assessment
1. The leaves of some plants change color in fall. Green foliage appears to turn to hues of
yellow and brown. Does the yellow color appear because carotenoids replace the green
chlorophylls? Explain your reasoning.
2. Examine the molecular structures of photosynthetic pigments in Figure 10.2.
Photosynthetic pigments are hydrophobic molecules located in thylakoid membranes.
Will these pigments dissolve in water?

Activity 2: Paper Chromatography of Plant Pigments

Paper chromatography is an analytical method that separates compounds based on
their solubility in a solvent. The solvent is used to separate a mixture of molecules that have
been applied to filter paper. The paper, made of cellulose, represents the stationary or
immobile phase. The separation mixture moves up the paper by capillary action. It is called the
mobile phase. The results of chromatography are recorded in a chromatogram. Here, the
chromatogram is the piece of filter paper with the separated pigment that you will examine at
the end of your experiment (see Figure 10.6).
We separate the compounds based on how quickly they move across the paper.
Compounds which are soluble in the solvent mixture are more concentrated in the mobile
phase and move faster up the paper. Polar compounds will bind to the cellulose in the paper
and trail behind the solvent front. As a result, the different compounds will separate according
to their solubility in the mixture of organic solvents we use for chromatography.

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Safety Precautions
● Work under a hood or in a well-ventilated space and avoid breathing solvents.
● Do not have any open flames when working with flammable solvents.
● Wear aprons and eye protection.
● Do not pour any organic solvent down the drain.
● Dispose of solvents per local regulations.
● Use forceps to handle chromatography paper that has been immersed in solvent and
wash your hands after completing this activity.
For this activity, you will need the following:
● Plant material: intact leaves of spinach and Coleus (one leaf of each plant per pair of
students)
● Filter or chromatography paper
● Ruler (one per group)
● Coins
● Pencils
● Colored pencils
● Beakers (400 mL) (Mason jars are an acceptable substitute)
● Aluminum foil
● Scissors
● Forceps
● Freshly prepared solvent mixture
o Petroleum ether: acetone: water in a 3:1:1 proportion
o If no hood or well-ventilated place is available, the mixture can be substituted
with 95 percent isopropyl alcohol. Note that, if isopropyl alcohol is used, the
pigment bands will smear. You may not be able to separate and identify the
chlorophylls or carotene from xanthophyll.
For this activity, you will work in pairs.

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Activity 2 Instructor Preparation and Teaching Notes
The following are recommendations for the Structured Inquiry for Activity 2:
● Ask students why chromatography is conducted using a nonpolar solvent rather than
water. Response should indicate that photosynthetic pigments are embedded in the
membrane and are not soluble in water.
● If students are using petroleum ether: acetone solvent, remind them to use the hood
and not to breathe the fumes. Make sure students know how to dispose of solvent.
● Emphasize that the pigment line must be above the liquid or it will get washed away.
Explain that the solvent will pick up the paper and carry the pigment with it. Pigments
that are more soluble will travel faster than pigments that are less soluble.
● Remind students to cover the chromatography container so liquid does not evaporate.
● Tell students to lay the chromatography paper on a paper towel when the experiment is
done. Emphasize that they must record the results immediately as the solvent line and
color bands will fade over time.
● Show a diagram of a chromatography strip, and to demonstrate how to calculate the Rf
value.

Structured Inquiry
Step 1: Hypothesize/Predict: Discuss with your lab partner what color pigments will likely be
present in the spinach leaves. Write your predictions in your lab notebook and draw a diagram
of how you think the pigments will separate out on the chromatography paper.
Step 2: Student-led Planning: Read Step 3 below. Discuss with your lab partner the setup of the
experiment. Then agree upon the dimensions of the filter/chromatography paper that you will
use. To allow good separation, the paper should not touch the walls of the container. The paper
must fit inside the container while being long enough for maximum separation. Write all your
calculations in your lab notebook.
Step 3: Follow the steps below to set up your filter paper and perform the chromatography
experiment.
1. Prepare the chromatogram by cutting a piece of filter paper. Transfer pigments from
spinach leaves as in Activity 1. A heavy application line will yield stronger colors when
the pigments separate, making it easier to read results. Allow the pigments to dry
between applications. Wet extracts diffuse on the paper and yield blurry lines.
2. Form a cylinder with the filter paper without overlapping the edges (to avoid edge
effects). The sample should face the outside of the cylinder. Secure the top and bottom
of the cylinder with staples.

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 3. Pour enough separation mixture to provide a mobile phase while staying below the
origin line on the chromatogram. The exact volume is not critical if the origin, the start
line where you applied the solvent, is above the solvent. See Figure 10.6.

Figure 10.6: Chromatography can be set up in a container such as a Mason jar. The start line of the sample, the
origin, is shown above the solvent.

4. Label the beaker with a piece of tape with your initials and your partner’s initials.
5. Lower the paper into the container with the band from the extraction in the lower
section. The paper must touch the solvent, but not reach the band of pigment you
applied. Why must the band be above the solvent line? Write your answer in your
notebook.
6. Cover the container tightly with a piece of aluminum foil.
7. Track the rising of the solvent front. Can you see a separation of colors on the paper?
8. When the solvent front is within 1 cm of the upper edge of the paper, remove the
cylinder from the beaker using forceps. Trace the solvent front with a pencil before it
evaporates and disappears! Draw the colored bands seen on your chromatography
paper in your lab notebook immediately. The colors will fade upon drying. If no colored
pencils are available, record the colors of the lines.
9. Let the paper dry in a well-ventilated area before making measurements because the
wet paper is fragile and may break when handled. This is also a precaution to avoid
breathing fumes from the chromatogram.
10. Discard solvent mixture per your instructor’s directions. Do not pour down the drain.

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Step 4: Critical Analysis: Open the dried cylinder by removing the staples. Measure distance
from the first pencil line to the solvent front, as shown in Figure 10.7. This is the distance
traveled by the solvent front. Measure the distance from the pencil line to the middle point of
each color band and the original pencil line. Record your results in your notebook in a table
modeled after Table 10.1. The retention factor (Rf) is the ratio of the distance traveled by a
colored band to the distance traveled by the solvent front. Calculate Rf values for each pigment
using the following equation:
Distance traveled by colored band
Rf = Distance traveled by solvent f ront

Figure 10.7: Chromatogram shows the distance traveled by the solvent front and the compounds separated by
chromatography.

Step 5: After determining the color of the band, tentatively identify each band. Did your results
support your hypothesis about the color of each band? Discuss which aspects of the
experiments may have yielded inconclusive results. How could you improve the experiment?

Guided Inquiry
Step 1: Hypothesize/Predict: What type of pigments are present in Coleus leaves and where are
the different colors located? Can you make a hypothesis based on the coloration of the
variegated leaves? Write your hypothesis down in your lab notebook. Would there be a
difference if you performed chromatography on pigment composition from different colored
regions of the leaves?
Step 2: Student-led Planning: Cut the chromatography/filter paper to the dimensions needed.
Apply pigments from different parts of the Coleus leaves following the procedure described
under Activity 1, keeping in mind that a darker line will yield stronger colors when the pigments
are separated, which will make it easier to read the results. Allow the pigments to dry between
applications. Wet extracts diffuse on the paper and yield blurry lines.
Step 3: When the solvent front reaches 1 cm from the top of the filter paper, stop the
procedure. Draw the pigment bands you see on the filter paper in your lab notebook. Clearly
indicate the color you observed for each band.

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Step 4: Let the cylinder dry and measure the distance the front traveled from the origin and the
distances traveled by each of the pigments. If the bands broadened during separation, take
measurements to the middle of each band.
Step 5: Critical Analysis: Calculate Rf for each of the bands and record them in a table in your
notebook. Compare the Rf you obtained with those of other groups. Are the Rf values similar?
What may have altered Rf values?

Assessments
1. Carotenoids and chlorophylls are hydrophobic molecules that dissolve in organic
solvents. Where would you find these molecules in the cell? What would happen if you
ran the chromatography in this lab with water as the solvent? [SP 6, Argumentation; SP
3, Questions and Methods]
2. All chlorophyll molecules contain a complexed magnesium ion. Your houseplant is
developing yellow leaves. What may cause this, and how can you restore your plant’s
health? [SP 1, Concept Explanation]
3. Seeds that grow under dim light are said to be etiolated, which describes their pale and
spindly appearance. They soon waste away after exhausting their food reserves. Can you
explain this observation? [SP 1, Concept Explanation]
[Answer Key]
1. Carotenoids and chlorophylls are embedded in the thylakoid membranes of chloroplasts
which, like all biological membranes, contain many phospholipids. If water is used as the
separation mixtures, chlorophylls and carotenoids would stay on the origin line.
2. The plants developed chlorosis, loss of green color, because of a magnesium deficiency
in the soil. The condition can be corrected by feeding the plant a fertilizer rich in
magnesium. Horticulturalists often add Epsom salts, which contains magnesium, to
garden soil.
3. Without the complete PAR, seedlings are deprived of the full spectrum of light and are
forced to use their food storage until their reserves are depleted. This will cause the
seedlings to wither and die. Plants growing under rocks and ledges show such spindly
appearance.

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 Lab 11: The Light Reaction of Photosynthesis
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
SYI-1.F.1 The folding of the
inner membrane increases
the surface, which allows
for more ATP to be
synthesized.
SYI-1.F.2 Within the
chloroplast are thylakoids
and the stroma.
SYI-1.F Describe the SYI-1.F.3 The thylakoids
SYI-1 Living systems are
structural features of a are organized in stacks
2.2 Cell Structure organized in a hierarchy
cell that allow called grana.
and Function of structural levels that
organisms to capture, SYI-1.F.4 Membranes
interacts.
store, and use energy. contain chlorophyll
pigments and electron
transport proteins that
comprise the
photosystems.
SYI-1.F.5 The
light-dependent reactions
of photosynthesis occur in
the grana.
ENE-1 The highly
complex organization of ENE-1.H.3 Energy-related
living systems requires ENE-1.H Describe the pathways are sequential
3.4 Cellular Energy constant input of role of energy in living to allow for a more
energy and the organisms. controlled and efficient
exchange of transfer of energy.
macromolecules.
ENE-1.I.2 The
ENE-1.I Describe the
photosynthetic processes
light-dependent reactions
ENE-1 The highly
that allow organisms to of photosynthesis in
complex organization of
capture and store energy. eukaryotes involved a
living systems requires
series of pathways that
3.5 Photosynthesis constant input of
ENE-1.J Explain how cells capture energy in light to
energy and the capture energy from light yield ATP and NADPH,
exchange of and transfer it to which power the
macromolecules. biological molecules for
production of organic
storage and use.
molecules.

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 ENE-1.J.1 During
photosynthesis,
chlorophylls absorb
energy from light,
boosting electrons to a
high energy level in
photosystems I and II.
ENE-1.J.2 Photosystems I
and II are embedded in
the internal membranes
of chloroplasts and are
connected through an
electron transport chain
(ETC).
ENE-1.J.3 When electrons
pass through the ETC, an
electrochemical gradient
of protons (H+) is
established across the
membrane.
ENE-1.J.4 The formation of
the H+ gradient is linked to
the synthesis of ATP.
ENE-1.J.5 The energy
captured in the
light-dependent reactions
is transferred to ATP and
NADPH, which power the
production of
carbohydrates in the
Calvin cycle.

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 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and models in applied contexts.
models presented in written format.
2.A Describe characteristics of a biological
concept, process, or model represented visually.
SP 2 Visual Representations
2.B Explain relationships between different
characteristics of biological concepts, processes,
Analyze visual representations of biological
or models represented visually in applied
concepts and processes.
contexts.
2.D Represent relationships within biological
models, including diagrams.
3.A Identify or pose a testable question based on
an observation, data, or model.
3.B Predict the results of an experiment.
SP 3 Questions and Methods 3.C Identify experimental procedures that are
aligned to the questions, including variables and
Determine scientific questions and methods. controls.
3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E Propose a new/next investigation based on
evidence from a previous experiment.
SP 4 Representing and Describing Data
4.A Construct a graph, plot, or chart.
4.B Describe data from a table or graph.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, including
equations in the curriculum and rates.
Perform statistical tests and mathematical
calculations to analyze and interpret data.
6.B Support a claim with evidence from biological
SP 6 Argumentation
principles, concepts, processes, and/or data.
6.C Provide reasoning to justify a claim.
Develop and justify scientific arguments using
6.E Predict the causes of effects of a change in
evidence.
components of biological systems based on data.

Sunlight is the main source of energy that powers life on Earth. Photoautotrophs
convert light energy into chemical energy through the process of photosynthesis. Land plants,
algae, and cyanobacteria are all photoautotrophs. The energy derived from the sun allows these
organisms to fix carbon, in the form of CO2, into sugars. In eukaryotes, photosynthesis takes
place in a specialized organelle, the chloroplast, and can be divided into three main phases:
1. Light capture by chlorophylls and other pigments and release of high energy electrons
from the pigment molecules

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 2. Electron flow through an electron transport chain embedded in the thylakoid
membranes with concomitant production of NADPH and ATP
3. Fixation of carbon into sugars through a series of reactions comprising the Calvin cycle
The light-dependent reaction refers to the first and second stages, although only the
first stage involves the absorption of light. The light-independent reaction describes the Calvin
cycle. At one time, carbon fixation was called the dark reaction, but it proceeds in darkness or
light. The primary light reaction occurs in the reaction centers; SPecial protein-chlorophylls at
the center of a photosystem (Figure 11.1).

Figure 11.1: Photosystem II (a) and photosystem I (b) are formed by a cluster of proteins associated with a
chlorophyll a pair embedded in the thylakoid membrane.

The photosystem (PS) is an aggregated array of pigments and proteins. Its primary
function is to use light energy to excite electrons that travel to the electron transport chain. In
cyanobacteria and eukaryotes, two photosystems, PSII and PSI operate in series (Figure 11.2).

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Figure 11.2: Electrons flow from PSII to PSI through several electron carriers embedded in the thylakoid membrane.
Electrons extracted from water reduce PSII to maintain the flow through the electron transport chain.

As can be seen in Figure 11.2, the electrons lost by the reaction center chlorophylls are
eventually replaced by electrons obtained by splitting water to form oxygen gas and protons.
The overall reaction of photosynthesis is summarized below.

A metabolic reaction can be studied by measuring the disappearance of reactants or the

accumulation of its products. In the first activity, you will predict the effect of environmental
conditions on photosynthesis by monitoring the disappearance of CO2. In the second activity,
you will follow the production of oxygen under several conditions that affect photosynthesis.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content for your
students:
● Discuss the various names used to describe the photosynthetic pathways, for example,
the light-independent reactions being called the dark cycle or the Calvin cycle.
● Have students show a brief video or animation summarizing the photosynthesis
pathways.

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 ● Provide students with a flowchart or diagram of carbon fixation in the light-independent
reactions. Have the students discuss its relationship to cellular respiration carried out by
the plant cells.
● Have the class hypothesize different ways that they could try to measure whether a
plant is carrying out photosynthesis.

In this lab, you will learn

● how to measure the dependence of CO2 fixation on the light-dependent reaction
● how to explain the chemical principles of pH indicators and the bicarbonate-CO2
equilibrium
● how to analyze the dependence of O2 production on the color of light spectrum and
other environmental variables

Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: Which gas is released by plants
during photosynthesis? Which gas is absorbed during photosynthesis? How does the
overall chemical equation for photosynthesis compare to the overall chemical equation
for respiration?
2. Answer the following questions in your notebook: What chemical reactions take place
when CO2 is released in water? What is the connection between an increase in CO2 in the
atmosphere and the acidification of oceans? Why are plants essential in mitigating the
effects of climate change?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 1: Influence of the Light Reaction on the Concentration of CO2

In this experiment, you will predict and test the effect of environmental conditions on
the process of photosynthesis. Gaseous carbon dioxide dissolves in water per the reaction
C O2 + H 2 O↔ H 2 CO3 ↔ H + + HCO3 .

Carbon dioxide and water combine to generate the unstable carbonic acid molecule that
separates into hydrogen and bicarbonate ions effectively lowering the pH of the solution. The
reaction is reversible. When CO2 molecules are consumed by photosynthesis, the reaction shifts
to the left. Hydrogen ions and bicarbonate ions combine to replace CO2 with the net effect of
increasing the pH of the solution.

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 A pH indicator is a chemical compound that changes color based on pH level. In this
experiment, you will use phenol red, which appears yellow at pH below 7, orange at neutral pH,
and bright red or pink at basic pH values (see Figure 11.3). How can the presence of phenol red
allow measurement of the consumption of CO2 by a plant submerged in water? Discuss the
method with your partner and write your answer in your notebook.

Figure 11.3: The pH indicator phenol red appears yellow at an acidic pH, pinkish orange at a neutral pH, and bright
red or pink at a basic pH.

Safety Precautions
● Be careful when blowing into the straw inside the reaction vessels. Do not suck up the
solution.
● Do not touch incandescent lamps, because they can be very hot.
● Wear aprons and eye protection.
For this activity, you will need the following:
● Plant material: Elodea or other aquatic plant
● Flasks
● Transfer pipettes
● Solution of phenol red
● New drinking straws
● Test tubes and test tube racks
● Lamp with LED bulb
● Beakers of water
● Hot plate
● Ice

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 ● Aluminum foil
● Acetate colored filter (at a minimum, use blue, red and green filters)
● Thermometer
● Meter stick
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for Structured Inquiry for Activity 1:
● Provide the class with a pH scale diagram that shows the pH of various substances that
are familiar with such as drain cleaner, lemon juice, and milk.
● Use healthy Elodea sprigs that have dark green leaves with few brown dead spots. The
sprigs should be at least 10 centimeters long and have leaves on at least 2/3 of the stem.
● Substitute colored cellophane if colored filters are not available.
● Caution students to be careful phenol red solution. It should not remain on the skin if
contamination occurs.
● Phenol red should not be disposed down the drain and must be placed in an appropriate
waste container.
● Warn students that phenol red solution will stain clothing and this it is difficult to
remove the stain through regular laundry washing. The stain remover used to eliminate
wine stains is effective at cleaning phenol red.
● Reinforce the precautions of working with a hot plate.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 1:
● Have students identify the skills, such as designing a setup to determine the presence of
carbon dioxide, they will need to carry out the guided inquiry. These skills were learned
in the structured inquiry.
● Place students in work groups. Encourage each group to assign specific tasks to group
members.
● Ask students to write their own paper written in their words. Make sure to check for
plagiarism by comparing writing of the answers between group members.

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Structured Inquiry
Step 1: Hypothesize/Predict: In this experiment, you will measure CO2 uptake in Elodea
exposed to light compared to a control Elodea in a control tube where light is blocked. Make a
hypothesis with your partner about the color changes you expect to see in the pH indicator
when the test tubes are exposed to light. Write down your hypothesis in your notebook. Discuss
what you predict will happen to the test tubes kept in the dark. Decide what you will do if you
observe no change in the test tubes exposed to light within the time allocated.
Step 2: Student-led planning: Set up the materials described in Step 3 below. If you have access
to a camera, you may take pictures of your set-up. Otherwise, draw the set-up in your lab
notebook indicating the colors in words or by using colored crayons. What is the control
treatment in this setup? Is it a positive control or a negative control? Explain your reasoning in
your notebook.
Step 3: Monitor the photosynthetic response by following CO2 uptake, as outlined in the
following steps.
1. Prepare test tubes for the experiment. First, label all tubes with a piece of tape and a
marker to identify the sample. The first tube will contain the sprig of Elodea exposed to
light. This is the experimental sample that will be labeled Light. The second tube will
contain a sprig of Elodea and will be wrapped in aluminum foil. Label this tube Dark
Control because it will serve as a control. The third tube will be labeled Light Control and
will contain water and indicator without plant material.
2. Prepare a dilute solution of phenol red (mixed with tap water) in sufficient quantity for
the test tubes. For example, if you set up test tubes that will contain 25 ml each, prepare
120 ml of solution to make it easier to aliquot equal amounts of solution. If the color is
close to red or pink, it means that the tap water is basic. In this case, it may be more
practical to use distilled water; since it may be difficult to blow enough CO2 to adjust the
pH. The reason for using distilled water is that freshly distilled water has a pH of 7.0. As
CO2 from the atmosphere dissolves in the distilled water, the pH drops below 7.0.
3. If the starting phenol solution is a light shade of pink, take a clean drinking straw and
gently blow bubbles into the solution until it turns bright yellow. What does the yellow
color indicate? Write your answer in your notebook.
4. Fill test tubes with water as indicated by your teacher (see Step 2). Then add the pH
indicator solution.
5. Cut leafy sprigs of Elodea with the scissors without crushing the material. To produce
comparable results, the sprigs should be similar in length and number of leaves.
6. Place the sprigs in the test tubes you prepared for the experiment. Top with water to
make sure that the sprigs are fully immersed.

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 7. Record the color of each solution in your lab notebook in a table like Table 11.1.
Remember to modify the table to accommodate all your data.
8. Wrap a piece of aluminum foil around the tubes if you are measuring photosynthesis in
the Dark Control tube. Make sure that there are no light leaks.
9. Place a lamp about 0.5 m from your experimental set up. Place a container of water
between the lamp and the test tube to absorb the heat radiating from the bulb if you
use an incandescent bulb, as shown in Figure 11.4. LED lamps radiate less heat making it
possible to bypass the use of a heat sink.
10. Start your watch and record the color of the tap water with a phenol indicator solution
every 30 minutes. To compare CO2 production in the presence and absence of light,
record the end-point color of the test tubes wrapped in aluminum foil after two hours of
incubation.
Table 11.1: Change in Phenol Red Color

Time Tube without

Tube with Elodea Tube with Elodea Tube without
Elodea exposed
(min) exposed to light in the dark Elodea in the dark
to light
0
30
60
90
120

Figure 11.4: Experimental set up to measure release of CO2 during photosynthesis.

186
Step 4: Critical Analysis: Record the colors you observe in the test tubes over time in the table
you prepared in your lab notebook. What happened to the Dark Control test tubes compared to
the Light Control tubes and what does this tell you about the validity of your experiment? Do
the experimental results support your hypothesis?

Guided Inquiry
Step 1: Hypothesize/Predict: With your partner, review the process of photosynthesis and
decide on an environmental condition you want to test. You may choose to investigate the
influence of temperature, light intensity, or light color. Can you dim the room lights to minimize
the effect of white light when experimenting with the effect of colored light? Decide with your
lab partner how you will test the environmental condition and what you predict will happen to
the pH in the solution. Include all necessary controls in your hypothesis. You should also devise
a positive and negative control for your experiment. Check with your instructor on the
availability of supplies to carry out your experiment.
Step 2: Student-led planning: Set up your experiment following the guidelines described under
Step 2 of structured inquiry. You may need to shut off the room lights and cover the windows so
there is only one light source. When measuring the effect of light intensity, use the equation
I=1/D2 where I is light intensity and D is distance. In every case, ensure that you measure only
one variable in your set up. The temperature can be kept constant by placing the tube in a
water bath. Prepare a table like Table 11.1 in your lab notebook to enter your data.
Step 3: Critical analysis: Record your measurements in your lab notebook. Analyze the results
and compare them to your hypothesis. Did the environmental variable affect pH changes as you
predicted? What do the changes in pH tell you about the changes in CO2 production? What
other factors may have contributed to the results? If you measured the effect of temperature,
explain which reactions in photosynthesis would be dependent on temperature? How would
you modify the experiment if you were to repeat it? Discuss your results with groups that tested
different environmental variables.

Assessments
1. In an experiment, the water was first boiled, which drove out all of the existing gases.
The water was then added to the test tube containing Elodea, which was then exposed
immediately to light. The photosynthetic activity of the Elodea was then monitored but
no photosynthetic activity was detected. How can you correct the situation by modifying
the composition of the Elodea environment? [SP 3, Questions and Methods]
2. Scientists report that the levels of carbon dioxide are rising in the atmosphere and
driving climate change. Another observation is that acidification of oceans is also a
consequence of climate change. Can you explain what link may exist between the two
phenomena and how it affects the marine ecosystem? [SP 1, Concept Explanation]

187
 3. Farmers report that planting corn plants too closely can stunt growth even when the
plants are heavily fertilized and receive plenty of light. Furthermore, the effect seems to
be reduced in recent years. What is the growth-limiting factor under these conditions?
What may be an explanation? How do plants respond to decreased availability of the
limiting factor? [SP 2, Concept Explanation; SP 6 Argumentation]
[Answer Key]
1. Photosynthesis would slow down considerably because little to no CO2 is present. There
is no recycling of NADPH and without a final electron acceptor, the electron transport
chain in the Z scheme would come to a halt. In a similar fashion, there would be no
recycling of ATP to ADP to be phosphorylated. The pH indicator would stay pink-orange
and not turn to yellow. If organisms such as water snails are introduced in the tube, they
will respire and release CO2 which, in turn, will be used for photosynthesis.
2. Excess CO2 dissolves in ocean water and combines with water to form carbonic acid.
Carbonic acid, in turn, dissociates into hydrogen ions and bicarbonate ions, driving the
pH down and acidifying ocean water. Acidification of oceans does not have a negative
impact on algae as measured so far, but it promotes overgrowth which may lead to
eutrophication and spreading of invasive species.
3. The plants compete for CO2. Plants open their stomata to increase the intake of CO2
losing water through evaporation. The levels of CO2 are rising in the atmosphere, which
increases plant growth and reduces opening of stomata in the short term. Because
climate change is accompanied by heat, drought, and other ecological disturbances, it
will negatively affect growth in the long term.

Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: Spirogyra are thin, thread-like green
algae that cluster up in rafts and float on the surface of ponds buoyed by bubbles of
gas. What gas buoys the network of algae? What happens when there is no light?
2. Answer the following questions in your notebook: If chlorophyll absorbs maximally in
the blue and red regions of the visible spectrum and carotenoids extend absorbance in
the blue-green region, which color of irradiation will be the most effective: red, green
or white?
3. Discuss the answers to questions 1 and 2 with the class.

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Activity 2: Dependence of Photosynthesis on Selected Wavelength in
the Light Spectrum
Several environmental factors influence the light-dependent reaction of photosynthesis.
In this activity, you will investigate the rate of the light-dependent reaction by measuring
indirectly the production of oxygen. Leaf disks are suspended in a solution of bicarbonate. A
small drop of dishwashing soap in the solution breaks the surface tension of the leaf disks and
allows the bicarbonate to penetrate inside the leaf. The bicarbonate in the solution provides a
source of CO2 for photosynthesis. The inner leaf spaces in a leaf are normally filled with air.
When a bicarbonate solution is forced into those air spaces, the leaf disks become heavier and
sink to the bottom of the solution.
As photosynthesis proceeds, the oxygen produced by PSII pushes liquid out of the air
spaces and eventually bubbles out of the disks, which float to the surface. By monitoring the
time it takes the disks to float, the rate of photosynthesis can be estimated under various
conditions. To compare results of experiments, you will use the estimated time for 50 percent of
the disks to float to the surface, labeled as ET50. This is a median value that allows you to
discount outliers, such as a floating disk that never sank in the first place or a disk stuck to the
bottom of the cup. Because ET50 values decrease when photosynthetic rates go up, you may
choose to plot 1/ET50 as a function of your independent variable.
Safety Precautions
● Wear goggles and an apron
● Use caution while working with the lamp as light bulbs become very hot
● Use caution while handling glass
For this activity, you will need the following:
● Plant material: intact leaves of spinach and Coleus, and light-colored leaves such as
iceberg lettuce, celery leaves or cabbage (one leaf per pair of students). Avoid fuzzy
leaves that trap air on their surface and do not sink easily.
● 0.2 M sodium bicarbonate solution (0.4 M, 0.6 M, and 0.8 M should be available)
● Two to three plastic 10 mL syringes
● Clear plastic cups
● Dilute dishwashing soap solution
● Single-hole punch
● Lamps
● Acetate colored filters (at a minimum, use blue, red and green filters)
● Aluminum foil

For this activity, you will work in pairs.

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Activity 2 Instructor Preparation and Teaching Tips
The following are recommendations for Structured Inquiry for Activity 2:
● Have students demonstrate oxygen bubbling on a specimen of Elodea growing under a
bright lamp. You can compare it to an Elodea that is shaded and was previously kept in
the dark.
● Demonstrate the syringe setup as shown in Figure 11.5. This will eliminate many errors
that can delay the experiment. Pictures of an appropriate initial setup and an incorrect
initial setup would be valuable for students.
● Use fresh leaf samples that have few yellowish or brown dead spots.
● Substitute colored cellophane if colored filters are not available.
The following are recommendations for the Guided Inquiry for Activity 2:
● Have the students identify the skills, such as designing a setup for determining changing
levels of carbon dioxide, they will need to carry out the guided inquiry. These skills were
learned in the structured inquiry.
● Place students in work groups. Encourage each group to assign specific tasks to group
members.
● Ask students to write their own paper written in their words. Make sure to check for
plagiarism by comparing writing of the answers between group members.

Structured Inquiry
Step 1: Hypothesize/Predict: Discuss with your partner how different the wavelengths of light
would affect the rate of photosynthesis. Write in your lab notebook the justification for your
predictions. Include the controls you will need to run a meaningful experiment.
Step 2: Student-led planning: In this guided inquiry, you will measure the ET50 in white light, in
the dark, and in different regions of the visible spectrum. Design your experiment so you change
only one variable at a time. Can you devise a set-up to minimize stray white light when you test
the effect of colored light? Some experimentalists build a cardboard box with an opening that
can be covered by a filter. In this case, run all your experiments under the same condition using
a transparent filter for white light and aluminum foil to block the light. Create a table for your
data.
Step 3: Set up of experiment:
1. Using a single-hole punch, punch 10-15 disks from an intact leaf being careful not to
damage the leaf in the process.
2. Transfer the disks to the barrel of a 10 mL syringe and replace the plunger.

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 3. Pull about 4–5 mL suspension solution in the syringe. Tapping gently on the barrel until
all disks float freely
4. Push the plunger to expel excess air.
5. Create a vacuum in the syringe by holding your index finger on the tip of the syringe
where the needle would be inserted and slowly draw the plunger down. You will feel the
vacuum pulling on your finger. Do not draw the barrel out of the syringe. The solution
should be flowing into the airspaces of the leaf disks. Wait two to three minutes and
release the vacuum. The disks should sink after you repeat this procedure two to three
times. See Figure 11.5.

Figure 11.5: Pulling the barrel of the syringe and capping the tip with a finger creates a vacuum inside the syringe.

6. Tap the syringe gently to re-suspend the disks and empty the content of the syringe into
a clear cup. Cover the disks with additional solution if needed to submerge them.
7. Continue with your experimental setup. Illuminate the clear cups with the lamp and
record the number of floating disks every minute for 20 minutes. For the dark control,
check the disks after 20 minutes.
Step 4: Critical analysis: Plot the number of floating disks for each condition as a function of
time and determine the ET50 for each condition. Compare the estimated photosynthetic rates as
1/ET50 at the wavelengths you tested. Do the results confirm your hypothesis? Plot your results
in a graph.

Guided Inquiry
Step 1: Hypothesize/Predict: Discuss with your partner which conditions would impact the rate
of photosynthesis in the disk. Conditions include the following:
1. The effect of varying CO2 concentration. This is achieved by using different
concentrations of a sodium bicarbonate buffer which releases CO2.
2. Plant material differences, such as comparing the deep green leaves of spinach to the
pale leaves of cabbage or celery

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 3. Light intensity differences, such as by changing the distance between the light source
and the cup
Recall that I=1/D2, where I is the intensity of light and D is the distance between the light source
and the target. You can use different areas of a variegated leaf from Coleus.
Choose a single variable you will study in your experiment. Write down in your notebook the
justification for your experiment and your prediction. How will it improve your understanding
of photosynthesis? Include the controls you will need to run a meaningful experiment. Once
you have determined a condition, discuss the feasibility of the experiment with your instructor
and be prepared to modify your experiment. After choosing a condition, design your
experiment carefully to limit the effect of a single variable.
Step 2: Student-led planning: Sketch your set-up in detail and ask your teacher for approval.
Modify your set-up per the feedback from your teacher if need be. Now you are ready to
assemble all the materials that you will need to perform the experiment. Follow the instructions
described in Step 3 under the structured inquiry. Count the number of disks that float over time
to estimate the ET50. Enter all data in a table like Table 11.2, which you will adapt to your
experimental conditions.
Step 3: Critical analysis: Plot the number of floating disks for each experimental condition as a
function of time and determine the ET50 for each condition. Plot the estimated photosynthetic
rates using 1/ET50 as a function of your independent variable, either bicarbonate concentration
or light intensity. Compare the 1/ET50 values for darkly pigmented leaves and pale leaves. In
each case, discuss the validity of the data and how you would improve on the experiment. How
reliable is it to use bicarbonate as a source of CO2? Can you compare light intensities without
actually measuring light flux? Is it a fair comparison to use the response of disks from different
types of plants? What else could have been at play?

Assessments
1. In an experiment, water is labeled with the radioactive O18 isotope. The labelled water is
then supplied to the leaf disks. In a second experiment, CO2 labeled with the isotope O18
is supplied to the disks. In which of the two experiments will you detect O18 in the
product of photosynthesis? Why is this so? [SP 3, Questions and Methods; SP 1, Concept
Explanation]

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 2. DCMU (3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea) is an herbicide that specifically
inhibits transfer of electrons from PSII. What would you predict would happen to the
production of oxygen in an Elodea twig submerged in a solution treated with DCMU?
How would an animal such as a water snail be affected by the presence of DCMU in the
same environment? You can assume that DCMU doesn’t affect the snail directly. [SP 6,
Argumentation]
3. What would happen to gas bubbles in Elodea if a test tube is exposed to light for a
prolonged period without aeration? What would happen if water snails are added to the
test tube? Illustrate your answer with a diagram showing gas and the interaction
between the Elodea and the water snail. [SP 6, Argumentation; SP 3, Questions and
Methods; SP 2, Visual Representations]
[Answer Key]
1. In the second experiment because CO2 molecules combine to form glucose during
photosynthesis, as opposed to water, which supplies hydrogen ions to the concentration
gradient in the thylakoid space and resupplies electrons to the photosystems.
2. When DCMU blocks electron flow, there is no electron acceptor for the electron ejected
from the reaction center chlorophyll. Chlorophyll must be in an oxidized state (missing
an electron) to accept electrons from H2O. No O2 will be produced if chlorophyll cannot
accept electrons. If there is no photosynthesis there will be less oxygen in the water,
which will adversely affect the snail and may cause it to get sick or die.
3. Upon prolonged exposure to light, all the CO2 gas is consumed and photosynthesis stops.
No oxygen is released. If snails are present in the water and breathing, they release CO2
that can be fixed by the plant. In turn, the snails breathe in the O2 released by the plant.
A diagram should show that CO2 is absorbed by the plant and released by the snail.
Oxygen is released by the plant and consumed by the snail.

Extension Activities
1. Measure the ET50 of disks from plants maintained overnight in the light and in the dark.
Do plants maintained in the dark perform photosynthesis faster, slower, or at the same
rate? Write your prediction with an explanation and discuss your results.

2. Compare ET50 at different temperatures, such as 10°C, room temperature, and 50°C.
Which steps of photosynthesis may be temperature dependent and how do they affect
the evolution of O2?

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 Lab 12: Photosynthesis: The Dark Reactions
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
SYI-1.D Describe the
SYI-1 Living systems structure and/or SYI-1.D 4 Chloroplasts are
2.1 Cell Structure:
are organized in a function of specialized organelles that
Subcellular
hierarchy of structural subcellular are found in photosynthetic
Components
levels that interact. components and algae and plants.
organelles.
SYI-1.F.2 Within the
chloroplasts are thylakoids
and the stroma.
SYI-1.F Describe the
SYI-1.F.6 The stroma is the
SYI-1 Living systems structural features of
fluid within the inner
2.2 Cell Structure and are organized in a a cell that allow
chloroplast membrane and
Function hierarchy of structural organisms to
outside of the thylakoid.
levels that interact. capture, store, and
SYI-1.F.7 The carbon fixation
use energy.
(Calvin-Benson cycle)
reactions of photosynthesis
occur in the stroma.
ENE-1 The highly
ENE-1.H.3 Energy-related
complex organization
pathways in biological
of living systems ENE-1-H Describe the
systems are sequential to
3.4 Cellular Energy requires constant role of energy in
allow for a more controlled
input of energy and living organisms.
and efficient transfer of
the exchange of
energy.
macromolecules.
ENE-1.I Describe the
photosynthetic ENE-1.I.1 Photosynthesis
processes that allow captures energy from the
ENE-1 The highly
organisms to capture sun and produces sugars.
complex organization
and store energy. ENE-1.J.5 The energy
of living systems
captured in the light
3.5 Photosynthesis requires constant
ENE-1.J Explain how reactions and transferred to
input of energy and
cells capture energy ATP and NADPH powers the
the exchange of
from light and production of carbohydrates
macromolecules.
transfer it to from carbon dioxide in the
biological molecules Calvin cycle.
for storage and use.

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 ENE-1 The highly
ENE-1.O Explain how
complex organization
the activities of
8.2 Energy Flow of living systems ENE-1.O.1 Photosynthetic
autotrophs and
Through Ecosystems requires constant organisms capture energy
heterotrophs enable
input of energy and present in sunlight.
the flow of energy
the exchange of
within an ecosystem.
macromolecules.

SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and models models in applied contexts.
presented in written format.
2.A Describe characteristics of a biological concept,
SP 2 Visual Representations process, or model represented visually.
2.B Explain relationships between different
Analyze visual representations of biological characteristics of biological concepts, processes, or
concepts and processes. models represented visually in applied contexts.
2.D Represent relationships within biological
models, including diagrams.
3.A Identify or pose a testable question based on an
observation, data, or model.
3.B Predict the results of an experiment.
SP 3 Questions and Methods 3.C Identify experimental procedures that are
aligned to the questions, including variables and
Determine scientific questions and methods. controls.
3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E Propose a new/next investigation based on
evidence from a previous experiment.
SP 4 Representing and Describing Data
4.A Construct a graph, plot, or chart.
4.B Describe data from a table or graph.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, including
Perform statistical tests and mathematical equations in the curriculum and rates.
calculations to analyze and interpret data.
6.B Support a claim with evidence from biological
SP 6 Argumentation
principles, concepts, processes, and/or data.
6.C Provide reasoning to justify a claim.
Develop and justify scientific arguments using
6.E Predict the causes of effects of a change in
evidence.
components of biological systems based on data.

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 Photosynthesis is the process that phototrophic organisms, like plants, algae,
cyanobacteria, and other bacteria, use to convert the energy from sunlight into chemical energy
that can then be used to make carbohydrates like glucose. The chemical equation for
photosynthesis in plants, algae, and cyanobacteria is shown in Figure 12.1.

Figure 12.1: The chemical equation for photosynthesis in plants, algae, and cyanobacteria. Overall, light energy is
used to fix CO2 into organic carbon in the form of glucose (sugar).

In eukaryotic photosynthetic organisms like plants and algae, photosynthesis occurs in

an organelle called a chloroplast (shown in Figure 12.2). Each chloroplast has both an inner and
an outer membrane and numerous membrane-bound discs, called thylakoids, organized into
stacks called grana (singular granum). Each thylakoid is composed of a folded thylakoid
membrane that surrounds an inner thylakoid space. The fluid-filled space within the chloroplast
that surrounds the thylakoids is called the stroma. In prokaryotic photosynthetic organisms like
cyanobacteria, the photosynthetic membranes are infoldings of the cell’s plasma membrane. In
either case, the photosynthetic membranes are packed with photosynthetic pigments like
chlorophyll, a type of photosynthetic pigment that appears green, which absorbs solar energy
to initiate photosynthesis.

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 Figure 12.2: (a) Illustration of chloroplast structure showing the organization of thylakoids and grana within the
chloroplast stroma. (b) Prokaryotic photosynthetic membranes are not contained with a distinct organelle, but
instead are infoldings of the plasma membrane.

Photosynthesis is composed of two sets of reactions: the light reactions (or

light-dependent reactions), which convert solar energy into chemical energy in the forms of ATP
and NADPH, and the dark reactions (or light-independent reactions), which use the chemical
energy formed from the light reactions to fuel the fixation of CO2 to produce glucose (as shown
in Figure 12.3). The light reactions occur in the thylakoid membranes, and the dark reactions
occur within the stroma of chloroplasts or the cytoplasm of photosynthetic prokaryotes. Note
that while the dark reactions do not require light to occur, they may take place in either the
presence or absence of light, which is why they are often referred to as the light-independent
reactions. Additionally, the dark reactions are dependent on the NADPH and ATP produced by
the light reactions.

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 Figure 12.3: Photosynthesis is composed of both the light reactions and the dark reactions. The light reactions
convert solar energy to chemical energy in the form of NADPH and ATP, which are then used by the dark reactions
to fix CO2 into carbohydrates like glucose.

The dark reactions refer to the various stages of enzymatic reactions called the Calvin
cycle that serve to fix CO2 using the energy of NADPH and ATP. The Calvin cycle can be split into
three stages: fixation of CO2 into organic carbon by the enzyme ribulose bisphosphate
carboxylase (RuBisCO), reduction of the organic carbon, and regeneration of the starting
molecules. The stages of the Calvin cycle are summarized in Figure 12.4.

Figure 12.4: The Calvin cycle contains three stages. In the first stage, CO2 molecules are fixed through the action of
RuBisCO into organic carbon molecules. In the second stage, the organic carbon molecules are reduced, using
NADPH and ATP. At this point, some of the reduced organic carbon leaves the cycle to form carbohydrates like the
sugar glucose. The remaining organic carbon is used during the third stage to regenerate the starting molecules of
the cycle, and ATP is used during this stage as well.

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 In photosynthetic organisms like plants, if the concentration of glucose exceeds the
organism’s immediate needs, it may be polymerized to form starch, a type of storage molecule
that is insoluble. By storing starches, the organism bypasses the problem of increased osmotic
pressure that may otherwise result when cells accumulate large concentrations of solute. This
starch can then be stored until the organism has a need for glucose monomers, at which point
the starch will be hydrolyzed to release individual glucose units.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Show a diagram of a chloroplast. Explain that energy from sunlight is captured in the
light reactions, which take place in the thylakoid membrane. This energy is used to make
sugars in the dark reactions, which take place in the stroma.
● Ask students where chlorophyll is located in the chloroplast, and what the role of
chlorophyll is. Response should indicate that chlorophyll is located in the thylakoid
membrane and captures energy from sunlight in the light reactions.
● Explain that besides chlorophyll, leaves contain other types of pigments. Some of these
pigments are accessories in photosynthesis. Others act as sunscreen that protects the
leaf from UV damage. The color of a leaf can vary depending on the type and amount of
pigment that is present.
● Ask students to think about how stressors, such as lack of water or drought, might affect
starch levels in plant tissue. Response should indicate that starch levels will drop when
the plant is stressed.

In this lab, you will learn

● how to test for the presence of starch in plant leaves
● how to determine the effect of light on starch accumulation

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: Why might the presence of starch
serve as a good indicator for the products of the dark reactions in plants?
2. Answer the following questions in your notebook: Leaves contain chlorophyll, but the
amount can vary. For example, stems and veins may contain less chlorophyll and appear
lighter in color. Some leaves are also variegated, with lighter and darker sections. Would
you expect starch to accumulate more in the lighter-colored or darker-colored sections
of a leaf? Why?
3. Discuss the answers to questions 1 and 2 with the class.

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Activity 1: Testing for the Presence of Starch in Plant Leaves
How might we test for the presence of starch in leaves? We could use a colorimetric
indicator for starch that changes color to detect any color change when starch is present.
However, this may be problematic since plant leaves, like those of spinach, are quite green due
to the presence of chlorophyll, and this may mask any color formation if we use a colorimetric
indicator for starch.
In this lab, to circumvent the problem mentioned above, you will first boil leaves in a
solution of 70 percent ethanol to remove chlorophyll. Following this, you will then stain the now
chlorophyll-free leaves with iodine, a dark red indicator that complexes with starch, turning
bluish-black. This bluish-black color can be seen in Figure 12.5, where a starchy seed has been
stained with a drop of iodine.

Figure 12.5: Iodine complexes with starch to form a bluish-black color, as seen here after a drop of iodine was
added to this cross section of a starchy seed.

Safety Precautions
● Ethanol is highly flammable and should not be exposed to open flames.
● Wear eye protection when working with solutions of 70 percent ethanol and iodine.
● Use caution when using iodine solution as it may stain skin and/or clothing.
● Use caution when handling hot liquids.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled fluids to prevent people from slipping.
For this activity, you will need the following:
● Spinach leaves
● Variegated leaves (examples include Coleus, Hosta, holly, clover)

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 ● 70 percent ethanol solution
● 0.01 M iodine solution
● Starch
● Glass test tubes
● Forceps
● Water bath
● Hot plate
● Beakers
● Petri dish
● Paper towels
● Hot mitts
● Container for collecting ethanol waste
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Make sure students know that a colorimetric indicator is a substance that changes color
in the presence of a certain chemical. Iodine is an indicator that turns purplish black in
the presence of starch.
● Ask students why ethanol, but not water, removes chlorophyll from leaves. Response
should indicate that chlorophyll, which is embedded in membranes, is hydrophobic and
is therefore soluble in hydrophobic solvents but not water.
● Remind students that ethanol is flammable. Tell them that iodine can stain clothing.
● Remind students to use the hotplate with care.

Structured Inquiry
Step 1: Set up a large beaker of boiling water using a hot plate. While waiting for the water to
boil, create a data table in which you can draw your spinach leaf four times and use the
following four categories: (1) before any treatment, (2) after boiling in water, (3) after boiling in
70 percent ethanol, and (4) after staining with iodine. Obtain a spinach leaf and draw the
appearance of the spinach leaf before treatment. Note the color.

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Step 2: Hypothesize/Predict: Based upon your knowledge of photosynthesis and starch
production in plants, predict where in the leaf you expect to find the most starch. Add your
prediction to the data table you created in Step 1.
Step 3: Once the water is boiling, add the spinach leaf to the boiling water for 30 seconds using
a pair of forceps. After 30 seconds, remove the spinach leaf from the water, place it on a Petri
dish, and look at the leaf. Is it uniformly green? Are some parts darker than others? Draw, in
your data table, the appearance of the spinach leaf after boiling in water.
Step 4: Student-led Planning: Obtain two test tubes. Add water to each, and add a spoonful of
starch to one. Add a drop of iodine to each and note the color.
Step 5: Place the boiled spinach leaf in a glass test tube using forceps. Add enough 70 percent
ethanol to the glass test tube to cover the leaf and immerse this tube in the boiling water bath
for 10 minutes or until all color from the leaf has been removed. Use forceps to remove your
leaf from the test tube and dip it in a test tube of water for one minute to rehydrate it. Place it
in a Petri dish. Draw a picture of your spinach leaf after boiling in ethanol.
Step 6: Critical Analysis: Use a dropper to apply iodine solution to the leaf. Record the intensity
of the bluish-black color formed as well as where in the leaf the color formed. Label the
starch-containing areas on your leaf drawing. Is the prediction you made in Step 2 supported by
your data?
Step 7: Collect any ethanol waste in the collection container.

Guided Inquiry
Step 1: Hypothesize/Predict: Look at the variegated leaves provided by your instructor and
think about leaf color. Are all leaves as green as spinach? Are all leaves entirely green? Pick two
different-colored areas from a variegated leaf and predict where starch is likely accumulated.
Write your ideas in your notebook.
Step 2: Student-led Planning: With your lab partner, create a table to record your data and test
your hypothesis from Step 1 using ethanol and iodine. Be sure to record the intensity of the
bluish-black color formed for each leaf (and/or section of leaf).
Step 3: Critical Analysis: Are the predictions you made in Step 1 supported by your data?
Discuss your answer with your lab partner and write it in your notebook.
Step 4: Collect any ethanol waste in the collection container.

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Assessments
1. Based on this experiment, what relationship might you expect between the leaf
greenness of various plants and their relative abilities to convert sunlight into chemical
energy in the form of starch? Make a graphical model of your prediction. How might the
growth of a greener plant differ from that of a less green plant? Explain. [SP 1, Concept
Explanation; SP 4, Describing and Representing Data; SP 6, Argumentation]
2. A student microscopically observed leaves from two different plants and noticed that
one had twice as many chloroplasts as the other. How do you predict the rates of starch
production will compare between these two plants? [SP 6, Argumentation]
3. In variegated leaves, explain how leaf cells lacking chlorophyll obtain free energy to
sustain their growth. How does this compare to the way that consumers (like humans
who eat plant products) obtain free energy to sustain their growth? [SP 1, Concept
Explanation]
[Answer Key]
1. Since more green pigmentation is associated with having more chloroplasts, greener
plants would be capable of more starch production during sun exposure. Thus, one
might expect that plants with greener leaves would grow faster and/or bigger. Such a
graph might look as follows:
2.

Having more chloroplasts would allow that plant to capture more free energy from the
sun and photosynthesize at a faster rate, producing starch at twice the rate as the plant
with half as many chloroplasts.
3. Plant cells that lack chlorophyll cannot photosynthesize to produce starch. However,
those cells within a leaf that do photosynthesize can provide enough starch to share
with and sustain non-photosynthetic cells. Consumers like humans and other organisms
that eat plant materials and other photosynthetic organisms obtain chemical free energy
from the starch within the ingested photosynthetic organisms to sustain their growth.

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 The strategy used by consumers is similar to that used by non-photosynthetic plant cells:
use the starch produced by photosynthetic cells as a chemical free energy source.

Activity 2: Pre-Assessment
1. Answer the following questions in your notebook: Explain how the dark reactions got
their name? Can they happen independently of exposure to the sun? Explain.
2. Answer the following question in your notebook: How may the amount of sun
exposure affect the dark reactions?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 2: Determining the Effect of Light on Starch Accumulation

While the name of the dark reactions suggests that they can happen in the dark, can
they? No, a better name for the dark reactions of the Calvin cycle is the light-independent
reactions. By light-independent we mean that the Calvin cycle does not involve the free energy
of the sun directly. However, for the Calvin cycle to occur, the light reactions—or more precisely,
the light-dependent reactions—carried out by photosynthetic pigments within thylakoid
membranes, must occur to convert the free energy from the sun into chemical free energy in
the form of NADPH and ATP. The dark reactions must be continually supplied with NADPH and
ATP from the light reactions in order to continue, even if the dark reactions do not directly
involve the sun.
One problem that some plants living in hot climates encounter is balancing their need
for CO2 to carry out the dark reactions with their need to retain water. While opening stomata
on leaf surfaces to get CO2 for carbon fixation, plants living in these conditions risk a major loss
of water, and become dehydrated. However, if they close their stomata to retain water, the O2
produced as a by-product of the light reactions can accumulate inside plant cells and serve as an
alternative substrate to RuBisCO when CO2 levels are low. This process, called photorespiration,
is harmful to the plants in that it significantly reduces carbohydrate production by the dark
reactions, using up NADPH and ATP without fixing CO2 (Figure 12.6), while also using O2 that
otherwise would be used as a terminal electron acceptor during respiration to make ATP.
Photorespiration is problematic for C3 plants, those lacking any mechanism to avoid this, which
are named as such because the first fixed carbon product contains 3 carbons.

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 Figure 12.6: Photorespiration occurs when cellular O2 levels are high and CO2 levels are low. When this occurs,
RuBisCO may bind to O2 instead of its typical substrate CO2. This results in the use of NADPH and ATP without the
production of carbohydrates.

Some plants have evolved mechanisms to overcome photorespiration (Figure 12.7). In

some plants, called the C4 plants, CO2 is first added to a 3-carbon molecule to produce a
4-carbon molecule in a mesophyll cell on the plant leaf’s exterior. This mesophyll cell then
serves as a CO2 supply for a more internal cell, called a bundle sheath cell. The mesophyll cell
releases CO2 from the 4-carbon molecule directly in the bundle sheath cell’s vicinity.
Interestingly, due to their distinct adaptive advantage, C4 plants evolved independently multiple
times in different plant lineages. One commercially significant C4 plant is corn.
Other plants, called CAM plants, evolved a pathway known as crassulacean acid
metabolism (CAM). CAM plants open their stomata at night, allowing them to retain their water
but also allowing the flow of CO2 into the plants. This CO2 is then added to a 3-carbon molecule
to produce a 4-carbon molecule, which serves as a CO2 source during the day, when the plant’s
stomata are closed. While these mechanisms are similar, C4 plants spatially separate the
incorporation of CO2 into the 4-carbon molecule in a cell different from the one carrying out the
Calvin cycle, while these two activities occur in the same cell, but at different times, in CAM
plants. Cacti and succulents are examples of CAM plants.

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 Figure 12.7: To avoid photorespiration, plants have evolved strategies to either spatially (C4 plants) or temporally
(CAM plants) separate the incorporation of CO2 into a 4-carbon molecule, which subsequently serves as a CO2
source, from the Calvin cycle.

Safety Precautions
● Ethanol is highly flammable and should not be exposed to open flames.
● Wear eye protection when working with solutions of 70 percent ethanol and iodine.
● Use caution when using iodine solution as it may stain skin and/or clothing.
● Use caution when handling hot liquids.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled fluids to prevent people from slipping.
For this activity, you will need the following:
● Spinach leaves
● Potted house plants including
● C3 plants: peace lily, golden pothos, or philodendron
● CAM plant: jade
● Thick black paper cut into strips
● Paper clips
● 70 percent ethanol solution
● 0.01 M iodine solution

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 ● Glass test tubes
● Forceps
● Water bath
● Hot plate
● Beakers
● Petri dish
● Paper towels
● Hot mitts
● Container for collecting ethanol waste
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Notes

The following are recommendations for the Structured Inquiry for Activity 2:
● Show a diagram of the Calvin cycle and photorespiration. Explain that photorespiration
serves no physiological function and wastes energy.
● Explain that C4 and CAM plants have independently evolved the ability to reduce the
occurrence of photorespiration. In C4 plants photosynthesis takes place in the interior of
the leaf where less oxygen is present. In CAM plants carbon dioxide is taken up at night
and stored as organic acid. C3 plants do not possess either of these adaptations and are
more susceptible to photorespiration.
● Ask students what environmental conditions would make C4 or CAM metabolism most
advantageous. Response should indicate that these two types of metabolism are most
advantageous in hot, dry conditions as the stoma must be kept closed to prevent water
loss, which causes carbon dioxide levels to fall and oxygen levels to rise.

Structured Inquiry
Step 1: Student-led Planning: Take a C3 plant and cover several of the leaves with black paper
strips, as shown in Figure 12.8, securing the strips with paper clips. Be sure the strip goes
around both the front and back of each leaf and that the black paper is secured tightly to the
leaves. Expose the plant to sun and water as normal for two to seven days prior to continuing
with the lab. Your teacher may have you move to the Guided Inquiry below at this time.

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 Figure 12.8: Cover several leaves of a potted plant with black strips, securing with paper clips.

Step 2: Hypothesize/Predict: You will now repeat the procedure you used previously to detect
starch production in leaves. Based upon your knowledge of starch production due to
photosynthesis, predict where you expect starch production to occur in a leaf that has been
partially covered with a black strip of paper. How do you expect starch production to compare
between the covered and uncovered sections of the leaf? Create a data table for drawing a leaf
that has been covered with a black paper strip. In your data table, include space for drawing
covered and uncovered sections of the leaf four times each and use the following four
categories: (1) before any treatment, (2) after boiling in water, (3) after boiling in 70 percent
ethanol and rehydration, and (4) after staining with iodine. Add your predictions regarding
starch production in the covered versus uncovered sections of the leaf to this data table.
Step 3: Prepare a large beaker of boiling tap water on a hot plate. While waiting for the water to
boil, draw the appearance of both the covered and uncovered sections of the leaf before
treatment. Once the water is boiling, add the leaf to the boiling water using a pair of forceps.
After 30 seconds, remove the leaf from the water and observe, drawing the appearance of each
section (the covered and the uncovered sections) in your data table.
Step 4: Remove the chlorophyll content from the leaf by boiling in 70 percent ethanol and
rehydrate it, drawing the appearance of both the covered and uncovered leaf sections in your
data table.
Step 5: Critical Analysis: Stain the leaf with iodine and draw a picture of the leaf in your data
table. Record the intensity of the bluish-black color formed in the leaf sections as well as the
locations in the leaf where the color formed, indicating these using arrows on your leaf drawing.
Are the predictions you made in Step 2 supported by your data?
Step 6: Collect any ethanol waste in the collection container.

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Guided Inquiry
Step 1: Hypothesize/Predict: Think about the differences between C3, C4, and CAM plants.
Predict how the differences in their CO2 fixation strategies prior to the Calvin cycle may affect
their production of starch in both the presence and absence of light. How might you test this?
Write your ideas in your notebook.
Step 2: Student-led Planning: With your lab partner, design an experiment to examine the
effect of CO2 fixation strategy (C3, C4, or CAM) on starch accumulation in leaves in both the
presence and absence of light. Once your teacher approves, create a table to record your data,
prepare your plants with the black paper strips, and expose them to the sun and water as
normal for two to seven days prior to your experiment, per your design.
Step 3: Treat your leaves to remove chlorophyll, rehydrate, and stain with iodine, recording data
on the relative amounts of starch found within your various leaves and sections of leaves. Be
sure to record the intensity of the bluish-black color formed for each leaf and each section of
leaf.
Step 4: Critical Analysis: Are the predictions you made in Step 1 supported by your data?
Discuss your answer with your lab partner and write it in your notebook.
Step 5: Collect any ethanol waste in the collection container.

Assessments
1. How does sun exposure affect the dark reactions’ conversion of the chemical free energy
from NADPH and ATP to that of carbohydrates? Explain. [SP 1, Concept Explanation]
2. Consider a C3 plant grown in hot, dry conditions compared to humid conditions. How do
you predict the acquisition of chemical free energy in the form of carbohydrates will
compare between these two conditions? Explain. Make a graphical model of your
prediction. How would plant growth be affected? [SP 6, Argumentation; SP 4,
Representing and Describing Data]
3. Compare how the acquisition of chemical free energy in the form of carbohydrates is
affected by humidity in C3 plants compared to C4 plants? [SP 6, Argumentation]
[Answer Key]
1. Reducing sun exposure will reduce the amount of NADPH and ATP made during the light
reactions. Thus, there will be less NADPH and ATP to fuel the dark reactions, and less
starch can be made as a result of reduced sun exposure.

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2. During hot, dry conditions, stomata are likely to be closed to prevent water loss from the
plant. As such, the amount of CO2 within the plant available for carbon fixation by
RuBisCO will be reduced. Thus, one would expect reduced carbohydrate production
under these conditions. By contrast, in humid conditions, water loss from the plant is no
longer a problem so plants would have their stomata open. As such, the amount of CO2
within the plant available for carbon fixation by RuBisCO would not be limiting and
RuBisCO activity, and hence carbohydrate production, would be expected to be higher
under these conditions. C3 plants would be expected to grow more under humid
conditions compared to hot, dry conditions. Additionally, rates of photorespiration
would be lower under humid conditions since the open stomata would allow for escape
of O2 from plant cells. A chart of relative carbohydrate production might look like this:

3. Under hot, dry conditions, C3 plants would be significantly hampered in their ability to
produce carbohydrates by the dark reactions, since internal CO2 levels would be low due
to closed stomata to prevent dehydration. Additionally, photorespiration triggered by
the inability of O2 to escape plant cells would be increased. However, C4 plants have
evolved a strategy to sequester even the very low amounts of internal CO2 into a
4-carbon molecule within mesophyll cells, which then serve to donate CO2 to more
internal bundle-sheath cells, which carry out the Calvin cycle to produce carbohydrates.
Thus, C4 plants avoid photorespiration and can fix CO2 even in hot, dry climates. While
one would still expect higher carbohydrate production by C4 plants under humid
conditions compared to dry conditions, the difference in carbohydrate production
between these two conditions is much less than in C3 plants. Because of the great
advantage that C4 plants have for growth in hot, dry conditions compared to C3 plants, C4
capabilities have evolved independently in multiple lineages of plants.

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 Lab 13: Mitosis and Meiosis
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
IST-1.B.1 In eukaryotes,
cells divide and
transmit genetic
information via two
IST-1.B Describe the
regulated processes.
events that occur in the
IST-1.B.2 The cell cycle
cell cycle.
is a regulated series of
IST-1 Heritable events for growth and
IST-1.B.C Explain how
4.6 Cell Cycle information provides cell reproduction and
mitosis results in the
for continuity of life. consists of interphase,
transmission of
mitosis, and
chromosomes from
cytokinesis.
one generation to the
IST-1.C.1 Mitosis
next.
transfers a complete
genome from a parent
cell to two genetically
identical daughter cells.
IST-1.D.1 A number of
internal controls of
checkpoints regulate
IST-1.D Describe the
progression through
role of checkpoints in
the cycle.
regulating the cell
IST-1.D.2 Interactions
IST-1 Heritable cycle.
4.7 Regulation of Cell between cyclins and
information provides
Cycle cyclin-dependent
for continuity of life. IST-1.E Describe the
kinases control the cell
effects of disruptions to
cycle.
the cell cycle on the cell
IST-1.E.1 Disruptions to
or organism.
the cell cycle may
result in cancer and/or
apoptosis.
IST-1.F Explain how
IST-1.F.1 Meiosis
meiosis results in the
IST-1 Heritable ensures the formation
transmission of
5.1 Meiosis information provides of haploid gametes in
chromosomes from
for continuity of life. sexually reproducing
one generation to the
diploid organisms.
next.

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 IST-1.G.1 Mitosis and
IST-1.G Describe the meiosis are similar in
similarities and/or the way chromosomes
differences between segregate but differ in
the phases and the number and
outcomes of mitosis genetic content of
and meiosis. daughter cells
produced.
IST-1.H.1 Separation of
homologous
chromosomes in
meiosis I ensures that
each gamete received a
haploid set of maternal
and paternal
IST-1.H Explain how the chromosomes.
IST-1 Heritable
5.2 Meiosis and process of meiosis IST-1.H.2 During
information provides
Genetic Diversity generates genetic meiosis I, homologous
for continuity of life.
diversity. chromosomes can
exchange genetic
material via “crossing
over.”
IST-1.H.3 Sexual
reproduction in
eukaryotes increases
variation.
SYI-3 Naturally
SYI-3.C.3 Certain
occurring diversity SYI-3.C Explain how
human genetic
among and between chromosomal
5.6 Chromosomal disorders can be
components within inheritance generates
Inheritance attributed to
biological systems genetic variation in
chromosomal changes,
affects interactions sexual reproduction.
such as nondisjunction.
with the environment.

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 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and models in applied contexts.
models presented in written format.
2.A Describe characteristics of a biological
concept, process, or model represented visually.
SP 2 Visual Representations
2.B Explain relationships between different
characteristics of biological concepts, processes,
Analyze visual representations of biological
or models represented visually in applied
concepts and processes.
contexts.
2.D Represent relationships within biological
models, including diagrams or photographs.
3.A. Identify a testable question based on
SP 3 Questions and Methods observation, data, or a model.
3.B. Predict the results of an experiment.
Determine scientific questions and methods. 3.D Make observations, or collect data from
representations of laboratory setups or results.

SP 4 Representing and Describing Data

4.A Describe data from a table.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, including
means and percentages.
Perform statistical tests and mathematical
calculations to analyze and interpret data.
5.A. Make a scientific claim.
SP 6 Argumentation 5.B Support a claim with evidence.
6.C Provide reasoning to justify a claim by
Develop and justify scientific arguments with connecting evidence to biological theories.
evidence. 6.E Predict the causes or effects of a change in
one or more components of a biological system
based on data and visual representations.

Cell theory states that all existing cells originated from preexisting cells. DNA is the
genetic material of cells, and in eukaryotic cells, it is packaged into chromosomes. You have two
copies of each chromosome: one from your mother (from the egg) and one from your father
(from sperm).
During mitosis, a cell splits into two daughter cells, with two copies of each
chromosome in each. In meiosis, four cells are produced—with only one copy of each
chromosome. The processes of mitosis (non–sex cell division or cell division of somatic, or body
cells) and meiosis (generation of sex cells or gametes) support the cell theory. The cell cycle
illustrated in Figure 13.1 shows the life cycle of somatic cells. Mitosis and meiosis share similar

213
processes; however, the outcomes are different and serve different purposes. Interphase (a
period between cell division and cytokinesis, or division of the cytoplasm into two separate
cells) represents the portion of the cell cycle in which DNA is not visible under the light
microscope. When a cell is signaled to replicate, the DNA is copied during the S-phase, or DNA
synthesis phase, in the middle of interphase. In this lab, you will observe cells in different
phases of mitosis and in interphase.

Figure 13.1: The cell cycle consists of interphase and the mitotic phase.

In this lab, you will learn

● how to observe the stages of mitosis in whitefish blastula cells
● how to identify and describe the stages of mitosis in whitefish blastula cells
● how to observe and describe the stages of mitosis in onion root tips
● how to read a karyotype
● how to determine karyotype abnormalities and identify an associated disorder or
syndrome

Instructor Preparation and Teaching Tips

● Assemble slides necessary for the lab: whitefish blastula cells and onion root tips. Order
slides (+10 percent additional) one month prior to lab.

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 ● Introduce the lab with the properties of life: growth, reproduction, homeostasis,
organization, metabolism, response to stimuli, and adaptation. Ask the students to
identify these properties, and then begin to expand on growth and reproduction at the
cellular level.
● Ask the students to think about how tall he or she was at five years old. Ask students to
think about how the difference in size was achieved.
● Draw out the cell cycle. Use the proportions from Figure 13.1 to set the stage for
students to understand the relative lengths of each stage of the cell cycle over a 24-hour
period.

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: List three reasons why organisms
need to produce new cells.
2. Answer the following question in your notebook: What cellular structures must be
replicated to ensure that new cells are functional after cell division?
3. Discuss the answers to questions 1 and 2 with a partner (think, pair, share) and then
the class.

Activity 1: Observe the Stages of Mitosis in the Blastula of a Whitefish

All organisms have the need to stay alive and reproduce. Mitosis addresses the need for
cell growth, maintenance, and repair. During interphase, the cell’s DNA is replicated so that
there are two copies of each chromosome, called the sister chromatids. In eukaryotes, all
chromosomes must be duplicated prior to mitosis and cytokinesis to ensure each new daughter
cell has the full complement of genetic information. Other structures, such as organelles, are
replicated during the G1 portion of the cell cycle (See Figure 13.1). As you can see in Figure 13.2,
mitosis is divided into specific phases. During prophase, the chromosomes coil up, and sister
chromatids become visible under a microscope. The nuclear membrane surrounding the
chromosomes also disappears. In metaphase, the sister chromatids align in the center of the
cell, attached to spindle fibers. During anaphase, the sister chromatids separate and move to
opposite poles of the cell. In telophase, the chromosomes arrive at the poles and begin to
decondense while the nucleus reforms. Figure 13.2 makes it look like the phases are very
distinct. The phases, however, transition without stopping. Notice also other important
structures, such as the spindle fibers and kinetochores on the centromeres of each
chromosome. Each pair of sister chromatids has a protein structure, called a kinetochore, which
becomes attached to spindle fibers. The spindle fibers pull the sister chromatids apart, toward
opposite ends of the cell.

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 Cancer is the uncontrolled growth and mitotic division of cells. Some chemotherapy
drugs, including taxanes such as Taxol from the yew tree and alkaloids from the vinca plant,
interfere with mitosis by binding to microtubules and preventing spindle fibers from separating
sister chromatids, thus leading to cell death.

Figure 13.2: Mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase.

Safety Precautions
● Be careful handling glass slides, as the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.

216
For this activity, you will need the following:
● Prepared slide of whitefish blastula cells or online images
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Walk through the basics of using a compound light microscope. All students should
begin by learning to carry the microscope to/from their lab sites with one hand below
the base and the other hand on the arm. Before using the microscope, the objective lens
should be placed under the lowest power; coarse and fine focus are next used to get the
image into view. Objective lenses can be rotated into place to view the images at higher
powers. Following the shift to higher objective lenses, emphasize to students that they
should use only the fine focus knob to clarify the image view; never use the coarse focus
after shifting from the lowest-power objective lens.
● Indicate to students that any images that are recorded in the lab notebook should be
given a descriptive caption that includes the contents of the image (e.g., whitefish
blastula cells) and the power used to view the image. Remind your students how to
determine viewing power: objective lens power multiplied by ocular lens power.
● Review the method used to scan through a slide image (see Figure 13.4).
● Practice identifying the various stages of mitosis, by visiting
[Link] Students will get
to see the onion root tip cells prior to the lab investigation and give them confidence in
identifying the different mitotic stages.

Structured Inquiry
Step 1: Hypothesize/Predict: Using Figure 13.1, predict the percent of time that a cell would
spend in each phase. Based on this prediction, how much time do you think cells will spend in
interphase as opposed to mitosis? Write your prediction in your notebook.
Step 2: Student-Led Planning: Look at Figure 13.2. In your notebook, make a table defining the
characteristics of the stages of mitosis, as well as interphase, that you can use to identify each
stage under the microscope. Note: There are four major phases of mitosis, plus prometaphase,
which is a transition phase between prophase and metaphase.
Step 3: Using the prepared slide, record the number of cells in each phase of the cell cycle. Use
the method shown in Figure 13.3 to count the cells. Record your count in a data table like that
shown in Table 13.1. Share your data with the class to create a group total count.

217
 Figure 13.3: Scan and count cells by using either a horizontal (black line) or vertical (blue line) back and forth
scanning technique with your eyes.

Table 13.1: Results of Cell Stage Identification

Phase or Stage Individual Totals Class or Group Totals Percent
Interphase
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Totals 100%

Step 4: In your data table, calculate the percentage of cells in each phase.
Step 5: Critical Analysis: Are the predictions you made supported by your data (observations
and calculations)? Do your results match the diagram as presented in Figure 13.1? Why or why
not?

Assessments
1. Explain why interphase could be the longest phase and mitosis and cytokinesis are
generally much shorter phases of the cell cycle. [SP 1, Concept Explanation]
2. Explain the importance of spindle fibers in mitosis and why the use of antimitotic drugs
that block spindle fiber formation are used to treat cancer. [SP 1, Concept Explanation]

218
[Answer Key]
1. Interphase is the functional period of a cell between divisions, representing a large
percentage of time and the number of cells seen in the prepared slide or images. A
smaller portion of the life cycle is devoted to mitosis and cytokinesis, in which two
daughter cells, with copies of the original genome as well as necessary organelles, are
formed.
2. Spindle fibers attach at the kinetochore on the centromeres of sister chromatids. The
spindle fibers (microtubules) are disassembled by the kinetochores, drawing the sister
chromatids apart and toward the opposite poles of the original cell during anaphase.
Any disruption of the assembly of the microtubules (spindle fibers) prevents that
separation of the sister chromatids. A tumor grows through unregulated mitosis, and
antimitotic drugs block this process, rendering tumor cells unable to produce more
genetically dysregulated cells.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: In a plant, where would you likely find
cells that are actively undergoing mitosis? Where would you likely find cells that aren’t
actively undergoing mitosis? Explain your answers.
2. Answer the following question in your notebook: Think about how a plant grows. In
which parts of the plant would many cells likely be undergoing mitosis?
3. Discuss the answers to questions 1 and 2 with a partner (think, pair, share) and then
the class.

Activity 2: Observe the Stages of Mitosis in Onion Root Cells

Plant cells also use mitosis for growth, maintenance, and repair. The plant’s cell wall, as
well as the nuclear material, makes observing mitosis much easier. Figure 13.4 shows some of
the similarities and differences between plant and animal mitosis. For example, during
cytokinesis an animal cell pinches apart into two daughter cells, while a plant cell develops a
new cell wall, called a cell plate, between the new daughter cells.

219
 Figure 13.4: Cytokinesis differences between plant and animal cells

Safety Precautions
● Be careful handling glass slides, as the edges may be sharp.
● Observe proper use of the microscope; avoid handling the electric cord with wet hands.
● Do not use the coarse adjustment knob of the microscope at higher magnifications.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
For this activity, you will need the following:
● Prepared microscope slides of stained onion root tips or online images
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● Orient your students to the root tip and otherwise, to give them a sense of location of
cells. Many differences exist between animal and plant cells; the most obvious in this
case is the shape of the cells, particularly as one moves toward the tip of the root.

220
 ● Introduce the terms associated with the root tip:
o Root cap: the tip of the root that protects the plant as it pushes into the soil
o Zone of division: the site where cells are actively undergoing mitosis
o Zone of elongation: the site where daughter cells are lengthening during growth
o Zone of maturation: location where daughter cells are differentiating into cells
that play a role in storage, protection, and conduction

Structured Inquiry
Step 1: Hypothesize/Predict: In your notebook, hypothesize what regions of the onion root tip
might differ in the number of cells undergoing mitosis. What is your reasoning behind this
hypothesis? Record your answers in your notebook.
Step 2: Student-Led Planning: In your notebook, make a drawing of each phase of mitosis, as
well as interphase, in a plant cell. Label, in each drawing, the defining features that you will look
for when identifying each stage under the microscope.
Step 3: Critical Analysis: Are the predictions you made in Step 1 supported by your
observations? Why do you think cells undergoing mitosis in the onion root cell are distributed
the way they are? What differences between plant and animal cells are visible under the
microscope slide?

Assessments
1. Describe some similarities and some differences between animal and plant mitosis. [SP
1, Concept Explanation]
2. Based on your observations, explain how onion roots grow at the cellular level. [SP 1,
Concept Explanation; SP 3, Questions and Methods]
[Answer Key]
1. Similarities include using the same phases of cell cycle. The phases of the cell cycle use
similar mechanisms. Differences include the use of a cell plate during cytokinesis of a plant
versus the use of a contractile ring in animals, causing the cell furrow between daughter
cells. Asters and centrioles (centrosomes) are visible in animal cells not in plants.
2. The root cap pushes through the soil because the zone of elongation just behind the root
cap continually provides new cells that push the root cap deeper into the soil. The zone of
maturation above the zone of elongation contains cells that provide support and are not
active in mitosis.

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Activity 3: Pre-Assessment
1. Answer the following question in your notebook: Looking at Figure 13.2 and Figure 13.9,
compare the outcomes of mitosis versus meiosis.
2. Answer the following question in your notebook: After meiosis, some daughter cells may
not contain the correct number of chromosomes. What failure in the meiosis process
could cause this to occur?
3. Discuss the answers to questions 1 and 2 with a partner (think, pair, share) and then the
class.

Figure 13.9: Overview of meiosis.

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Activity 3: Meiosis and Fertilization
Meiosis is the process in sexually reproducing eukaryotes that forms sex cells or
gametes, which include sperm and eggs (ova). To avoid doubling the number of chromosomes
in each generation, reduction division (halving the number of chromosomes) in gamete
production is necessary. Meiosis uses very similar mechanisms to mitosis. There are, however,
several significant differences. The source cells for meiosis are found in the reproductive organs
of animals (gonads: ovaries and testes) and plants (flowers: ovaries and anthers). Major
differences between mitosis and meiosis include the association of homologous chromosomes
(sister chromatids of the same chromosomes) attached as a tetrad group of four, as well as
crossover of chromosome regions during prophase I of meiosis I. Crossover provides one source
of genetic variation in offspring.
Humans have 23 different chromosomes. Note that we have a diploid (2N) set of
chromosomes: a full set of 23 chromosomes received from the sperm and another set of 23
from the ovum (egg) during fertilization. See Figure 13.10. During metaphase I of meiosis I,
homologous chromosomes are separated. The chromosome number is reduced from diploid
(2N) to haploid (1N) by the end of meiosis I, with each cell retaining duplicate sister chromatids.
Meiosis II results in four haploid (1N) cells in sperm, each with only one set of each of the 23
chromosomes. In female humans, meiosis results in uneven division of the cytoplasm and the
formation of two polar bodies (nuclei only) that are not involved in fertilization. One note
about human fertilization: meiosis II is completed only after the sperm has entered the ovum.
There are several malfunctions that can occur during meiosis. Nondisjunction occurs
when the sister chromatids in tetrads separate unevenly. Nondisjunction results in one cell
getting an extra chromosome while another cell is missing a chromosome. Nondisjunction is
associated with certain human genetic disorders. For example, Down syndrome is usually
caused by nondisjunction that results in three copies of chromosome 21. Translocations can
occur when chromosomes exchange genetic information with nonhomologous chromosomes.
For a more detailed list, see this web page:
[Link]
Karyotypes (chromosome spreads) are made by stopping cells in mitosis with a chemical
and then dyeing with Giemsa stain. A picture is taken through a microscope and then digitally
enlarged to see the chromosomal banding, or G-bands. Dark and light banding patterns help
identify chromosomes and alterations to normal chromosomes. The chromosome spreads can
be seen in Figure 13.10. Human females have two X chromosomes, and males have one X and
one Y.

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 Figure 13.10 Sorted human male and female chromosomes.

Safety Precautions
None
For this activity, you will need the following:
● Images of karyotypes
For this activity, you will work in pairs.

224
Activity 3 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry for Activity 3:
● Begin the teaching of meiosis with a discussion of the cells associated with sexual
reproduction in animals: eggs and sperm. The combination of the egg and sperm during
fertilization forms the foundation of genetic material found in the resulting offspring.
Ask: Do these cells, the egg and sperm, have the same amount of genetic material as the
cells discussed in Activities 2 and 3? Why or why not?
● Introduce terminology associated with meiosis (boldface words in the introduction to
Activity 3), and highlight similarities between meiosis and mitosis. Direct the students’
attention to Figure 13.9 and indicate that, similar to mitosis, the cell replicates its DNA,
and that material is allocated to daughter cells. Further indicate that unlike mitosis, the
division process occurs twice in meiosis, such that a) the steps of mitosis are repeated in
meiosis; and b) the final cell product contains only half of the total number of
chromosomes.
● Introduce the idea that the analysis of genetic material provides insight into an
organism’s phenotype. One method used to assess whether and where genetic
abnormalities exist in human patients is to examine its chromosomal composition or
karyotype. Karyotyping can be one of the first steps taken to understand the origin of
developmental issues. It may not be immediately clear upon a person’s
symptoms/presentation what abnormalities exist. Irregularities in the centromere
location (the ‘pinched-in’ site on each chromosome) or banding locations and patterns
(also called ‘G-bands’) can inform doctors about the possible health conditions that exist
in patients.

Structured Inquiry
Step 1: Hypothesize/Predict: A karyotype shows the number and appearance of chromosomes
in the nucleus of a cell. Predict what you would look for in an abnormal karyotype. Record your
predictions in your notebook.
Step 2: Examine the three karyotypes shown in Figure 13.12, Figure 13.13, and Figure 13.14.
Compare these karyotypes to the normal karyotypes shown in Figure 13.10. Can you tell if
the individual is female, male, or indeterminate (does not have a normal distribution of sex
chromosomes)? Record any abnormalities in your notebook, and research the meaning of the
changes in chromosome number or appearance.

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Figure 13.12: Karyotype 1.

Figure 13.13: Karyotype 2.

Figure 13.14: Karyotype 3.

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Step 3: Critical Analysis: What are the implications of nondisjunction in a karyotype? Using
Karyotype 1 (Figure 13.12) as an example, explain the implications of the nondisjunction
malfunction during meiosis for the chromosome count of the daughter cells.

Assessments
1. This Robertsonian translocation involves a fusion of the long arms of two
non-homologous chromosomes during meiosis. A patient possesses a Robertsonian
translocation among chromosome 14 onto chromosome 13. No genetic material is
missing from either chromosome. Explain why it is possible that there is no
abnormality in the person but could be in the patient’s offspring. [AP 1, Concept
Explanation; SP 6, Argumentation]

2. Crossing over of chromosomes during prophase I is common. How does this process
increase the genetic diversity of the daughter cells? [SP 1, Concept Explanation]
[Answer Key]
1. It is possible that, if the translocation doesn’t disrupt a necessary gene or other
important genetic region, the person with this karyotype would be phenotypically
normal. Any sperm has a chance of receiving either an extra chromosome 14 or a
missing chromosome 14. If the sperm was missing the chromosome, the resulting
diploid genotype would be incomplete and the offspring would likely be nonviable.
However, a sperm containing an extra chromosome 14 would likely be viable, as it is not
missing any genetic information.
2. Crossing over allows genetic information from inherited chromosomes to mix. For
example, if the person forming gametes has a chromosome from one parent with genes
coding for brown hair and no freckles, and a homologous chromosome from the other
parent with genes coding for red hair and freckles, crossover could produce a
chromosome with genes coding for red hair but no freckles.

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 Lab 14: Mendelian Genetics
This investigation addresses the following Require Course Content and Science Practices from the AP
Biology CED:

REQUIRED COURSE CONTENT

Enduring
Topic Learning Objective Essential Knowledge
Understanding
EVO-2.A.1 DNA and RNA
are carriers of genetic
information.

IST-1.I.1 Mendel’s laws of

segregation and
EVO-2.A Explain how independent assortment
EVO-2 Organisms are shared, conserved, can be applied to genes
linked by lines of descent fundamental processes that are on different
from common ancestry. and features support chromosomes.
5.3 Mendelian Genetics common ancestry. IST-1.I.2 Fertilization
involves the fusion of two
IST-1 Heritable IST-1.I Explain the haploid gametes,
information provides for inheritance of genes and restoring the diploid
continuity of life. traits as described by number of chromosomes.
Mendel’s laws. Rules of probability can
be applied to analyze
passage of single gene
traits, and patterns of
inheritance often can be
predicted from data,
including pedigrees.
IST-1.J.1 Patterns of
inheritance of many traits
do not follow ratios
predicted by Mendel’s
laws, e.g., linked traits.
IST-1.J.1 Sex-linked traits
often can be predicted
5.4 Non-Mendelian IST-1 Heritable IST-1.J Explain deviations from pedigree analysis.
Genetics information provides for from Mendel’s model of IST-1.J.3 Many traits are
continuity of life. the inheritance of traits. often the product of
multiple genes or results
from physiological
processes.
IST-1.J.4 Some traits result
from non-nuclear
inheritance, such as
mitochondrial DNA.

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 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.A Describe biological concepts, and/or
processes.
Explain biological concepts, processes, and
1.C Explain biological concepts, processes, and/or
models presented in written format.
models in applied contexts.
2.B Explain relationships between different
SP 2 Visual Representations characteristics of biological concepts, processes,
or models represented visually in applied
Analyze visual representations of biological contexts.
concepts and processes. 2.D. Represent relationships within biological
models, including mathematical models and
diagrams.
3.A Identify or pose a testable question based on
observation, data, or a model.
3.B State the null and alternative hypotheses, or
SP 3 Questions and Methods predict the results of an experiment.
3.C Identify experimental procedures that are
Determine scientific questions and methods. aligned to the question.
3.D Make observations or collect data.
3.E Propose a new/next investigation based on
evidence/data from previous experiments.
SP 4 Representing and Describing Data
4.A Describe data from a visual representation,
including patterns.
Represent and describe data.
5.A Perform mathematical calculations, including
SP 5 Statistical Tests and Data Analysis
equations in the curriculum, ratios, and
percentages.
Perform statistical tests and mathematical
5.C Perform chi-square hypothesis testing.
calculations to analyze and interpret data.
5.D Use data to evaluate a hypothesis or
prediction.
6.A Make a scientific claim.
6.B Support a claim with evidence from biological
principles, concepts, processes, and/or data.
6.C Provide reasoning to justify a claim by
connecting evidence to biological theories.
SP 6 Argumentation 6.D Explain the relationship between
experimental results and larger concepts,
processes, or theories.
6.E Predict the causes of effect of a change to a
component(s) in a biological system based on a
visual representation or data.

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 A genome is the entire genetic complement of a cell, which is composed of DNA. This
DNA contains all the hereditary information of the cell, including the genes that code for
proteins. These proteins are responsible for doing the work of the cell and are the building
blocks of cell structure. The central dogma of genetics states that information flows from DNA,
to RNA to protein. The genes within DNA contain the instructions on how to make proteins.
Humans and other diploid organisms have two versions of most genes in every cell. These two
versions are called alleles. One allele was provided by the organism’s mother and the other by
its father. The genotype consists of the information present in the DNA of the organism. It also
denotes the alleles for a specific gene. The protein or trait expressed by the genotype is called
its phenotype.
Gregor Mendel, a German monk, (shown in Figure 14.1) crossed garden peas to better
understand the inheritance of traits. The traits he examined in peas followed what is now called
a Mendelian pattern of inheritance. In Mendelian inheritance, one allele is dominant, and the
other allele is recessive. The dominant allele is always expressed, even if it is paired with a
recessive allele in a heterozygous genotype (one dominant and one recessive allele). The only
way a recessive trait will be expressed in Mendelian inheritance is if a genotype is homozygous
(both alleles are the same) for the recessive alleles. From his experiments, Mendel determined
two principles. The first is the principle of segregation, which states that the two alleles
separate from each other during the formation of sex cells (gametes). The second law is the
principle of independent assortment. This principle states that genes for different traits are
distributed from a parent to offspring independent of other genes. Therefore, no gene following
Mendelian inheritance influences whether another gene will be inherited or not. Most genes
and traits in humans and other organisms follow far more complicated inheritance patterns
involving different dominance patterns, no dominance, or the linkage of genes that violates the
principle of independent assortment. However, studying Mendelian genetics provides an
excellent introduction to the basic principles of inheritance.

Figure 14.1: Photograph of Gregor Mendel.

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Instructor Introduction Notes
The following are recommendations for introducing this lab’s background content to your
students:
● Ask students to define, in their own words, the terms defined in the introduction to
clarify any confusion.
● Ask students to give examples of the principles of Mendelian inheritance in class.
● Review an example of a polymorphic trait, such as eye color, to emphasize that many
traits do not follow Mendelian genetics. Point out that examples of single-gene
inheritance in humans are rare. One example is the ability to taste the bitter compound
PTC (phenylthiocarbamide). Another example is an ordinary form of sickle-cell anemia.
Students’ Misconceptions
● Address the following common misconceptions:
o The dominant allele is also the most frequent in the population. Give the blood
group O as an example or the dominant achondroplasia (dwarfism) in the human
population.
o The dominant allele is always normal or beneficial, and the recessive allele is
always harmful. In Huntington’s disease, for example, the dominant allele is
lethal.
● Stress that the so-called “good” or “bad” genes do not have any moral connotation and
refer simply to the ability of the individual to lead a healthy life. With proper care and
suitable medication, many harmful genes can be controlled. Give as an example
phenylketonuria (PKU), a rare condition in which the amino acid phenylalanine is not
metabolized. It is treatable by following a strict diet low in phenylalanine.

In this lab, you will learn

● the basic principles of inheritance, the definition of genotype and phenotype, and the
assumptions of Mendelian inheritance
● how to handle, identify, and cross Mendelian traits in the fruit fly Drosophila
melanogaster and predict offspring genotypes using a Punnett square
● how to statistically test the probability that the difference between an observed and an
expected result is due to chance

Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: How would you know if a trait
follows a Mendelian inheritance pattern, assuming you know and can track the
genotypes and phenotypes of an organism as it produces offspring?

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 2. Answer the following questions in your notebook: What is a Punnett square, and what
does it show? Using a Punnett square, predict the offspring of a cross between two
heterozygous parents for gene A (Aa × Aa).
3. Discuss the answers to questions 1–3 with the class.

Activity 1: Single Trait Inheritance

Based on the principles set forth by Mendel, we can predict what genotypes and
phenotypes offspring will have based on the genotypes and phenotypes of their parents. One
efficient way to do this involves using a Punnett square. A Punnett square is a grid where all the
alleles of one parent are provided as the column headers while all of the alleles from the second
parent are provided as the row headers (Figure 14.2). When the alleles from the two parents
are combined in the grid, the internal squares predict the genotypes of their offspring. In
addition, multiple Punnett squares can predict offspring genotypes across several generations.
The first filial generation (F1) is the offspring that results from crossing the original, parental
generation. The second filial generation (F2) is the offspring that results from crossing F1
individuals.

Figure 14.2: A Punnett square is used as a visual representation of crossed traits and the results of the crosses.
Capital R represents the dominant trait, and lowercase r represents the recessive trait. The first square is a cross
between the two dominant traits R and R. RR is the result.

Safety Precautions
● Do not let the flies fly away.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled fluids to prevent other people from slipping.
● Wash your hands with soap and water after completion of the activity.
For this activity, you will need the following:
● Fruit flies
● Glass vials
● Fly food

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 ● Paint brushes for manipulating flies
● Foam plugs
● Stereomicroscope
● Fly anaesthetizing substance
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● The fly anaesthetizing substance could be a commercial anaesthetizing substance,
carbon dioxide or simply placing flies in a freezer for a short period of time
● Inspect a few flies before class to make sure that they carry the phenotypes that they
are supposed to carry and that there was no confusion in the order.
● Have students sort a large number of flies in a limited amount of time. Most students
have no experience identifying phenotypes or assigning sex to flies. Knowing in advance
what they are supposed to see will help them work faster.
o Select and store a few specimens that will be preserved for prelab display. Store
them in the freezer.
o Set up a demonstration stereomicroscope showing at least one male and one
female for comparison as well as samples of the phenotypes if possible.
o Ask students to practice focusing stereomicroscopes before proceeding with flies,
because they will depend on rapid and accurate visualization of traits and sexes.
o Have color photographs of a male and a female fly ready as well as the
phenotypes that will be scored. Although looking at actual specimens is better
for the students, showing them pictures will help them recognize the phenotypes
that they follow through the crosses. Many photographs are available online.
● Show a picture of the life cycle of a fruit fly to explain which stages the students will be
using in their experiments. The life cycle of a fruit fly from egg to adult is about 11 days
at 72 °F (22 °C). Many diagrams of the fruit fly life cycle are available online. (See
[Link]
● Review the entire procedure for immobilizing flies with an anesthetic, sorting with a
paintbrush, and transferring the flies to a vial before class. Assume that students will
take longer to sort flies, and adjust your timing accordingly.
● Determine the sex of the fruits fly. The accuracy of the entire experiment depends on
the ability to determine the sex of the flies and distinguish virgin females.
● Tell students to gently turn flies on their backs with a brush. They should notice that
males tend to be smaller and have a darker and more rounded tip of the abdomen. A
second trait of males is the presence of the tarsal sex combs on the first pair of legs,
which most students will find difficult to identify. Consider having a practice exercise in
which students are asked to reliably identify the sex of a few flies, and check their work.

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 ● Ask students to collect virgin females by one of the following methods:
o The visual method, which students can easily follow without coming to class at
odd hours. Virgin females are larger and lighter in color than their older
counterparts that have been fertilized. Virgin males are also light in color but
have distinctive genital traits.
o The removal method, which requires waiting until mature pupae are present in
the culture in the vial. At that point, dispose of all adult flies by tapping them into
a clean, dry vial with a foam plug and discard them in the morgue. After all adults
have been removed from the vial, wait about 10 hours and collect the females,
which are in all likelihood virgins.
● Summarize the manipulation necessary to cross flies for the experiments:
o Remind students to work quickly.
o Prepare the vials with media and label with the date and parental genotypes. Tell
students to mark the vials with their initials.
o Select about four to six virgin females with the first genotype tested and the
same number of males of the second genotype.
o When transferring files, advise students to position the vial on its side and to
delicately place the flies along the plastic side of the tube, not directly on the
medium at the bottom where they could get stuck. Close the vial with the foam
plug. Let the vial remain on its side until the flies start to move around again.
● Tell students that if a fly wakes up and flies away, it means that the whole sample was
not anaesthetized long enough, and all the flies will wake up soon. They must apply
more anesthetic to the whole sample and ignore the one fly that got away rather than
chase the lone escaped fly and lose the whole sample.
● Have ready a fly morgue. A simple homemade disposal bottle can be prepared by filling
bottles with a mixture of water, ethanol, apple cider vinegar, and a drop of dish
detergent to break the surface tension. See
[Link]

Tips
● Remind students that they must work quickly, because flies will wake up and fly away.
Several methods of anesthetizing flies are possible. Ether is not advisable for use in a
classroom because it is highly flammable, volatile, and toxic to flies if applied too long.
Commercial anesthetic compounds are available. The use of CO2 is convenient but
expensive because CO2 pads must be purchased. Leaving flies for 8 to 12 minutes in a
freezer should immobilize them long enough for scoring.
● Plan to check the cultures every few days for possible fungal infections, which would
invalidate the results.

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The following are recommendations for the Guided Inquiry for Activity 1:
● Emphasize that students should use Punnett squares and not try to guess genotypes
and phenotypes.
● Review backcrosses if necessary.

Structured Inquiry
Step 1: Hypothesize/Predict: In this activity, you will perform crosses using the fruit fly
Drosophila melanogaster. Your teacher will indicate the phenotypes that you will monitor in
your crosses and will show you photographs of the traits (e.g., eye color vermillion/sepia, body
color gray/ebony, normal wings/curly wings). You will begin by crossing a wild-type parent with
a recessive parent. Knowing the dominant and recessive traits of the parent, predict the
genotype and phenotype of the first-generation offspring using Punnett squares (see Figure
14.2). Draw the two Punnett squares in your notebook. Add your predictions to the first data
table. You will fill the second one with your actual data later.
Step 2: Student-Led Planning: Listen carefully to your teacher’s instructions on how to handle,
anesthetize, determine the sex of, and count your flies. Practice, with your partner, how best to
use the stereomicroscope. After you understand the procedure, draw a table in your notebook
to record your data (sex and phenotype of parent, first generation, and second generation.)

Figure 14.3: A typical vial of Drosophila melanogaster. The food medium is at the bottom of the vial, with fly larvae
(maggots) on its surface. The fruit fly pupae can be seen adhered to the inner wall of the vial.

Step 3: Cross the wild-type parent and recessive parents as described by your teacher in Step 1.
Then incubate your vials. After the flies have laid eggs, anaesthetize and re-collect the parent
generation. Remove all the adult flies.

235
Step 4: After seven days, anesthetize the adult flies from the vials prepared in Step 3. Separate
the male and female flies and count the phenotypes present, for each sex. Record your counts
in your notebook.
Step 5: Repeat Steps 3 and 4 for the F2 generation. Record your counts in your notebook.
Step 6: Critical Analysis: Are the predictions you made in Step 1 supported by your data? Why
or why not? What methods could you use to improve your results? Discuss with your partner
and then write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: Predict the original cross and the phenotypic frequencies of an
already-crossed group of flies. The phenotypic frequency can be found by dividing the number
of flies of a particular trait by the total number of flies. Using flies you already have available,
examine the flies and record the sex and traits. Prepare data tables (to record sex and dominant
and recessive traits) and Punnett squares.
Step 2: Student-Led Planning: Plan how you will cross your flies. After your teacher has
approved, make your crosses for at least two generations. Record your data in your lab
notebook.
Step 3: Critical Analysis: Are the predictions you made in Step 2 supported by your data? Why
or why not? What methods could you use to improve your results? Discuss with your partner
and then write your answers in your notebook.

Assessments
1. You are analyzing the inheritance of a new gene and find that it does not have a clear
dominant and recessive allele. Instead, both alleles seem to be expressed when both are
present. Does this gene follow a Mendelian inheritance pattern? Why or why not? [SP 1,
Concept Explanation]
2. Answer the following about the assumptions of Mendelian inheritance: [SP 1, Concept
Explanation; SP 3, Questions and Methods]
a. Do most traits in humans follow a Mendelian inheritance pattern? Why or why
not?
b. How could you determine that a trait does or does not follow the assumptions of
Mendelian inheritance?
[Answer Key]
1. This does not follow a Mendelian inheritance pattern, because Mendelian inheritance
assumes that there is only one dominant allele and that, when present, the dominant
allele prevents the expression of the second allele (the recessive allele).

236
 2.
a. Most traits in humans do not follow Mendelian inheritance, because many follow
different patterns of dominance or could involve linked genes that violate the
principle of independent assortment.
b. Answers will vary. Sample answer: One would know that a trait does not follow
the assumptions of Mendelian inheritance by performing crosses. If the ratio of
observed offspring genotypes differs from that expected from Mendelian
inheritance, then that provides evidence that the trait does not follow the
assumptions of Mendelian inheritance. Typically, the ratios are compared
statistically, such as using a Chi-squared test, to determine the probability that
they are different.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: What does the chi-square test tell
you?
2. Answer the following question in your notebook: Explain the difference between
expected and observed data.
3. Answer the following question in your notebook: What is a null hypothesis?
4. Discuss the answers to questions 1, 2, and 3 with the class.

Activity 2: Test for Independence (Chi-Square Test)

The chi-square test is an independence test for the likelihood that an observed
distribution is due to chance. Chi-square is calculated using the following equation

where X is the chi-square test statistic, c is the significant level of the test (we will use 0.05), O is
the observed value for variable i, and E is the expected value for variable i.
To interpret the data, use a standard chi-square table, as provided by your teacher. On
the left side of the table is the degree of freedom (df) which is calculated by subtracting 1 from
the number of categories in the data. Across the top is the probability (p-value) or the
probability that the observed value matches the expected value. It is used to determine
whether the null hypothesis should be accepted or rejected. The null hypothesis states that
there is no significant difference between the groups being measured.

237
Safety Precautions
● None
For this activity, you will need the following:
● Standard chi-square test table
● Calculator
For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● Review with students the rules of statistics applied to genetics. The probability of two
independent events is equal to the product of the probabilities of each. The probability
of events that exclude each other is equal to the sum of probabilities.
● Ask students to provide examples, and have some of your own ready.
● Ask students if the number of flies of a certain phenotype after each cross should be
identical or close to the ones they predicted.
● Ask students how they would decide that the results do not support their hypotheses.
● Ask students to define null hypothesis and interval of confidence. Draw on the board a
diagram of the bell curve showing the distribution of results and outliers.
● Ask students what could be the possible reasons for divergence between predicted and
measured outcomes. Let the students discuss the answers among themselves, and write
reasons on the board.
The following are recommendations for the Guided Inquiry for Activity 2:
● Have students practice the chi-square test on two sets of data, one from a dihybrid cross
for independently segregated alleles and one for linked alleles. The null hypothesis in
each case is that the traits follow a Mendelian inheritance pattern. This exercise should
build confidence in students that they can recognize whether to accept or reject a null
hypothesis.
● Emphasize the need for working with many flies. At least 100+ flies should be scored for
each cross.

Structured Inquiry
Step 1: Use chi-square analysis to determine if the data from the Activity 1 Structured Inquiry is
independently assorted. Watch the video ([Link]
to learn how to perform a chi-square test. Create a data table for your analysis, and show your
calculations in your notebook.

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Step 2: Hypothesize/Predict: Predict whether you will accept or reject your null hypothesis (i.e.,
if they are independently assorted). What is your alternative hypothesis? Add your predictions
to the data table you created in Step 1.
Step 3: Student-Led Planning: Using the data you collected in the previous activity, perform a
chi-square test. Work with your partner.
Step 4: Critical Analysis: Based on your chi-square test, will you accept or reject your
hypothesis? Why or why not? What methods could you use to improve your results? Discuss
with your partner, and then write your answers in your notebook.

Guided Inquiry
Step 1: Now, use chi-square analysis to determine if the data from the Activity 1 Guided Inquiry
is independently assorted. Create a data table for your analysis, and show your calculations in
your notebook.
Step 2: Hypothesize/Predict: Predict whether you will accept or reject your null hypothesis (i.e.,
are your observed results significantly different from your expected results?). What is your
alternative hypothesis? Add your hypotheses to the data table you created in Step 1.
Step 3: Student-Led Planning: Using the data you collected in the previous activity, perform a
chi-square test. Work with your partner. Your expected data would be the number of flies that
should possess the different phenotypes based on the ratios predicted by Mendelian
inheritance for each cross.
Step 4: Critical Analysis: Based on your chi-square test, will you accept or reject your
hypothesis? Why or why not? What does this tell you about the validity of your data? Discuss
with your partner, and then write your answers in your notebook.

Assessments
1. What does the chi-square test tell you about a set of observed versus expected results?
[SP 3, Statistical Tests and Data Analysis]
2. Describe another situation, outside the realm of science, where a chi-square test would
be useful. [SP 3, Statistical Tests and Data Analysis]
3. What would change the degree of freedom in a chi-square test? [SP 3, Statistical Tests
and Data Analysis]
[Answer Key]
1. The chi-square test is a statistical test used to determine the probability that the
difference between an observed and expected result is real or due to chance.
2. Answers will vary. One example is in business, where an accountant could examine if
the difference between observed and expected profits are significant or not.

239
3. The degree of freedom is determined by the number of data categories. Therefore,
adding a category to an experiment would increase the degree of freedom by one.

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 Lab 15: DNA Restriction Analysis
This investigation addresses the following Required Course Content and Science Practices
from the AP CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
IST-1.K.1 DNA, and in
IST-1.K Describe the
some cases RNA, is the
structures involved in
primary source of
passing hereditary
heritable information.
information from one
Prokaryotes and
IST-1 Heritable generation to the next.
Topic 6.1 DNA and RNA eukaryotes can contain
information provides
Structure plasmids.
for continuity of life. IST-1.L Describe the
IST-1.L.1 DNA exhibits
characteristics of DNA
specific nucleotide
that allow it to be used
base pairing: A pairs
as the hereditary
with U, and C pairs with
materials.
G.
IST-1.P.1 Genetic
engineering
IST-1.P Explain the use
IST-1 Heritable techniques, including
of genetic engineering
Topic 6.8 Biotechnology information provides electrophoresis, can be
techniques in analyzing
for continuity of life. used to analyze and
or manipulating DNA.
manipulate DNA and
RNA.

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 SCIENCE PRACTICES
Science Practices Skills
SP 1 Concept Explanation
1.A Describe biological concepts and/or
processes.
Explain biological concepts, processes, and
1.C Explain biological concepts, processes, and/or
models presented in written format.
models in applied contexts.
2.A Describe characteristics of a biological
concept, process, or model represented visually.
SP 2 Visual Representations 2.B Explain relationships between different
characteristics of biological concepts, processes,
Analyze visual representations of biological or models represented visually in applied
concepts and processes. contexts.
2.D Represent relationships within biological
models, including diagrams.

3.A Identify or pose a testable question based on

an observation, data, or model.
3.C Identify experimental procedures that are
SP 3 Questions and Methods aligned to the question, including variables and
controls.
Determine scientific questions and methods. 3.D Make observations, or collect data from
representations or laboratory setups or results.
3.E Propose a new/next investigation based on
evidence/evaluation from a previous experiment.

4.B Describe data from a table, including trends

SP 4 Representing and Describing Data
and/or patterns and relationships between
variables.
Represent and Describe Data
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculating, including
Perform statistical tests and mathematical
equations in the curriculum.
calculations to analyze and interpret data.

SP 6 Argumentation 6.A Make a scientific claim.

6.A Support a claim with evidence.
Develop and justify scientific arguments using 6.B Provide reasoning to justify a claim.
evidence.

242
 Bacteria and archaea are unicellular organisms and do not possess organized immune
systems like multicellular organisms. However, they do possess restriction endonucleases,
which are enzymes that defend against viral invasion by cutting through viral DNA. By cutting
genes, these DNA scissor enzymes destroy viral function inside the bacteria. The bacteria
protect against auto-digestion of their own genetic information by adding methyl (–CH3) groups
to their DNA sequences that would otherwise be targeted by endonucleases. These sequences
of nucleotides are called restriction sites. Scientists have developed ways to use restriction
endonucleases to select certain strands of DNA and cut them away. This is incredibly useful for
isolating a specific gene on a DNA strand that contains thousands of genes!
Figure 15.1 shows an example of a restriction endonuclease called EcoR1. EcoR1
recognizes a specific palindrome on the DNA strand. A palindrome is a section that reads the
same way frontward and backward. For example, the restriction enzyme, EcoR1, recognizes the
sequence GAATTC on both sides of the double helix. Each type of restriction enzyme recognizes
a different set of palindromes. The restriction enzymes cut through the sugar-phosphate
background, leaving sticky ends, where complementary DNA strands can be attached (a process
known as annealing). This allows different DNA strands to be joined together, so DNA can be
recombined.

Figure 15.1: Restriction enzymes cut the sugar-phosphate backbone of DNA at specific restriction sites. The cut
DNA can then be recombined with a different DNA strand at the restriction sites.

243
 Restriction enzymes are often used in research and forensics. Restriction fragment
length polymorphism (RFLP) was the original procedure used in DNA fingerprinting. Crime scene
DNA was isolated and restricted into fragments using restriction enzymes. The fragments were
then separated into bands using electrophoresis, a method where DNA fragments are
separated out by weight using an electric field. The separated bands of DNA were then
compared to determine if different samples of DNA showed the same banding pattern. If the
banding patterns were similar, it suggested that the DNA came from the same person.
Therefore, RFLP can be used to match the DNA found at crime scenes with those of victims or
suspects. Another use of restriction enzymes is in recombinant DNA technology, where DNA
from one organism can be removed and inserted into the DNA of another organism.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Note that New England Biolabs ([Link] sometimes provides free
restriction enzymes to educators. Ask for the education outreach department
● Explain that bacteria use restriction enzymes to cut up viral DNA. Show a diagram of a
restriction site. Explain that a restriction enzyme nicks only one strand of the DNA, but
because the sequence is palindromic, two nicks are made. Show the sticky ends that are
produced after two cuts are made.
● Show the structures of cytosine and methylated cytosine. Explain that bacteria
methylate their own DNA so it is not cut apart by restriction enzymes.
● Show a diagram of the use of restriction enzymes in recombinant DNA technology.
Explain that two different fragments cut by the same restriction enzyme can be
annealed together because they have complementary sticky ends.
● Show a diagram that explains how restriction fragment length polymorphism (RFLP), or
DNA fingerprinting, is used in forensic analysis. Explain that restriction enzymes cut DNA
into small fragments, but the pattern of cutting varies from person to person. RFLP
analysis can be used to determine who was present at a crime scene, and to assess
genetic relationships.
● Explain that electrophoresis is a technique that can be used to separate fragments of
DNA. In electrophoresis DNA is loaded into a gel and an electric field is applied. The
DNA, which has a negative charge, moves through the field, but small pieces move
through the gel faster than large pieces. Thus, electrophoresis separates DNA on the
basis of size.

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In this lab, you will learn
● how to predict where restriction enzymes will digest a sample of DNA into fragments
● how to digest samples of DNA with restriction enzymes
● how to separate DNA using electrophoresis

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: How could we measure the size of an
unknown fragment of DNA?
2. Answer the following question in your notebook. What would be suitable controls for
an experiment involving restriction enzymes? (Hint—Think about how you would know
the sizes of your fragments also.)
3. Discuss the answers to questions 1 and 2 with a partner and then share with the class.

Activity 1: Digestion of DNA

In this activity, you will determine the size of pre-digested DNA (pre-cut with restriction
enzymes). As mentioned above, DNA can be separated out by size using gel electrophoresis. In
gel electrophoresis, DNA fragments are inserted into a gel medium called agarose. The DNA will
be loaded onto a gel using loading dye, which helps visualize the movement of the charged
molecules through the agarose gel. The loading dye also contains a viscous sugar solution to
ensure that the DNA sample sinks into the small depressions in the gel, called wells. The loading
dyes do not actually stain the DNA during electrophoresis. To judge the size of a DNA fragment,
molecular size markers, or just markers, are inserted into the gel next to the DNA fragments.
The marker contains DNA fragments of several known sizes. They are used to estimate the
length of an unknown DNA fragment. Visualizing DNA in the gel is problematic since it is
transparent to the eye. Therefore, after the DNA has run through the gel, you will use a DNA
stain to help visual the DNA.
Safety Precautions
● Wash your hands before and after these activities.
● Never eat or drink in the lab. This lab involves the use of poisonous buffers, DNA stains,
and other chemicals
● Wearing aprons, gloves, and goggles are highly recommended. The DNA stain can stain
hands and clothing, and can harm the skin.
● Use caution with hot agarose.
● If any solutions get into your eyes, flush with water.

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 ● Use caution with electrical connections:
○ Set gel in a box and load samples with the electrodes disconnected.
○ Turn off the power supply before connecting or disconnecting the gel box.
○ Turn off power supply and unplug all leads before handling the gel box and
removing the gel.
● Dispose of used materials as indicated by your instructor.
For this activity, you will need the following:
● Pre-digested DNA samples and control sample
● Two 20 µL micropipette with µL pipette tips
● Waste container for used pipette tips
● Practice gels with practice loading dye (50 percent glycerol with methylene blue)
● 1 percent agarose in TAE buffer gel or premade agarose gel
● Masking tape (1-inch wide) (Not needed if gel trays have adjustable walls)
● Loading dye
● 1X TAE buffer
● Micro-centrifuge (optional)
● Electrophoresis gel box and power supply
● Staining trays
● DNA stain
● Lambda DNA
● Various restriction enzymes (availability determined by instructor)
For this activity, you will work in pairs or groups.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Inform students that the DNA stain is poisonous and must only be handled with gloves.
● Model use of the micropipette and explain how to read it. Show students where the
stops are for filling and ejecting, and explain that liquid should be drawn in slowly to
avoid bubbles. If students have not used micropipettes before, give them plenty of time
to practice with water.
● Show how to inject dye into a practice well. Explain that the dye must be ejected slowly
or it will shoot out of the well. Give students plenty of opportunity to practice.

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 ● Model how to prepare and pour the gel, and how to set up the electrophoresis chamber.
Tell students to watch the direction of movement of dye after they turn on the power. If
the dye moves in the wrong direction, they will need to cut the power and reverse the
leads. Tell students not to touch the apparatus when a current is running through it.
● Experiment using a microfuge and explain how to use it, and emphasize that the
microfuge must be balanced. A loud noise indicates that it is not balanced and should be
shut off immediately.
● Explain that DNA size is measured in base pairs. A base pair is a nucleotide and its
complementary partner on the other DNA strand.
● Explain that the molecular size markers, which are sometimes referred to as a ladder as
they have the appearance of rungs of a ladder, are DNA fragments of known size. A plot
of the size of each fragment in base pairs versus distance traveled in the gel yields a
standard curve that can be used to determine the size of an unknown fragment.
However, a DNA fragment travels at a rate that is inversely proportional to the logarithm
of its size, and for this reason a linear plot will produce a curved line that is hard to read.
Instead, students will plot their data on semi-log paper, which has a logarithmic scale on
one axis and a linear scale on the other axis. On a logarithmic scale, each line represents
a 10-fold increase in value. A plot of DNA size on the semi-log scale (the y-axis) versus
migration distance (the x-axis), yields a straight line, which is much easier to read.
Provide students with a sample plot so they know what the data should look like.
● Show students a lambda phage restriction map, such as the one shown here:
[Link]
eractive%20Tools/DNA%20Sequences%20and%20Maps/Lambda_map.pdf. Explain that
lambda phage DNA is 48,502 base pairs long and the number next to each restriction
site is the distance from base pair one. Explain that some restriction enzymes only cut
the DNA once; the site that is cut is called a unique site. Show students how to calculate
the fragment sizes that will result when DNA is cut at one or more sites.

Structured Inquiry
Creating the Agarose Gel
Step 1: Pay close attention to your teacher, who will demonstrate how to use the micropipette
using the process below:
1. Determine the volume required by twisting the thumbwheel dial to the required
volume. Note that numbers are read from top to bottom. There may be a red line
indicating a decimal point in the display. For example, 015 with a red line between
the 1 and 5 would read 1.5 µL.
2. Attach (seat) a clean pipette tip by gently pushing the micropipette into the top of
the tip. Be gentle removing the tip to avoid spilling the whole box.

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 3. Press the push button (plunger) on top to the first (soft) stop. Insert the disposable
pipette tip (only the tip) into the liquid to be transferred. Slowly release the push
button to suck up the liquid. Note—If you have pressed the push button too hard
and gone to the harder blow-out setting you will pull up more than the desired
volume.
4. Insert the pipette tip into the microfuge tube or other area where you need to move
the material.
5. Press the push button past the first stop to the second (hard) stop to transfer (blow
out) the liquid. Do not release the push button until you have removed the tip from
the tube.
6. Eject the disposable tip into the appropriate waste container. Do not reuse tips,
unless instructed, to avoid cross contamination.
Step 2: Your teacher has prepared 1 percent agarose gel solution that he or she will pour into
your gel tray. Prepare your gel tray. Some trays have casting gates that can be locked before
pouring the molten agarose. Other trays have open sides that must be taped with masking tape
(Figure 15.2). Make sure the masking tape is tightly applied to the open edges of the gel tray.
Place the comb across the tray as demonstrated by your instructor.

Figure 15.2: If your gel trays need to be taped, apply the tape straight to form a wall that will hold the gel. Be sure
to insert the comb straight into the gel. All the comb teeth should penetrate an equal distance into the gel. Once
the gel is fully set, remove the comb slowly by pulling straight up on both ends of the comb.

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Step 3: Your teacher will now pour the warm 1 percent agarose into your tray. If there are
bubbles, use a clean micropipette tip to move the bubbles away from the comb. Wait until the
gel has become opaque to ensure the agarose has solidified and is ready for use. This usually
takes about 20 minutes at a room temperature of 21 °C. Once the gel is fully set, remove the
comb slowly by pulling straight up on both ends of the comb (see Figure 15.2).
Loading, Running and Staining the Agarose Gel:
Step 1: Your instructor will supply you with a practice gel in a Petri dish. Fill the dish with tap
water.
Step 2: Obtain a microfuge tube of practice loading dye. Draw up 5–10 µL of dye. Hover the tip
over the well, just touching the surface of the liquid. The tip should make a slight dimple in the
water surface. Slowly depress the plunger of the micropipette, releasing the loading dye into
the well. The dye should fill the well. Pull the tip out of the liquid before releasing the push
button.
Step 3: (Note—amounts and procedures might differ based on DNA supplies.) Obtain the
pre-digested DNA samples and controls in microfuge tubes from your instructor. Add 2 µL of
loading dye to your samples. Make sure you use a new pipette tip for each. Cap each tube and
pulse spin in the micro-centrifuge (alternately spin and then stop). To avoid damage and injury,
let the rotor come to a complete stop. Do not stop the rotor with your fingers. When you
remove your sample from the centrifuge, tap it on the table 3 times to help the heavier
constituents settle to the bottom. Alternately you can tap the microfuge tube with your finger,
as shown in Figure 15.3 to coax the DNA and loading dye together into the bottom of the
microfuge tube.

Figure 15.3: Centrifuge and tapping method to move small amounts to the bottom of microfuge tubes. If no
centrifuge is available, flick the bottom of the microfuge tube with your finger at least 20 times to thoroughly mix
its contents.

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Step 4: Student-led planning. Place your gel in the gel box as shown by your teacher (see Figure
15.6). The wells should be oriented at the black terminal end (negative side). DNA is negatively
charged. Based on this, which way will the DNA travel across the gel? Create a diagram in your
notebook showing the expected direction of travel.
Step 5: Add enough 1X TAE buffer to top your gel by 2 mm. Depending on the size of your gel
box, this may be approximately 275 mL.
Step 6: Student-led planning. Load your gel, as practiced in Step 2, with the pre-digested DNA
samples you prepared in Step 3, and control DNA, in the order indicated by your instructor. The
samples contain the loading dye that you practiced with in Step 2. Generally, the standard,
which you will use to determine the size of your DNA samples, are placed into the left-most
well. The other samples are loaded to the right of the standard, as shown in Figure 15.4. How
can you ensure that the DNA will not run off the gel? Write your answer in your notebook.

Figure 15.4: Place your sample tray so that the wells are located closest to the negative electrode of the gel box.

Step 7: Place the top on the gel box with the red terminal aligned with the red side and the
black terminal aligned with the black side (Figure 15.7). Plug the cords into the power supply.
Again, double check: red with red, black with black. If the terminals are misaligned, your gel will
run backwards and off the gel, yielding no results.

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Figure 15.5: Be sure that the red and black wires are plugged into the corresponding colors on both the gel box top
and the power supply (red to red and black to black).

Step 8: Turn on the power supply and run your loaded gel for the time described by your
teacher. Generally, gels run for 20 minutes at 200 V using 0.25X TAE or 30 minutes at 100V using
1X TAE buffer.
Step 9: After the proper amount of time, the bands of loading dye on your gels should be
approximately 2–3 cm from the opposite edge of the gel. Remove the top of the gel box. Then
remove the gel tray, draining extra liquid back into the box. Carefully slide the gel off the tray
into a staining tray. Your teacher will stain your gel, which allows you to visualize the bands of
DNA fragments, and instruct you on when you can observe the results. Clean up your lab table.
Step 10. Once your gel is ready, your teacher will place it on a light table for better viewing.
Place a millimeter ruler next to the gel and take a picture of the gel using your phone camera.
Be sure to take a picture from directly above the gel, as you will use your photo to take
measurements in the next step.
Step 11: Using the photograph of the gel and ruler you took in the previous step, measure (in
millimeters) the distance migrated by all bands in each lane from the front of the well to the
forward edge of each band, as shown in Figure 15.6 and Figure 15.7. Create a data table for your
data in your notebook.

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 Figure 15.6: Diagram of a gel that has been run.

Figure 15.7: Example of a DNA Gel with Ruler. The standard lane is on the far left. This lane is used to determine
the size of the DNA fragment, in base pairs.

Step 12: Using the standard in the leftmost row of samples, estimate the size, in base pairs, of
the DNA bands in each well. Add your data to the table you created in Step 11.
Step 13: Your teacher will provide you with semilog graph paper with DNA size, in base pairs, on
the y-axis and distance travelled, in mm, on the x-axis. Plot the data from the table you created
in the previous two steps on the semilog graph paper.

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Step 14: Critical Analysis: Was there a control in this activity? If so, what does the control reveal
about how well the gel worked? What evidence does the gel provide to support the action of
restriction enzymes? How did the fragments sort in the gel, size-wise? Why did they sort this
way? Discuss with your partner and then write your answers in your notebook.

Guided Inquiry
Step 1: Figure 15.8 shows the structure of the bacteriophage Lambda, a virus that invades the
bacteria Escherichia coli. The genome of Lambda is 48,502 base pairs long. Figure 15.25 shows
the genes located on Lambda that provide the information on making new viruses.

Figure 15.8: The Lambda phage is a virus that invades, and reproduces in, the cells of bacteria.

Step 2: Hypothesize/Predict: Examine this link to find the complete list of restriction sites for
the Lambda bacteriophage:
[Link]
e%20Tools/DNA%20Sequences%20and%20Maps/Lambda_sites.pdf. In addition, your instructor
will provide a list of available restriction enzymes. Figure 15.9 shows a partial restriction map for
Lambda. Based on the introduction to this activity, your pre-assessment questions and the
restriction sites for the Lambda bacteriophage, predict where the restriction enzymes available
in your lab will cut the Lambda DNA. How large will each fragment likely be, in base pairs? Draw
and record the predicted DNA fragment sizes in your notebook.
Step 3: Student-led planning: Test the hypothesis you made in Step 2 by setting up a restriction
digest as you did in the Structured Inquiry. Your instructor will guide you in how to set up the
solutions that you will analyze in a second agarose gel. Example amounts may include, 4 µL of
Lambda DNA, 6 µL of buffer, and 1 µL of specific restriction enzyme in a microfuge tube. Don’t
forget to provide controls for your experiment. Also, be sure to spin or tap down liquids to the
bottom of the micro tube.

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Step 4: Incubate your samples at 37 °C for 30 minutes in a water bath. Then, analyze your
samples using gel electrophoresis and the same procedure you used in the Structured Inquiry.
You may need to store your digested samples in the refrigerator if you cannot immediately run
the gel. Record all your procedures in your notebook
Step 5: Critical Analysis: Was there a control in this activity? If so, what does the control show?
What evidence does the gel provide to support the action of the restriction enzymes you used?
How well did your estimation of the fragment sizes on the map match your results? Explain.
Discuss with your partner and then write your answers in your notebook.

Assessments
1. Look at the plasmid (an extrachromosomal piece of DNA) in Figure 15.11. If you digested
this plasmid with restriction enzymes BamHI, HindII, and ScaI, how many fragments
would appear in a gel? How long (# of base pairs) would each fragment be? Draw a gel
showing the digestion. [SP 2, Visual Representations; SP 5, Statistical Tests and Data
Analysis]

Figure 15.9: Partial restriction map for the Lambda phage.

2. Explain how electrophoresis can separate out molecules, such as DNA, by size using
electrical charge and gravity. Would electrophoresis work on an electrically neutral
molecule? Explain your answer. [SP 1, Concept Explanation]

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[Answer Key]
1. 2023, 1732, 784

2. Electrophoresis separates out molecules of different size by applying a constant

electrical charge. The molecules have an electrical charge and are attracted to the
opposite electrical charge generated by the electrophoresis chamber. However, due to
gravity, the larger fragments will not travel as far across the gel as the smaller fragments
if the electrical field is applied for the same amount of time to all fragments.

References/Further Investigation
Using Restriction Enzymes: [Link]
DNA Fingerprinting: [Link]
Why Do you Have to Wear Gloves when Handling DNA:
[Link]
[Link]
Agarose Sieving:
[Link]
ufficiently-resolve-my-dna-sample/
Practice Loading Gel Video: [Link]
How to Load a Gel: [Link]
Blank Semi-Log Graph Paper:
[Link]
How to do a Semi-Log Graph and Trendline in Excel:
[Link]

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 Lab 16: Bacterial Transformation
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
IST-1.K.1 DNA, and in
IST-1.K Describe the
some cases RNA, is the
structures involved in
primary source of
passing hereditary
heritable information.
information from one
Prokaryotes and
IST-1 Heritable generation to the next.
Topic 6.1 DNA and RNA eukaryotes can contain
information provides
Structure plasmids.
for continuity of life. IST-1.L Describe the
IST-1.L.1 DNA exhibits
characteristics of DNA
specific nucleotide
that allow it to be used
base pairing: A pairs
as the hereditary
with U, and C pairs with
materials.
G.
IST-1.P.1 Genetic
engineering
IST-1.P Explain the use techniques, including
IST-1 Heritable
of genetic engineering bacterial
Topic 6.8 Biotechnology information provides
techniques in analyzing transformation, can be
for continuity of life.
or manipulating DNA. used to analyze and
manipulate DNA and
RNA.
EVO-3.A.1 Populations
of organisms continue
to evolve.
EVO-3.A Explain how
EVO-3 Life continues to EVO-3.A.2 All species of
evolution is an ongoing
Topic 7.8 Continuing evolve within a organisms have evolved
process in all living
Evolution changing environment. and continue to evolve;
organisms.
evidence includes the
development of
resistance to antibiotics
in bacteria.

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 SCIENCE PRACTICES
Science Practices Skills
SP 1 Concept Explanation
1.A Describe biological concepts and/or
processes.
Explain biological concepts, processes, and
1.C Explain biological concepts, processes, and/or
models presented in written format.
models in applied contexts.
2.A Describe characteristics of a biological
concept, process, or model represented visually.
SP 2 Visual Representations 2.B Explain relationships between different
characteristics of biological concepts, processes,
Analyze visual representations of biological or models represented visually in applied
concepts and processes. contexts.
2.D Represent relationships within biological
models, including diagrams.

3.A Identify or pose a testable question based on

an observation, data, or model.
3.C Identify experimental procedures that are
SP 3 Questions and Methods aligned to the question, including variables and
controls.
Determine scientific questions and methods. 3.D Make observations, or collect data from
representations or laboratory setups or results.
3.E Propose a new/next investigation based on
evidence/evaluation from a previous experiment.

4.B Describe data from a table, including trends

SP 4 Representing and Describing Data
and/or patterns and relationships between
variables.
Represent and Describe Data
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculating, including
Perform statistical tests and mathematical
equations in the curriculum.
calculations to analyze and interpret data.

SP 6 Argumentation 6.A Make a scientific claim.

6.A Support a claim with evidence.
Develop and justify scientific arguments using 6.B Provide reasoning to justify a claim.
evidence.

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 Biologists view the role of deoxyribonucleic acid (DNA) in an organism the same way a
chef views a recipe. Like a recipe, DNA contains the information for a cell’s ingredients, as well
as instructions for how cell components should function. The ingredients of a cell, or its
molecular structure, are encoded in the DNA’s genes. Proteins are particularly important
ingredients because they form many structural components of an organism. Some proteins are
also enzymes, which speed up chemical reactions within the cell. A cell’s instructions are the
regulatory genes that coordinate the functions of other genes. Unlike a recipe, DNA is written in
a universal language called the genetic code that all cells can interpret.
The discovery of the genetic code led to the hypothesis that the DNA of one organism
could possibly be transferred into another organism. Therefore, one organism could use, or
express, the genes of another organism. This hypothesis was successfully tested by Herbert
Boyer and Stanley Cohen in 1973. They modelled a natural process called transformation, in
which bacterial cells take up DNA molecules from the environment and incorporate them into
their genomes. Boyer and Cohen first removed bacterial genes for antibiotic resistance from
one type of bacterium by cutting up the bacterial DNA using restriction enzymes. They then
inserted the antibiotic resistance genes into the DNA of a plasmid. A plasmid is a small ring of
DNA, some of which can be integrated into the DNA of a bacterium. Bacteria that can take up
plasmids and incorporate their DNA are called competent bacteria. In the Boyer and Cohen’s
experiment, the bacteria were made competent by stressing them using a heat shock
treatment. The bacteria then incorporated the plasmid, including the antibiotic resistance gene,
into their own DNA, allowing the bacteria to become resistant to antibiotics. Furthermore,
whenever a transformed bacterium reproduced, it passed on the resistance gene to all of its
offspring, eventually forming an entire colony of antibiotic-resistant bacteria. The experimental
protocol is summarized in Figure 16.1. Using this technique, the researchers created the first
recombinant DNA organism. In fact, Boyer and Cohen learned how to modify the DNA
information to produce unique characteristics in organisms.

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Figure 16.1: In Boyer and Cohen’s bacterial transformation, the bacteria were made competent by stressing them
using a heat shock treatment. The plasmid then becomes part of the bacterium’s genome, including the antibiotic
resistance gene. This allows the bacteria to become resistant to antibiotics.

In this lab you will learn

● how to perform a gene transfer in bacteria using a plasmid
● how to inoculate and grow transformed cell cultures on microbial media
● how to measure gene expression of transformed cells by detecting the presence of
active reporter genes

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Activity 1: Pre-Assessment
1. Answer the following question in your notebook: What techniques are used to transfer
a gene from one bacterium into another?
2. Answer the following question in your notebook: Explain one way that scientists can
control the expression of genes in a bacterial cell.
3. Be prepared to discuss the answers to questions 1 and 2 with the class.

Activity 1: Transformation of E. coli Cells

In this lab, you will carry out a version of the classical experiment conducted by Boyer
and Cohen. You will insert a plasmid into a strain of bacteria that is receptive to taking up and
expressing the plasmid. Just like in the Boyer and Cohen experiment, this plasmid contains a
gene for antibiotic resistance. However, it is linked to a green fluorescent protein gene and a
regulatory gene that is activated by the expression of antibiotic resistance. Bacteria that are not
transformed typically die from being exposed to ampicillin. Your procedure involves two stages
(Figure 16.2). Stage 1 represents the transformation stage, where you will induce the cells to
take up the green-fluorescent and antibiotic-resistance genes. Stage 2 represents the analysis
stage, where you will grow the bacteria to ensure you achieved transformation.

Figure 16.2: The transformation process, in Stage 1.

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 The transformation being performed in this activity requires that cells be in a competent
state. Competence is a condition in which the bacterial cells can pass DNA easily through their
cell walls and cell membranes. This is achieved by placing the cells in a calcium chloride solution
(CaCl2) and exposing them to a heat shock. Heat shock is the process of subjecting a cell to a
higher temperature than ideal environmental temperature for a short period. The role of
calcium chloride in the competence process is to counteract the electrostatic repulsion between
the plasmid DNA and the bacterial cellular membrane. This allows the plasmid to come in
contact with the cell membrane. The heat shock produces pores in bacterial cell membranes
and permits the plasmid DNA to pass into the cell as shown in Figure 16.3. In this stage of
transformation, you will make competent cells and then store them in the refrigerator until you
complete the transformation process.

Figure 16.3: Heat shock transformation of E. coli.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Review the three major mechanisms of horizontal gene transfer (HGT) in bacteria,
transformation, transduction, and conjugation.
● Ask students what are the main evolutionary consequences of horizontal gene transfer.
They should conclude that bacteria can diversify much faster than can be predicted by
mutations alone.
● Ask students what would be the consequence of HGT if many plasmids carried antibiotic
resistance genes.
● Ask students for examples of recombinant DNA technology which may be familiar. For
example, recombinant human insulin is used by diabetics. Factor VIII is used to treat
hemophilia. Be prepared to answer questions on genetically modified organisms in food,
but don’t allow controversy to take over class time.

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 ● Remind students that the transformation lab requires organizational skills because all
the steps are crucial and must be performed as directed and following timing of steps
matters.
● Ask students to prepare a flow chart of the procedure and go over it with the class.
● Advice students to divide the tasks before starting the lab. Good coordination of effort
is essential for a successful lab.
Safety Precautions
● Use caution when handling biological specimens.
● Wear safety goggles throughout the duration of the laboratory activity.
● It is highly recommended that you wear rubber gloves and a lab apron when handling
any chemicals. In addition, use aseptic techniques in handling bacterial cultures to avoid
infection.
● Immediately inform your teacher of spills.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent people from slipping.
● Immediately wash your hands if they directly contact any chemicals or cultures. It is
important to wash your hands after the laboratory activity to avoid any possible
contamination of other surfaces with chemical residues or biological materials that
might be on your hands.
For this activity, you will need the following:
Materials:
● Surgical gloves
● Safety goggles
● Waterproof marker
● Micro tube rack
● Eight disposable 1 mL calibrated plastic needle-point pipettes (or adjustable
micropipettes, if available)
● Eight disposable plastic inoculating loops
● 1 polystyrene warm-beverage cup filled with crushed ice
● 20 µl micropipette (one per class)
● Micropipette tips for class (for 20 µl micropipette)

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 ● 42 oC water bath (one per class)
● Floating test tube holder (one per class)
● 37 °C incubator
● Parafilm
● Blue LED light box (one per class)
● Refrigerator
● Biological hazardous waste container
Chemicals and Media:
● One Luria broth media Petri plate
● One Luria broth + Amp media Petri plate
● One bottle of sterile Luria broth (one per class)
● Squirt bottle with 10 percent bleach solution
● Squirt bottle with 70% ethanol solution
● Crushed ice
● Micro tube filled with 50 mm CaCl2
● Micro tube filled with Luria broth (two per group)
Living materials:
● One streak plate of E. coli per group (Strain MM294 or competent E. coli cells)
● One pGREEN or pGLO plasmid solution (0.005 µg/µl; one per class)
Note—These materials may be provided for the class as separate materials or as components in
a transformation kit. Your instructor will tell you how these materials will be distributed to your
group.
For this activity, you will work in groups of 3–4.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Preparing competent cells is a complicated procedure even for experienced molecular
biologists. Consider ordering competent cells or, at least, prepare ahead of the lab some
competent cells to test and transform them to test the procedure.
● To streak cells for students, follow procedures from a standard microbiology manual.

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● Troubleshoot:
o Nothing grew: the loop was not cooled enough before touching bacterial
samples.
o No single colonies: the loop was not flamed between contacts with bacterial
samples or not streaked over an area large enough to dilute the culture.
● If pouring your own plates, follow a standard protocol remembering that ampicillin is
added aseptically after the LB agar solution has been autoclaved and cooled to 50 °C.
● If an autoclave is not available, prepare LB agar broth in a flask and boil for about 10
minutes mixing well the content and being careful not to allow the solution to boil over.
Cool to about 50 °C, add ampicillin if required, and pour agar in Petri plates. Let the Petri
plates solidify, and, then, turn them upside down for storing. Leaving plates at room
temperature for two to three days decreases the amount of condensation.
● Demonstrate all aseptic techniques.
● Consider running a lab practice session to give students an opportunity to handle
pipettes, aseptic techniques, handling of loops and Petri plates if enough material is
available. Students should be reminded to look at the loop to make sure that they picked
a sample, and the loop is not empty.
● Remind students that plates must be labeled along the edge of the bottoms with their
initials, date, and experimental conditions. Lids should not be labeled. Labeling plates in
the center will interfere with the reading of results.
● Remind students to keep plates covered unless they are actually spreading cultures. Ask
them what would happen if they left the plates open. Students forget that
microorganisms are ubiquitous. Ask them if something would grow on the LB+amp
plates. This is an opportunity to remind students that not all microorganisms are
bacteria and that mold will contaminate plates.
● Emphasize that speed and accuracy are paramount to the success of the transformation
lab. If students are not familiar with experiments that last over several days, they may
not realize that the consequences of mistakes made on day 1 will not be apparent until
day 3.
● Give students the following advice to spread cultures evenly on the surface of the Petri
plate:
o Spread culture with a loop using a back and forth motion and turning the plate
90° twice.
o Incubate plates inverted to allow condensation to drip on the lid, but do not
invert the plates immediately after spreading the culture. It is best for the
instructor to invert all the plates after the activity is completed by the students.
● If an incubator at 37 °C is not available, plates can be left at room temperature for about
72 hours until colonies reach optimal size.
● Turn off the lights in the room to observe the GFP glow and let eyes adapt to darkness
first. Pictures will require a long exposure time.
● Remind students that gloves are considered biohazard material after they are used with
cultures. They should be disposed of accordingly.

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 ● If an autoclave is not available, use 10 percent freshly diluted bleach to disinfect Petri
plates, loops, and other items that came into contact with bacteria.
o Bleach should be used in the hood or, if not possible, containers/pans should be
covered when not in use.
o Students should wear goggles, gloves and aprons to protect their clothes.
o Once loops and pipets have been allowed to soak in bleach for 15 minutes, they
can be disposed of as regular trash. Cover the surface of Petri plates with bleach
and let it sit with the disinfectant for about 1 hour. Always check local regulations
before proceeding.
o If there is exposure, take first aid measures at once
o If eyes and face are exposed, and eyes should have been protected with goggles,
rinse using eyewash station or faucet for at least 15 minutes or as directed by
safety protocol.
o If skin is exposed, rinse with faucet water for 15 minutes.
The following are recommendations for the Guided Inquiry for Activity 1:
● Help students recognize that there is a lawn of bacteria. Students may have difficulty
recognizing the difference between an empty plate and a lawn where no individual
colonies are visible.
● Ask the students why an experiment is only as good as its controls. Ask students why
three control plates are necessary. It is important to show that the cells are viable,
that the LB/amp plate does inhibit growth if the resistance genes are not
transformed in the cells, and that plasmid transformation is a low efficiency process.
Without ampicillin in the medium, transformed cells would be outnumbered and
barely detectable.

Structured Inquiry
Stage 1: Inducing Competency and Transforming E. coli:
Step 1: Hypothesize/Predict: You will grow your bacteria on media containing ampicillin.
Describe what you would expect if your transformation is successful. Write your ideas in your
notebook.
Step 2: Use the waterproof marker to label two micro tubes with a short group name. Also label
one micro tube – and one +. Place the labeled micro tubes in the micro tube rack. Have a
Styrofoam cup filled with crushed ice ready.
Step 3: With the plastic needle-point pipette, transfer 250 µl of the 50 Mm CaCl2 solution to
each micro tube. Dispose of the needle-point pipette.

265
Step 4: Remove one sterile plastic loop from the package and scrape up one large colony from
the E. coli streak plate. Transfer the E. coli to the – micro tube and swirl the loop in the CaCl2
solution. Make sure you see no residual bacteria on the loop. Dispose of the loop in the
biological hazardous waste container. Close the cap on the – micro tube and place it in the cup
containing the crushed ice.
Step 5: Repeat Step 3, this time placing the E. coli into the + micro tube and returning the micro
tube to the rack instead of placing it in ice.
Step 6: Remove one sterile plastic loop from the package and dip the loop part only into the
pGREEN solution. Swirl the loop in the solution in the + micro tube. Close the cap on the + micro
tube and place it in the cup with the ice. Dispose of the loop in the biological hazardous waste
container.
Step 7: After 15 minutes on ice, carry the ice bath containing the – and + micro tubes to the 42
o
C water bath. Place both micro tubes in the floating micro tube holder and let them sit for 90
seconds. Remove both micro tubes from the 42 oC water bath and immediately return them to
the ice bath.
Step 8: If instructed by your teacher, place your – micro tube and + micro tube in the micro tube
rack and store the racks in a designated refrigerator. The competent bacteria can be stored for
no more than two days.
Step 9: Clean up:
● Return the E. coli plate to the teacher for storage or disposal.
● Empty the ice from the insulated beverage cup and return the cup.
● Return the micro tube filled with 50 mm CaCl2 to your teacher.
● Return any other materials to the teacher.
● Obtain the 70% ethanol solution bottle and squeeze some ethanol onto your workspace.
Wipe down the table with paper towels and place the used towels in the trash. Then
wipe down the tables again with paper towels wetted with tap water. Dry the table with
a fresh set of paper towels.
Step 10: Critical Analysis: Discuss with your team the reason for adding the pGREEN plasmid
solution to only one micro tube. How would your results likely be affected by leaving the
bacteria in the 42 oC water bath for more than 180 seconds or less than 90 seconds? Record
your answers in your lab book. Explain what likely happened during your experiment if you see
no bacterial growth.

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Stage 2: Culturing the Bacteria
Step 1: Retrieve your micro tube rack from the freezer. Let your rack sit until it reaches room
temperature.
Step 2: Using a disposable needle-point pipette or other pipette, add 250 µL of Luria broth
solution to the micro tube. Close the lid on the micro tube. Dispose of the needle-point pipette
in the biohazard waste container. Let the micro tube incubate in the rack for 10 minutes at room
temperature. Be sure to follow all teacher instructions.
Step 3: While the micro tube is incubating, use the waterproof marker to label the lower half of
the two Luria broth + Amp media Petri plates with your group name and the date. Then label
one plate – plasmid and the other plate + plasmid.
Note— Look at Figure 16.5 before continuing to the remaining steps. The figure shows you how
to add the contents of your micro tubes to the Luria broth media plates. You will need to use a
separate needle-point pipette and separate plastic sterile loop for each plate. It is important to
use the following steps to inoculate each Luria broth media plate with the bacteria in the micro
tubes:
1. Place the Luria broth + Amp media plates on the table with the lower plate down.
2. Remove the upper plate.
3. Inoculate the plate with the E. coli.
4. Immediately replace the upper plate.
5. Do not move the Luria broth media plates until it is time to place them in the incubator.

Figure 16.5: Illustration of process for steps 7–10.

267
Step 4: After the 10-minute incubation period, use a pipette to transfer 100 µL of your micro
tube solution to the media in the lower half of the – plasmid Luria broth+Amp plate. Use a
plastic sterile loop to spread the liquid evenly across the surface of the media. Dispose of both
the needle-point pipette and the plastic sterile loop in the biohazardous waste container.
Step 5: Use a new disposable needle-point pipette to transfer 100 µL of your + micro tube
solution to the media in the lower half of the + plasmid Luria broth + Amp plate. Use a plastic
sterile loop to spread the liquid evenly across the surface of the media. Dispose of both the
needle-point pipette and the plastic sterile loop in the biohazardous waste container.
Step 6: Let the plates sit at room temperature for 10 minutes before moving.
Step 7: Carefully carry your four plates for placement in a 37 oC incubator. Leave the plates in
the incubator for 24 hours. If an incubator is not available, store the plates at room temperature
for at least 72 hours.
Step 8: Clean up:
● Return all remaining materials to the teacher.
● Place your micro tubes containing the bacteria into the biohazardous materials
container or in an area designated by your teacher.
● Obtain a squirt bottle with 70% ethanol solution and squeeze some ethanol onto
your workspace. Wipe down the table with paper towels and place the used towels
in the trash. Then wipe down the tables again with paper towels wetted with tap
water. Dry the table with a fresh set of paper towels.
Step 9: The E. coli you placed on the Luria broth media plates have now had time to multiply. As
they multiplied, the E. coli with the pGREEN plasmid replicated the plasmid as well as their own
DNA. Create a drawing of the bacterial growth on each plate in your notebook, noting which
growth is spotty versus extensive. Your goal in this inquiry is to identify which plate shows the
presence of transformed E. coli that are expressing the green fluorescent protein reporter gene.
Remember, the green fluorescent protein reporter gene is telling you that the ampicillin
resistance gene is also being expressed.
Step 10: Collect your four incubated Luria broth media plates and carefully carry them to your
workspace. You should have the plates labeled as followed from Activity 1:
● Luria broth (LB) media Petri plate – plasmid
● Luria broth with ampicillin (LB + Amp) media Petri plate – plasmid
● Luria broth (LB) media Petri plate + plasmid
● Luria broth with ampicillin (LB + Amp) media Petri plate + plasmid

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Step 11: Carefully remove the Parafilm from each plate and dispose of the Parafilm in the
biohazard container. (The Parafilm may have some bacterial contamination.)
Step 12: Record your findings in your notebook. Draw the growth patterns of each plate. You
may also want to take a photograph of each plate.
Step 13: One at a time, remove the lid from each plate and illuminate the bacterial plates with
the blue LED light boxes. Record your findings in your notebook.
Step 14: Clean up:
● Dispose of the four Petri plates in the biohazardous materials container or place in
an area designated by your teacher.
● Return the LED light boxes to the teacher.
● Obtain the squirt bottle with 70% ethanol solution and squeeze some ethanol onto
your workspace. Wipe down the table with paper towels and place the used towels
in the trash. Then wipe down the tables again with paper towels wetted with tap
water. Dry the table with a fresh set of paper towels.
Step 15: Critical Analysis: Based on your results, which plate or plates contained bacteria that
were expressing the ampicillin resistance gene on the pGREEN plasmid? Compare your data to
the data from the other students in class. Did any of the groups have data different from yours?
If there was a difference, explain what could have caused this variation. Record your answers in
your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: In your initial experiment, you tested for transformation by
growing your transformed and untreated bacteria on media containing the antibiotic ampicillin.
What was the role of the ampicillin in determining whether the bacteria were transformed?
Explain how you were able to distinguish between the control bacteria that you didn’t
transform and the transformed bacteria. What would occur if you placed your non-transformed
and transformed bacteria on LB plates that did not contain ampicillin? Write your hypothesis in
your notebook.
Step 2: Student-led Planning: Write out the steps to test your hypothesis about culturing
non-transformed E. coli and transformed E. coli having the pGREEN vector on plain LB plates.
Remember to keep in mind how you will safely dispose of your lab waste. Submit your
experimental procedure to your teacher. Once your teacher approves of your procedure,
perform your experiment and record your data in an appropriate format. Write your results in
your notebook.
Step 3: Carry out the procedure you devised in Step 2. Your setup will be as shown in Figure
16.6.

269
 Figure 16.6: Experimental treatments for Guided Inquiry.

Step 4: Critical Analysis: Compared to the growth on the LB + Amp plates, what additional
information did you discover by growing the non-transformed and transformed bacteria on the
plain LB plates? How would this information be useful to scientists who are conducting
transformation experiments? What is the value of using the GFP reporter gene in assessing the
success of cell transformation? Write your results in your notebook.

Assessments
1. Explain why ampicillin must be placed in the Luria broth medium used to grow the
transformed cells? [SP 1, Concept Explanation; SP 3, Questions and Methods]
2. Explain why the heat shock step is essential for transformation to occur? [SP 1, Concept
Explanation; SP 3, Questions and Methods]
3. In this activity, you induced competence in E. coli cells. However, certain environmental
conditions that stress bacteria can induce a natural competence. What is the value of
competence to survival under stressful conditions? [SP 1, Concept Explanation; SP 6,
Argumentation]
4. Describe how your results would most likely change if the ampicillin-resistance gene
underwent a severe mutation within the bacteria? [SP 6, Argumentation]
5. Draw the gene sequence that shows why expression of the ampicillin-resistance gene
also causes the expression of the green fluorescent protein. [SP 1, Concept Explanation;
SP 2, Visual Representation]
6. Heat shock transformation does not guarantee that all bacteria will take up plasmids or
that all bacteria will have the same number of plasmids. How could you detect this in
your results? [SP 2, Concept Explanation; SP 3, Questions and Methods]

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[Answer Key]
1. Ampicillin is the antibiotic that will induce expression of the ampicillin-resistance gene
that was inserted in the pGREEN plasmid.
2. The heat shock process permits the cell membrane of E. coli to take in the plasmid by
making pores in the cell membrane. Otherwise, the plasmid will remain outside the E.
coli cell.
3. Competence encourages E. coli to take in DNA that is present in the environment. This
process could provide novel genes that improve survival during stressful conditions.
4. If the ampicillin resistant gene was severely mutated, it likely will not be expressed and
the bacteria will die from the ampicillin before being able to reproduce. Therefore, the
plates with ampicillin will have no bacterial growth.
5. A drawing should show the ampicillin-resistance gene and green fluorescent protein
gene in line with the regulatory gene that induces the ampicillin-resistance gene. For
example: ampicillin-resistance regulatory gene followed by ampicillin-resistance gene
followed by green fluorescent protein gene.
6. The intensity of the fluorescent protein gene is an indicator of transformation efficiency.
So, E. coli on the LB+Amp + plasmid plate would not glow under blue light if the bacteria
died from the ampicillin and cells with fewer plasmids would have fainter colonies than
cells with more plasmids.

Extension Activities 3:
1. Carry out a study to determine whether exposure of the pGREEN plasmid to ultraviolet
light affects the expression of the plasmid.
2. Perform a DNA extraction on E. coli cells to determine whether you can isolate the
pGREEN plasmid from the transformed cells on the LB plate + plasmid and the LB+Amp
plate + plasmid. Then use the polymerase chain reaction and electrophoresis to confirm
the results.

271
 Lab 17: Hardy-Weinberg Equation
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRE COURSE CONTENT
Topic Enduring Understanding Learning Objective Essential Knowledge
EVO-1 Evolution is
characterized by a
7.1 Introduction to change in the genetic EVO-1.C Describe the EVO-1.C.1 Natural
Natural Selection makeup of a population causes of natural selection is a major
over time and is selection. mechanism of evolution.
supported by multiple
lines of evidence.
EVO-1 Evolution is
characterized by a EVO-1.E Describe the EVO-1.E.1 Natural
change in the genetic importance of selection acts on
7.2 Natural Selection makeup of a population phenotypic variation in a phenotypic variations in
over time and is population. populations.
supported by multiple
lines of evidence.
EVI-1.H.1 Evolution is
also driven by random
EVO-1.H Explain how
occurrences, including
EVO-1 Evolution is random occurrences
mutation, genetic drift,
characterized by a affect the genetic
and migration/gene
change in the genetic makeup of a population.
flow.
7.4 Population Genetics makeup of a population
EVO-1.J.1 Mutation
over time and is EVO-1.J Describe the
results in genetic
supported by multiple change in the genetic
variation, which
lines of evidence. makeup of a population
provides phenotypes on
over time.
which natural selection
acts.
EVO-1.K.1
Hardy-Weinberg is a
model for describing and
predicting allele
EVO-1 Evolution is
frequencies in a
characterized by a EVO-1.K Describe the
non-evolving population
change in the genetic condition under which
7.5 Hardy-Weinberg under certain
makeup of a population allele and genotype
Equilibrium conditions, such as a
over time and is frequencies will change
large population.
supported by multiple in populations.
EVO-1.K.2 Allele
lines of evidence.
frequencies in a
population can be
calculated from
genotype frequencies.

272
 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.C Explain how biological concepts or processes

SP 2 Visual Representations are represented visually related to larger
biological principles, concepts, processes, or
Analyze visual representations of biological theories.
concepts and processes. 2.D Represent relationships within biological
models, including mathematical models.

3.A Identify or pose a testable question based on

SP 3 Questions and Methods
an observation, data, or a model.
3.B State the null and alternative hypotheses, or
Determine scientific questions and methods.
predict the results of an experiment.

SP 4 Representing and Describing Data

4.B Describe trends and/or patterns in data.
Represent and describe data.

SP 5 Statistical Tests and Data Analysis

5.A Perform mathematical calculations, including
mathematical equations from the curriculum.
Perform statistical tests and mathematical
5.D Use data to evaluate a hypothesis, including
calculations to analyze and interpret data.
rejecting or failing to reject the null hypothesis.
SP 6 Argumentation
6.B Support a claim with evidence from biological
Develop and justify scientific arguments using principles, concepts, processes, and/or data.
evidence.

Population genetics is the field of biology that studies genetic variation in populations
over time. Change that occurs in a population over time is known as evolution. It involves
changes in the proportion or frequency of specific alleles in a given population from one
generation to the next. These changes can be due to factors such as recombination, selection,
and mutations. An example of evolution in a population due to selection might involve alleles
that code for traits that convey survival benefits that are favored over other alleles that code for
traits that are less beneficial. The favorable allele would gradually increase in frequency over
time while the unfavorable allele would diminish and potentially disappear.

273
 Allele frequency is defined as the proportion of alleles in the population that are the
particular type in question. It is represented as a fraction of 1. For example, if an allele makes up
65 percent of the total population and another makes up 35 percent, the frequencies will be
0.65 and 0.35, respectively. The frequencies of all the different alleles for one particular gene
will always add up to 1.0. One allele is usually designated as p (typically the more common or
dominant allele) and the other is usually designated as q (typically the less common or recessive
allele). Therefore, p + q= 1.0. According to the Hardy-Weinberg model, there will be no change
in allele frequency in a sexually reproducing population (genetic equilibrium) if:
● There is no mutation.
● No migration occurs that alters the population.
● Random mating between individuals in the population occurs.
● The population is large enough so that changes in allele frequencies will not be due
to chance.
● No selection occurs.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● Brainstorm the definition of evolution with the students. What does it mean to them? At
what scale do we measure evolution? Can it happen quickly, or does it take a long time
to happen?
● Define population genetics. Why do we need to know how populations change over
time? What are some practical applications of understanding the changes in genetic
structure over time?
● Define allele, recombination, selection, and mutations. How do these things relate?
Discuss the definition of allele frequency and why the students think p+q=1. Make sure
they can make sense of the equation.
● Discuss the Hardy-Weinberg Model and how it applies to determining allele frequency.
Ask the students to brainstorm as to why each of the requirements need to be met in
order to have genetic equilibrium. What would happen if something happened to
change one of the requirements?

274
In this lab, you will learn
● how genetics affects the sense of taste
● about the components of the Hardy-Weinberg equation
● how to use the Hardy-Weinberg equation to calculate allele frequencies of dominant
and recessive alleles

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Have the students think about sample size and small populations. How would the class
compare to the entire United States? North America? The whole human population?
Have them think about how certain allele frequencies might vary from region to region.
● Have the students discuss why it’s unlikely for Hardy-Weinberg Equilibrium to ever be
reached with most alleles. Have them try to think of ways that it could be reached.
● Discuss how the equation is derived from p+q=1. Why do we need to think about
homozygous and heterozygous individuals? Are there cases where you will never have a
heterozygous individual? How about a homozygous individual?
● Have the students discuss the evolutionary advantages and disadvantages of having the
ability to taste bitter. What regions of the world might have more people with the ability
to taste bitter? What places might not?

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: Why might the allele frequencies
from a sample population not match those from the general population?
2. Answer the following questions in your notebook: Would you expect allele frequencies
of a given allele to remain constant among humans in the United States? Would you
expect the conditions of Hardy-Weinberg to still be met in the next 50 years within the
United States? Why or why not?
3. Discuss the answers to questions 1 and 2 with the class.

275
Activity 1: Hardy-Weinberg Equation: Determining PROP Tasters vs.
Non-tasters
If the Hardy-Weinberg model applies, then the frequencies of genotypes designated as
either pp, pq, or qq will not change over time. Hardy Weinberg equilibrium can be written as an
equation and can be used to estimate the proportions of these alleles. The Hardy-Weinberg
equation is
p2 + 2pq + q 2 = 1.0 ,
where p2 represents the homozygous individual for the dominant allele, pq represents the
heterozygous individual, and q2 represents the homozygous individual for the recessive allele.
The ability to taste certain bitter compounds is genetically determined. In this lab, we
will conduct a simple experiment to study gene proportions using the class as a sample
population. We will be using a chemical called PROP (6-n-propylthiouracil), shown in Figure 17.1
and found in bitter tasting foods, like the Brussels sprouts shown in Figure 17.2. Individuals that
can taste this compound are homozygous dominant (TT; highly sensitive- supertaster) or
heterozygous (Tt; moderately sensitive- medium-taster). Those individuals unable to taste are
homozygous recessive (tt; non-taster).

Figure 17.1: Chemical structure of PROP (6-n-propylthiouracil).

Figure 17.2: Supertasters have less preference for and tend to avoid bitter-tasting foods like Brussels sprouts.

276
Safety Precautions
● PROP is used to treat hyperthyroidism. You should not be exposed to high amounts of it.
The taste strips only have very small amounts.
For this activity, you will need:
● Control taste strip
● PROP taste strip
For this activity, you will work in groups of 4.

Structured Inquiry
Step 1: Hypothesize/Predict: Based on your previous experience with bitter tasting foods,
hypothesize about whether you are a supertaster, medium-taster, or a non-taster. Predict your
reaction to PROP. Write your prediction in your notebook.
Step 2: Student-led Planning: In your notebook, create a data table to enter your group’s
reactions to the control and PROP taste strips. Create another table to show the reactions of the
entire class. You should indicate whether the individual believes that the strips are extremely
bitter (supertaster), bitter (medium-taster), or cannot be tasted (non-taster).
Step 3: Place a control taste strip on your tongue. Allow the saliva to saturate the strip. Discuss
with your partner what the strip tasted like. Record your reaction. Classify the taste as
extremely bitter, bitter, or not bitter.
Step 4: Repeat Step 3 and place the PROP taste strip on your tongue.
Step 5: Critical Analysis: Record the reactions of all group members in your data table. Are the
predictions you made in Step 1 supported by your data? Why or why not? What methods could
you use to improve your results? Discuss with your group and then write your answers in your
notebook.

Guided Inquiry 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Guided Inquiry 1:
● Revisit the discussion on sample size. Have them discuss how their group might compare
to the whole class. Then have them discuss how it would compare to other regions.
● Have them think about why there is variation across populations. Why is this variation
important? What other traits can they think of that might vary across different groups of
people? Why are these variations important? What are some applications of
understanding the variation?

277
Guided Inquiry
Step 1: Hypothesize/Predict: Predict the gene frequencies of the dominant and recessive PROP
tasting genotypes based on the proportion observed in your group. How do you think it
compares to the entire class? Write your ideas in your notebook.
Step 2: Student-led planning: Obtain data from the other groups in the class. Create a table in
your notebook to place allele frequencies for the class.
Step 3: Use the class data and the Hardy-Weinberg equations, p + 1 = q and
p2 + 2pq + q 2 = 1.0 , to calculate allele frequencies for the supertasters, medium-tasters, and
non-tasters.
Step 4: Critical Analysis: Determine whether your prediction is supported. Are there more
individuals with the dominant phenotype? With the recessive phenotype? How does this
compare to your group’s data? Is either population (group or class) in Hardy-Weinberg
equilibrium? Discuss your answer with your group members and write your responses in your
notebook.

Assessments
1. What are some reasons that the values for p and q for your class might differ from the
frequencies reported for the entire North American population of humans, in terms of
PROP tasting? [SP 1, Concept Explanation; SP 6, Argumentation]
2. What are the limitations of Hardy-Weinberg equilibrium? [SP 1, Concept Explanation]
3. A population of bears has individuals with black fur (dominant) or brown fur (recessive).
Black bears have the genotype BB or Bb. Brown bears have the genotype bb. The
frequency of the BB genotype is 0.59. [SP 5, Statistical Tests and Data Analysis]
a. What is the frequency of heterozygous bears?
b. What is the frequency of the B allele?
c. What is the frequency of the b allele?

278
[Answer Key]
1. The population within the class is much smaller than the entire North American
population. Therefore, the class population would likely differ in its proportion of the
various tasters from a larger population due to random chance.
2. The Hardy-Weinberg equilibrium is an idealized model that has the following
assumptions:
● There is no mutation.
● No migration occurs that alters the population.
● Random mating.
● The population must be large enough so that changes in allele frequencies will not
be due to chance.
● No selection occurs.
3. a. 2pq= 0.356, b. p = 0.768, c. q = 0.232

279
 Lab 18: Natural Selection Simulation
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Topic Enduring Understanding Learning Objective Essential Knowledge
EVO-1.C.1 Natural
selection is a major
mechanism of evolution.
EVO-1.C.2 According to
Darwin’s theory,
individuals with more
favorable phenotypes
EVO-1 Evolution is EVO-1.C Describe the
can better compete for
characterized by a causes of natural
limited resources and
change in the genetic selection.
7.1 Introduction to survive, thus passing
makeup of a population
Natural Selection traits to subsequent
over time and is EVO-1.D Explain how
generations.
supported by multiple natural selection affects
EVO-1.D.1 Evolutionary
lines of evidence. populations.
fitness is measured by
reproductive success.
EVO-1.D.2 Fluctuating
biotic and abiotic
environments can affect
the rate of evolution.

EVO-1.E.1 Natural
selection acts on
phenotypic variations in
populations.
EVO-1 Evolution is EVO-1.E.2 Environments
characterized by a change and apply
EVO-1.E Describe the
change in the genetic selective pressure to
importance of
7.2 Natural Selection makeup of a population populations.
phenotypic variation in a
over time and is EVO-1.E.3 Some
population.
supported by multiple phenotypic variations
lines of evidence. significantly increase or
decrease fitness of the
organism in particular
environments.

280
 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
Explain biological concepts, processes, and
models in applied contexts.
models presented in written format.

2.A Describe the characteristics of a biological

concept, processes, or model represented
visually.
2.B Explain relationships between different
SP 2 Visual Representations characteristics of biological concepts, processes,
or models represented visually in applied
Analyze visual representations of biological contexts.
concepts and processes. 2.C Explain how biological concepts represented
visually relate to larger biological principles,
concepts, processes, or theories.
2.D Represent relationships within biological
models, including diagrams.

SP 3 Questions and Methods

3.A Identify or pose a testable question based on
Determine scientific questions and methods. an observation, data, or a model.

SP 4 Representing and Describing Data

4.B Describe trends and/or patterns in data.
Represent and describe data.

SP 5 Statistical Tests and Data Analysis

Perform statistical tests and mathematical

5.A Perform mathematical calculations.
calculations to analyze and interpret data.

SP 6 Argumentation
6.A Make a scientific claim.
Develop and justify scientific arguments using 6.E Predict the effects of a change in one or more
evidence. concepts in a biological system based on data.

281
 Evolution is the change in a population over time. One of the major driving forces of
evolution is the process of natural selection, in which organisms that are better adapted to a
particular environment tend to survive and reproduce. The concept of natural selection was first
brought to light by Charles Darwin and Alfred Wallace in the 1850s. Their observations
illuminated several requirements or conditions that must be met for natural selection to occur:

● Organisms must have variation, or differences in their physical and behavioral

characteristics.
● Organisms must be in competition for resources.
● Some organisms must be better adapted, or suited, to their environment than others.
● Some organisms must have greater reproductive success.

These concepts lead to what is known as the survival of the fittest. This means that some
organisms have adaptations that allow them to better survive in a particular environment.
These organisms are able to successfully compete for resources and then produce offspring that
are viable and able to pass their genes onto the next generation. These are said to be selected
for. Those individuals that disappear over time have been selected against.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:

● More than a million live species on Earth. An explanation for the diversity in living things
itself is a useful way to introduce this topic.
● Understanding evolution is the key to understanding diversity. The mechanism of
evolution is natural selection, and though students may have a general understanding of
how evolutionary change works, they need to understand the range of evolutionary
changes, from individuals with certain traits to how the traits in a whole population can
change.
● Ask the students to discuss the relationship between Darwin and Wallace’s work. How
did they come to their conclusions? Were their ideas accepted right away? What more
modern data help support their ideas?

In this lab, you will learn

● how one organism is naturally selected over another
● how natural selection occurs over multiple generations
● how environmental factors affect natural selection

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Activity 1 Instructor Preparation and Teaching Tips
The following are recommendations for Activity 1:

● Encourage the students to think like Darwin or Wallace. What were their observations
that helped them come to their conclusions?
● Have them think about all the influences we have around us—climate, weather,
geography, and even human-caused issues. What traits might be an advantage for
certain scenarios? What traits might be a disadvantage? Are there any traits you can
think of that might help in one scenario but be harmful in another?
● Students may struggle with the concept of time, as it can take generations, even after a
large catastrophic event, for natural selection pressures to become evident. You might
want to discuss catastrophic events versus slower-acting pressures, such as niche
partitioning in an island system.
● Throughout the activity, ask students to think about different traits that modern plants
and animals have that are perfectly adapted to our current climate but might be a
disadvantage when the climate changes significantly in the future. How might these
species adapt, or will they not be able to survive the change?

Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: Variations in physical characteristics
can either be advantageous or harmful for organisms. Give an example of when an
organism’s physical traits might be harmful. Give an example of when they might be
beneficial. Describe how the frequency of harmful and beneficial traits changes over
generations in a population.
2. Answer the following question in your notebook: Why would the frequency of physical
characteristics in a population change over several generations?
3. Answer the following question in your notebook: If a flood occurred, affecting a
population of raccoons, describe how you think some members of that population might
be able to survive better than others. (Take being rescued out of the equation.)
4. Answer the following question in your notebook: Name at least two environmental
changes that could occur that might affect the survival of organisms within a particular
ecosystem.
5. Discuss the answers to questions 1–4 with your class.

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Activity 1: Survival of the Fittest
In natural selection, any inherited trait that makes survival likely for an individual is an
adaptation. Those individuals that are best adapted to a given environment are more likely to
survive and reproduce. In this activity, you will simulate natural selection occurring in a
population over time. You will begin with a random number of pipe cleaners representing
individual organisms with a particular phenotype. Brown or green pipe cleaners will represent
brown or green caterpillars, like the one shown in Figure 18.1.

Figure 18.1: Green caterpillars can easily blend in with their environment, increasing their chances of survival.

Natural selection depends on the environment, as environmental factors drive natural

selection. For example, a pond suddenly becomes a river as a result of flooding within a region.
The flooding results in the loss of many animals, and only a few of the strongest survive. Natural
disasters, climate change, chemicals that trigger mutation, and pH levels are all examples of
environmental changes that might affect diversity or variation within a population. In this
activity, you will examine how survival of an organism, as determined by a variety of selective
forces, affects the evolution of a simulated population.
Safety Precautions
● None
For this activity, you will need the following:
● 30–50 pipe cleaners or chenille sticks (green and brown assortment)
● Paper plates
For this activity, you will work in groups of four.

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Structured Inquiry 1 Instructor Preparation and Teaching Tips
The following are recommendations for the Structured Inquiry 1:

● Discuss and define selective advantage. Selective advantages are characteristics of a

particular organism that enable it to survive better than others in the population.
● What might be some selective advantages of a brown caterpillar over a green one in
grass? On dirt? What about on pavement? Think of a number of scenarios that could
provide an advantage to an organism.
● How might you test this concept in nature? Ask students how they might measure
selective advantage when studying organisms in their natural habitats.

Guided Inquiry 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Guided Inquiry 2:

● Review what a phenotype is and why certain phenotypes can have an advantage in
one scenario over another. Can the students think of any organism that may have one
phenotype in one habitat and another in a different habitat?
● How might selecting for certain phenotypes lead to speciation? How might
phenotypic variation guide adaptation to a catastrophic event?

Structured Inquiry
Step 1: Collect an assortment of 30–50 pipe cleaners, roughly half of each color. Place five of
each color on a plate that will represent the habitat. Set the others aside. These will be
offspring.
Step 2: Hypothesize/Predict: Hypothesize about which caterpillars might survive better. Make a
prediction that describes what will happen if a predator takes caterpillars at random, regardless
of color, and then the remaining caterpillars only mate with caterpillars of the same color.
Predict how the population will change over multiple generations if neither color confers an
advantage.
Step 3: Student-Led Planning: You will record the number of individuals displaying each
phenotype for 10 generations (i.e., the number of brown and green caterpillars). Create a table
that will allow you to monitor each generation. Then, for each generation, first, determine the
initial percentage of each color and record this information in your notebook. Remove three
caterpillars of random colors from the habitat plate. To make this random, close your eyes and
randomly remove three caterpillars. (These are the ones that died.) Set them aside in a dead
caterpillar pile. Close your eyes again and randomly select three caterpillars to mate.
Temporarily remove them from the habitat plate to make recording data easier. Then pair them
with their same-colored mates from the habitat plate. The offspring will be the same color as
the parents and will be chosen from the extra caterpillar plate (not the dead caterpillar pile). For
example, if you randomly selected two green caterpillars and one brown caterpillar, and those

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were able to pair and mate, select two greens and one brown from the extra caterpillar pile as
offspring for the pairs. Record your results in your data table. Put the parents and offspring on
the habitat plate. Repeat for nine more generations, or until you run out of caterpillars.
Step 4: Critical Analysis: Are the predictions you made in Step 2 supported by your data? Why
or why not? What methods could you use to improve your results? Discuss with your group and
write your answers in your notebook.

Guided Inquiry 1
Step 1: Using 30–50 green and brown pipe cleaner caterpillars, repeat the steps in the
Structured Inquiry. This time, you will choose a selective advantage. Discuss with your group
what the selective advantages might be and record your responses in your notebook.
Step 2: Hypothesize/Predict: In green grass, which color would have a selective advantage?
Why? Hypothesize about which caterpillar might survive better, then make a prediction that
describes the future population of the caterpillars. How do you think the population will change
over 10 generations?
Step 3: Student-Led Planning: With your group, decide on one trait that will confer a selective
advantage to one color or the other. For example, if you decide that brown caterpillars are more
visible to predators, choose two brown caterpillars to die. Then, choose the caterpillars that will
mate. Color will not affect the ability to mate, so close your eyes to choose two caterpillars that
will mate. Pair them with mates. Then add two additional caterpillars, of the same color that
mated, to represent the offspring of the mating. This signifies the end of this generation. Repeat
for nine more generations and record the number of green versus brown caterpillars at the start
and end of each generation.
Step 4: Critical Analysis: Record the number of individuals displaying each phenotype for five
more generations (i.e., the number of brown and green caterpillars). Are the predictions you
made in Step 2 supported by your data? Why or why not? What methods could you use to
improve your results? Discuss with your group, and then write your answers in your notebook.

Guided Inquiry 2
Step 1: Student-Led Planning: Pick another phenotype that could apply a selective advantage
(e.g., reproductive success or protection against predators). Describe your phenotype to your
teacher, including what advantage it gives to individuals that possess that phenotype and how
that advantage will be simulated with the pipe cleaners. After your teacher approves, describe
your phenotype in your notebook.
Step 2: Hypothesize/Predict: Predict how natural selection would occur with the new
phenotype and hypothesize how it will affect the phenotypic frequencies of the population
after 10 generations. Use the same simulation procedure from Guided Inquiry 1. How would the
frequency of brown and green change after a natural disaster? Write your hypothesis in your
notebook.

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Step 3: Think about how you will simulate change in selection that occurs after a natural
disaster. For example, you might decide that damage from a fire is brief, so for one or two
generations, only green caterpillars die. Then things go back to normal. Or maybe the damage is
severe and lasts for five generations. Change your selection based on your decision and
continue. Write your plan in your notebook.
Step 4: Critical Analysis: Make a graph that depicts changes over the 10 generations. How does
the percentage of each phenotype change over time? Discuss your answer with your group, and
write it in your notebook.

Assessments
1. What factors might contribute to changes in a population? [SP 1, Concept Explanation;
SP 6, Argumentation]
2. In the Arctic, there are white rabbits and brown rabbits. Which would be more likely to
survive if there is extensive snow cover? As the climate changes (i.e., the temperature
increases), what would you expect to happen? [SP 1, Concept Explanation; SP 6,
Argumentation]
3. In a population of caterpillars, a mutation has suddenly arisen that makes green
caterpillars less “tasty” to their predators. What would you expect to occur in the
population over time? [SP 1, Concept Explanation; SP 6, Argumentation]
4. Observe what happened in Figure 18.2 and explain how it represents an example of
natural selection. [SP 3, Visual Representations; SP 1, Concept Explanation]

Figure 18.2: Describe the effect of natural selection that occurs in the two photographs.

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[Answer Key]
1. Some factors that contribute to changes in a population are natural selection due to
competition for resources, beneficial and harmful mutations, environmental changes,
and greater reproductive success within populations.
2. The white rabbit would have a better chance at survival than the brown rabbit, as the
white rabbit can more easily blend in with the white snow. The brown rabbit would be
more subject to predation in the snow. If temperatures increase and it does not snow as
much, brown fur would blend in with the bare soil, making the rabbit less obvious prey.
A white rabbit would be more noticeable to a predator.
3. Predators would begin to avoid the green caterpillars because of their distaste for the
newly mutated caterpillar, and they will find another source of food. As a result, over
time, a greater percentage of the caterpillars would be green.
4. This image shows how organisms interact in an herbivore-plant relationship. Over time,
the cactus with the spines will have greater reproductive success than the spineless
cactus. Therefore, the frequency of cactus with spines will increase over time.

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 Lab 19: Plant Transpiration
This investigation addresses the following Required Course Content and Science Practices
from the AP Biology CED:
REQUIRED COURSE CONTENT
Topic Enduring Understanding Learning Objective Essential Knowledge
SYI-1.A Explain how the
properties of water that SYI-1.A.3 The hydrogen
1.1 Structure of Water SYI-1 Living systems are
results from its polarity bonds between water
and Hydrogen Bonding organized in a hierarchy
and hydrogen bonding molecules result in
of structural levels that
affects its biological cohesion, adhesion, and
interact.
function. surface tension.

ENE-1.C.1 Organisms
ENE-1 The highly ENE-1.C Explain how
have evolved highly
complex organization of specialized structures
efficient strategies to
living systems requires and strategies are used
2.3 Cell Size obtain nutrients and
constant input of energy for the efficient
eliminate wastes to and
and the exchange of exchange of molecules
from the environment.
macromolecules. to the environment.
ENE-2.I.1 Growth and
homeostasis are
maintained by the
constant movement of
molecules across
ENE-2 Cells have membranes.
membranes that allow ENE-2.I Explain how ENE-2.I.2
them to establish and osmoregulatory Osmoregulation
2.8 Tonicity and
maintain internal mechanisms contribute maintains water balance
Osmoregulation
environments that are to the health and and allows organisms to
different from their survival of organisms. control their internal
external environments. solute composition and
water potential, with
water moving from
areas of high water
potential to areas of low
water potential.
ENE-3 Timing and
coordination of ENE-3.D Explain how the ENE-3.D.1 Organisms
biological mechanisms behavioral and/or respond to changes in
8.1. Responses to the involved in growth, physiological response their environment
Environment reproduction, and of an organism relates to through behavioral and
homeostasis depend on changes in internal or physiological
organisms responding to external environment. mechanisms.
environmental cues.

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 SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.A Explain biological concepts and/or processes.
Explain biological concepts, processes, and 1.C Explain biological concepts, processes, and/or
models presented in written format. models in applied contexts.

2.B Explain relationships between different

SP 2 Visual Representations characteristics of biological concepts, processes,
or models represented visually in applied
Analyze visual representations of biological contexts.
concepts and processes. 2.D Represent relationships within biological
models, including diagrams.

3.A Identify or pose a testable question based on

an observation, data, or a model.
3.B State the null and alternative hypotheses that
SP 3 Questions and Methods are aligned to the questions, including variables
and controls.
Determine scientific questions and methods. 3.D Make observations, or collect data from
representations of laboratory setups or results.
3.E Propose a new/next investigation based on
evaluating evidence from a previous experiment.

SP 4 Representing and Describing Data 4.A Construct a graph, plot, or chart.

4.B Describe data from a table or graph, including
Represent and Describe Data describing trends and/or patterns.

SP 5 Statistical Tests and Data Analysis

5.A Perform mathematical calculations, including
applying equations in the curriculum, means, and
Perform statistical tests and mathematical
rates.
calculations to analyze and interpret data.
5.C Use data to evaluate a hypothesis/prediction.
6.A Make a scientific claim.
SP 6 Argumentation
6.B Support a claim with evidence from biological
principles, concepts, processes, and/or data.
Develop and justify scientific arguments using
6.E Predict the effects of change in one of more
evidence.
components in a biological system based on data.

Plants take in water from their roots. The water travels upward to all the plant’s other
parts, ending up in the leaves. Excess water evaporates from small openings on the undersides
of the leaves called stomata. Stomata can open and close to regulate the loss or conservation of
water, depending on the needs of the plant or in response to environmental conditions. The
process of water evaporating from stomata is called transpiration.

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 Have you ever wondered how water moves upward against gravity without some kind of
pump? The simple answer is that water moves from areas of high water potential to areas of
low water potential. Water potential is the tendency of water to move from one area to
another. In plants, the leaves tend to have lower water potential than the roots, so the
movement is upward from under the ground toward the leaves. Different factors can affect
whether water potential is high or low, including negative pressure (suction) caused by
transpiration and the concentrations of solutes in the water and/or the surrounding soil.
When a plant does not have sufficient water moving through its structures and into its
leaves, it can experience water stress, which can affect all processes within the plant. Water is
the solvent through which plant materials move. Various things can cause water stress,
including drought, high rates of transpiration, and high salinity in the soil.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● It may be helpful to begin the lab by giving the students a brief overview on the topic of
plant transpiration. You can use real-life plant samples or a model of a plant to make the
process more understandable to the students.
● Since the students will be working with a potometer, it would be useful to also
demonstrate how to properly set up and use this instrument for the experiment.
● Upon finishing the overview of the topic, be sure to instruct students on the proper way
to dispose of their plant samples and how to disassemble their potometer.
● Fresh, pre-cut plants should be available for this experiment. Have the proper quantity
of plants you will need for your class size. Proper storage of plant samples should also be
considered prior to this exercise to minimize premature decay of the plant material prior
to the experiment.

In this lab, you will learn

● how to measure the rate of transpiration
● how to test and predict the way in which an environmental change affects the rate of
transpiration
● how to observe patterns of and predict percentages of open versus closed stomata
under different conditions

Activity 1: Pre-Assessment
1. Answer the following questions in your notebook: What environmental conditions do
you think will most likely cause stomata to open? Which will most likely cause stomata
to close? Explain your answer.

291
 2. Answer the following question in your notebook: Why do you think the suction caused
by transpiration is considered a negative-feedback mechanism?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 1: Measurement of Transpiration as Water Loss under

Selected Conditions
You will conduct this first activity in three parts. First, you will assemble an apparatus
called a potometer, illustrated in Figure 19.1. A potometer consists of a plant cutting stuck into
one end of a U-shaped tube and a graduated cylinder stuck into the other end. This device
creates water potential, which will allow you to measure transpiration. In the second part, you
will collect data for transpiration under a set of control conditions. In the third part, you will
choose from a list of variables to change the conditions for the plant and then you will collect
transpiration data again using the modified apparatus.

Figure 19.1: When transpiration takes place, water evaporates from open stomata. As the water potential
difference increases between the bottom of the stem and the leaves, the stem draws in water from the tube, and
the water level drops in the pipette.

Safety Precautions
● Use care with all glassware.
● Clean up any spilled water immediately to avoid slippage.
● Insert the pipette carefully to avoid breaking the tip.
● Do not allow electric cords to get wet.
● Be careful when handling hot lamps.

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For this activity, you will need the following:
● Ring stand
● Plant stalk with leaves
● Incandescent lamp
● Clamps
● Plastic tubing
● Basin half-filled with water
● Calibrated 1.0-mL pipette
● Basin
● Syringe
● Petroleum jelly
● Fluorescent light or water tank with incandescent light
● Fan
● Water sprayer
● Plastic bag
● Timer
For this activity, you will work in pairs.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Since this may be the first-time students have worked with a potometer, it may be
beneficial to briefly lecture on the use of this instrument as well as to demonstrate how
to properly use the instrument and take measurements prior to conducting the
experiments. Since calibrated pipettes will also be used, remind the students of the
importance of lab safety (e.g., students should never pipet anything by mouth).
● Since students will be measuring the level of water every five minutes, it will be helpful
to have timers on hand to assist them in taking their measurements accurately.
● After introducing students to the topic, have them create their data tables prior to
beginning the lab exercise to ensure that they are including the appropriate criteria in
their lab reports for later analysis.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 1:
● Students could remain in groups of two while doing the Guided Inquiry, but you may
wish to have each student write their own hypotheses regarding the proposed outcomes
of the experiment. This is to ensure that each student contributes equally and submits
their own original content.

293
 ● You will want to check the students’ hypotheses and results from the initial experimental
design during the Structured Inquiry before allowing them to proceed with performing
their experiment to ensure that they are on the right track and will not waste
unnecessary materials.
● Since multiple variables will be tested in the Guided Inquiry portion of the exercise, have
an ample supply of materials (e.g., fans, syringes, water sprayers, plastic bags, etc.) that
they will need to conduct this portion of the exercise. They will also likely need advice on
how to determine what controls to use for their experiment.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 3, clarify to students how to analyze their results. Additionally, providing a
grading rubric for this activity with criteria describing each of the mandatory parts of this
activity can help guide students to include the proper components in their experiment.

Structured Inquiry
Step 1: Set up the potometer, using Figure 19.1 as a guide. To reduce the formation of bubbles,
cut the twig and insert it into the water-filled plastic tubing while underwater. Add the pipette
and adjust water level as necessary so the starting point is high enough to allow for readable
decreases during the experiment. If there are any air bubbles, you can use the syringe to
remove them. Use clamps to attach the potometer to the ring stand, making sure the heights of
the plant relative to the pipette are similar to those in Figure 19.1.
Step 2: Hypothesize/Predict: After you insert the stem into the tube, how much water do you
think will move out of the pipette? Write your predictions in your notebook.
Step 3: Student-Led Planning: You will start with measuring changes in the water level for the
plant under the typical light conditions of the room. Before you begin, make a data table to
collect information about the water level and the change in water level as a function of time.
You will take data every five minutes, for a total of 20 minutes.
Step 4: When you are ready, insert the stem, making sure it is submerged, and take an initial
reading of the water level at time zero and subsequent readings every five minutes. Then,
record the water loss in your data table every five minutes, for a total of 20 minutes. After the
20-minute period, remove the leaves from the photometer and set them aside, as you will need
to measure the area of the leaf measured in the next step.
Step 5: Critical Analysis: Discuss with your partner how the first trial worked and if any
adjustments need to be made to the setup to ensure more accurate results. Are the predictions
made in Step 2 supported by the data? Can your results be improved? After the data collection,
you will need to calculate a rate of transpiration per minute. To do this, you will take a water
level reading every five minutes for 20 minutes for each part of the experiment. Then, you can
graph the water measurements by time to estimate the rate of transpiration per minute. After
this, you will need to determine the area of the leaf surface measured. Since there are multiple
leaves of different sizes, you will trace each leaf on graph paper and add up the total area. The

294
final result of the experiments should show the rate of transpiration. Discuss with your partner
and write your answers in your notebook.

Guided Inquiry
Step 1: Hypothesize/Predict: Choose from the following list of variables to alter the conditions
for the plant:
● Use fluorescent light (if only an incandescent light is available, place a tank of
water between the plant and the light source to absorb heat).
● Apply wind using a fan.
● Apply high moisture by spraying the plant with water and then enclosing it in a
plastic bag.
How do you think your chosen variable will affect the rate of transpiration? Does the
adjustment accurately mimic conditions in a natural environment? Why or why not?
Step 2: Student-Led Planning: How will you adjust your setup to incorporate your chosen
variable? Create a plan with your partner. Then, have your teacher approve your plan before
altering the setup. Once approved, alter your setup and create data tables for your experiment
in your notebook.
Step 3: Collect the transpiration data using the potometer as in the Structured Inquiry. Record
the measurements in your data table.
Step 4: Use the information in the Structured Inquiry to find the total surface leaf area of your
plant sample and use it to calculate a final rate of transpiration for both trials. Graph your data
in your notebook.
Step 5: Critical Analysis: How did the typical conditions compare with the altered conditions in
terms of the rate of transpiration? Was the difference significant? Discuss your answers with
your partner and write them in your notebook.

Assessments
1. One cutting has more leaves than another. Given this information, can you predict how
the rate of transpiration would compare between the cuttings? [SP 3, Questions and
Methods; SP 6, Argumentation]
2. Explain whether your prediction in the Guided Inquiry was accurate. Include a reason
based on how the variable affected transpiration. [SP 1, Concept Explanation}
3. How might drought or flooding induced by climate change affect transpiration of plants,
and how, in turn, would this affect the ecosystem? Give an example. [SP 6,
Argumentation]

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[Answer Key]
1. No, without knowing the surface area of the leaves on each plant, the estimated rate
using only time and water changes is not reflective of how much transpiration would
actually take place for a given plant.
2. Student answers will vary but should include accurate descriptions of how transpiration
would be affected by the variable. For example, wind should cause an increase in
transpiration because it would increase evaporation from the leaf.
3. Climate change can cause drought in areas, which would lead to water stress in a plant
and cause transpiration to slow down, further reducing the amount of moisture in the
ecosystem. Climate change can also cause flooding, which would lead to water
saturation for plants, increasing transpiration significantly. Additionally, flooding may
drown the plant entirely, which it could not survive. Reduction of plants in an ecosystem
would have many detrimental effects, including loss of food sources and habitats for
many other species.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: What environmental conditions could
be applied to a plant that would cause it to open more of its stomata?
2. Answer the following question in your notebook: Do you think there is a condition that
would result in mostly closed stomata?
3. Discuss the answers to questions 1 and 2 with the class.

Activity 2: Comparison of Opening or Closing of Stomata under

Selected Conditions
Plants will open or close stomata based on many factors. Stomata help regulate the
water needs of the plant and exchange gases with the environment. If there is too much water
inside the cells, the stomata will open to release the pressure. If water pressure is low, the
stomata will remain closed to retain moisture. However, to keep water moving up from the
roots and into the leaves, some stomata do need to open to build the negative pressure that
allows roots to draw up water. Special cells, called guard cells, act like doors to open or close
stomata.

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Figure 19.2: Transpiration takes place through open stomata, (a) generally scattered across the underside of leaves.
(b) A single stoma. (c) A cross section of a stoma shows the two small guard cells above the large open air space
within the leaf tissue. The guard cells contain darkened areas.

Safety Precautions
● Avoid directly inhaling nail polish fumes.
● Handle the microscope carefully and always carry it with two hands.
For this activity, you will need the following:
● Scissors
● Clear nail polish
● Empty microscope slides
● Microscope
● Leaves
● Clear cellophane tape
● Forceps

For this activity, you will work in pairs.

Activity 2 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 2:
● It may be useful to include a brief review on the different parts of a plant that are
involved in plant transpiration. This can be given as part of the initial lecture before
Activity 1, or you can introduce this topic prior to beginning Activity 2.
● Since students will be using a microscope for this exercise, it is recommended to
describe to the students how the structure of stomata will look to assist them in their
observations. Additionally, remind students of the proper use and care of a microscope
(e.g., how to properly carry a microscope as well as how to clean and store it upon
completion of the experiment).

297
 ● Since scissors will be used for this exercise, remind students of the tenets of lab safety.
Also, you may want to consider having masks on hand to minimize the inhalation of the
nail polish.
● Since slides will be used, it will be beneficial to demonstrate for students how to prepare
slides of their samples. Additionally, have a sharps biohazard container on hand for
proper disposal of broken slides.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 2:
● Students could remain in groups of two while doing the Guided Inquiry, but you may
wish to have each student write their own hypotheses regarding the proposed outcomes
of the experiment. This is to ensure that each student contributes equally and submit
their own original content.
● You will want to check the students’ hypotheses and results from the initial experiment
during the Structured Inquiry before allowing them to proceed with performing their
new experiment, to ensure that they are on the right track and will not waste
unnecessary materials.
● Since the students will be placing the leaves under different environmental/test
conditions, multiple variables will need to be provided to assist them in this portion of
the Guided Inquiry. Therefore, be sure to have an ample supply of the additional
materials that they will need to conduct this portion of their exercises. Remind them
that they will also need controls to use for this experiment.
● Instruct students on how to set up their data table prior to conducting their experiment
so that it can be already prepared when they need to record their results.
● In Step 3, clarify to students how to analyze their results. Additionally, providing a
grading rubric for this activity with criteria describing each of the mandatory parts of this
activity can help guide students to include the proper components in their experiment.

Structured Inquiry
Step 1: Prepare slides of leaf samples at normal room conditions. To do this, you will cut a
sample of a leaf and coat both sides of the leaf with clear nail polish. Allow it to dry completely
by holding the sample and waving it gently in the air. Then, create wet mounts of the leaf
samples and observe them under the microscope.
Step 2: Hypothesize/Predict: What percentage of open to closed stomata do you expect to
find? Record your hypothesis in your notebook.

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Step 3: Student-Led Planning: Prepare a data table to count the stomata. Then, place the slide
under the microscope and count the number of stomata. Record your data in your table. If
there is time, consider making more than one sample. Draw sketches of the samples.
Step 4: Critical Analysis: Is it simple or complicated to count the stomata? Is it possible to count
the same ones more than once? How did you ensure this did not happen? What did the results
show when the leaves from the two different trials were compared?

Guided Inquiry
Step 1: Hypothesize/Predict: Choose a condition to which you will expose a leaf sample for 20
minutes. How do you think the altered condition will affect the stomata? Record your ideas in
your notebook.
Step 2: Student-Led Planning: Prepare a slide of the leaf sample by cutting a leaf and coating
both sides with clear nail polish. Allow the leaf sample to fully dry before placing it on the
microscope slide. Prepare a data table and count the number of closed and open stomata. Draw
sketches of the samples.
Step 3: Critical Analysis: Compare your results to the results from the Structured Inquiry. What
conclusion can you draw from the data? Write your answer in your notebook.

Assessments
1. In a certain habitat, the plants can keep their stomata open for most of the day. What
does this suggest about the characteristics of the habitat? [SP 1, Concept Explanation]
2. Could a researcher estimate how many plants in an enclosed area that have their
stomata open using the concentration of carbon dioxide in the air? Explain your answer.
[SP 3, Questions and Methods; SP 1, Concept Explanation]
3. How could a research scientist use the methods in this lab to determine how to care for
a given species of plant? [SP 3, Questions and Methods]
[Answer Key]
1. If plants can keep their stomata open for most of the day, this suggests that they live in a
very humid environment, such as a cloud forest, where the air isn’t dry enough to cause
the interior of the plant to desiccate.
2. Open stomata would take in more carbon dioxide from the environment than closed
stomata. Therefore, the researcher should find a lower concentration of carbon dioxide
in the air if most of the plants had their stomata open and a lower concentration if most
of the plants had their stomata closed.

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3. A scientist could observe the average number of stomata open or closed under different
environmental conditions to determine how much water the plant needs and the
temperatures at which it can keep its stomata open and perform gas exchange.

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 Lab 20: Biodiversity in Leaf Litter
This investigation addresses the Required Course Content and Science Practices from the AP
Biology CED:
REQUIRED COURSE CONTENT
Enduring
Topic Learning Objective Essential Knowledge
Understanding
ENE-3 Timing and
coordination of
ENE-3.D Explain how the
biological mechanisms ENE-3.D.1 Organisms
behavioral and/or
involved in growth respond to changes in
physiological response
8.1 Responses to the reproduction, and their environment
of an organism is related
Environment homeostasis depend through behavioral and
to changes in the
on organisms physiological mechanisms
internal or external
responding to (such as phototaxis).
environment.
environmental cues.

ENE-4.A.1 The structure

of a community is
measured and described
in terms of species
composition and species
ENE-4.A Describe the diversity, including the
structure of a application of Simpson’s
community according to Diversity index.
its species composition ENE-4.B.2 Interactions
ENE-4 Communities
and diversity. among populations
and ecosystems change
determine how they
on the basis of
ENE-4.B Explain how access energy and matter
interactions among
interactions within and within a community.
8.5 Community populations and
among populations ENE-4.B.4 Competition,
Ecology disruptions to the
influence community predation, and
environment.
structure. symbioses, including
parasitism, mutualism,
ENE-4.C Explain how and commensalism can
community structure drive population
relates to energy dynamics.
availability in the ENE-4.C.1 Cooperation or
environment. coordination between
organisms, populations,
and species can result in
enhanced movement or
access to matter and
energy.

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 SYI-3 Naturally
occurring diversity SYI-3.G Explain how the
SYI-3.G.1 The diversity of
among and between addition or removal of
species within an
components within any component of an
8.6 Biodiversity ecosystem may influence
biological systems ecosystem will affect its
the organization of the
affects interactions overall short-term and
ecosystem.
with the environment. long-term structure.

EVO-1 Evolution is SYI-2.B.1 The distribution

characterized by of local and global
SYI-2.B Describe human
change in the genetic ecosystems changes over
8.7 Disruptions to activities that lead to
make-up of a time.
Ecosystems changes in ecosystem
population over time SYI-2.B.2 Human impact
structure and/or
and is supported by accelerates change at
dynamics.
multiple lines of local and global levels.
evidence.

SCIENCE PRACTICES
Science Practice Skills
SP 1 Concept Explanation
1.C Explain biological concepts, processes, and/or
models in applied contexts.
Explain biological concepts, processes, and models
presented in written format.
SP 2 Visual Representations
2.D Represent relationships within biological models
including mathematical models, diagrams, and flow
Analyze visual representation of biological concepts
charts.
and processes.
3.A Identify or post a testable question based on an
observation, data, or model.
SP 3 Questions and Methods
3.D Make observations, or collect data from
representations of laboratory setups or results.
Determine scientific questions and methods.
3.E Propose a new/next investigation based on
evaluation of a previous experiment.
SP 4 Representing and Describing Data
4.A Construct a graph, plot, or chart.
Represent and describe data.
SP 5 Statistical Tests and Data Analysis
5.A Perform mathematical calculations, including
mathematical equations in the curriculum (such as
Perform statistical tests and mathematical
the Simpson Diversity index).
calculations to analyze and interpret data.
6.A Make a scientific claim.
SP 6 Argumentation
6.B Support a claim with evidence from biological
principles, concepts, processes, and/or data.
Develop and justify scientific arguments using
6.E Predict the causes or effects of a change in, or
evidence.
disruption to, one or more components in a
biological system based on data.

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 According to recent records, Earth is inhabited by an estimated 8.7 million different
species of organisms. All of these organisms live within a region of Earth called the biosphere.
The biosphere is defined as the regions of Earth’s surface, atmosphere, and hydrosphere that
can sustain living organisms. A variety of ecosystems make up the biosphere. Ecosystems are
composed of abiotic and biotic components that influence the biodiversity of the ecosystem.
Biodiversity commonly refers to the variety and variability of life in a given ecosystem or on
Earth as a whole.
Biodiversity is typically measured by species richness and species evenness. Species
richness represents the number of different species in an ecosystem. Species evenness
represents how close in number each species is in an ecosystem. The diversity index is
calculated from species richness and species evenness.
In effect, the diversity index describes the biodiversity of an ecosystem. In simple terms,
an ecosystem, such as the Antarctic ice sheet that is dominated by one or two species, is less
diverse than an ecosystem, such as a tropical rain forest in which several different species have
a similar abundance. However, two ecosystems with identical types of species may not have the
same biodiversity. This is possible if one environment has a higher proportion of Species A and a
lower proportion of Species B compared to another environment. Another complication of
measuring biodiversity is that the biodiversity of a particular environment can vary over time,
depending on seasonal changes in weather and other factors.
Leaf litter brings life to the uppermost layer of soil known as topsoil. Leaf litter
contributes to the soil’s humus component and provides energy, moisture, nutrients, and
shelter to animals, bacteria, fungi, and protists. As leaves and other dying plant parts fall to the
ground, they undergo the process of decomposition. Decomposition involves the recycling of
energy and nutrients present in the cells and tissues of all organisms. During this process, dead
cells and tissues are broken down by both the environment and organisms into molecules.
Organisms that break down leaf litter and other dead matter are called decomposers or
detritivores.
The amount and composition of leaf litter vary greatly between different types of
ecosystems. For example, the leaf litter found in a wet area of a forest would likely contain more
ash and cypress leaves than a dry area. In contrast, a drier section of the forest may have more
oaks and linden leaves. Differences can even arise within an ecosystem, depending on the time
of year and local variations in the terrain.
In this activity, you will be collecting samples of leaf litter from different locations. Your
teacher will instruct you on where to collect the leaf litter samples. Based on what you learned
about leaf litter variation, it is important to take notes on the area from which you collect the
leaf litter. In addition, describe the climate of each area as well as the environmental conditions
at the time you collect the leaf litter. You will then be asked to construct a Berlese funnel to
isolate any organisms living in the leaf litter.

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 The Berlese funnel was designed specifically for trapping and separating insects from
ground litter. It was developed by Italian entomologist Antonio Berlese in 1927 and works by
taking advantage of the negative phototactic responses of leaf litter organisms. A diagram of a
Berlese funnel is shown in Figure 21.1. It consists of a funnel containing a mesh and a lamp
above the funnel. Leaf litter is placed in the Berlese funnel on top of the mesh screen. A
container filled with a preservative (or water) is placed under the lower opening of the funnel.
When the lamp is turned on, the heat and light produced by the lamp dry out and warm up the
leaf litter. This in turn produces an unfavorable environment for the leaf litter organisms and
drives them to the bottom of the funnel. The organisms then fall through the mesh into the
container where they can be collected.

Figure 21.1: Design of a Berlese funnel.

In this activity, you will build a Berlese funnel to remove organisms from your leaf litter samples.
Then, you will use your data to mathematically calculate the biodiversity of the leaf litter using a
diversity index.

Instructor Introduction Notes

The following are recommendations for introducing this lab’s background content to your
students:
● If your students have not yet had an ecology introduction, it may be helpful to show a
diagram and explain the different levels of ecological organization (organism, population,
community, ecosystem, biome, and biosphere).

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 ● Species richness and evenness can be introduced using a hypothetical data set. For
example, a table containing four species can be set up, with the species listed as the row
headers. Then, two columns could be set up, labelled Area 1 and Area 2 (or similar). Fill
in the population sizes of the different species so that the numbers are more similar
(greater species evenness) than the second area. You can then state that the two areas
have similar species richness (because they have the same number of species) but differ
in species evenness. If you drop one species to zero population in one of the areas, then
the two areas will have different species richness.
● A diagram of a typical soil surface community may be shown to help students visualize
the habitat they are about to study, as well as soil cores from different types of soil to
show how the soil layers differ among different ecosystems. Point out the layers
students are sampling (leaf litter plus part of the uppermost layer of soil).
● A drawn diagram, on the board, of how the Berlese funnel works is beneficial. You can
explain the different parts of the funnel, and students could draw and label the diagram
in their own notes as you explain each component.

In this lab you will learn

● how to collect and separate organisms from leaf litter
● how to use images or a key to identify leaf litter and soil organisms
● how to use a mathematical formula to calculate the diversity index as a means of
explaining the biodiversity of leaf litter environments

Activity 1: Pre-Assessment
1. Answer the following question in your notebook: Explain why organisms living in leaf
litter display negative phototaxis.
2. Answer the following question in your notebook. Leaf litter typically contains organisms
that live in the upper layers of the topsoil. Explain the environmental factors (both of the
physical environment and of other species) that would determine the number and types
of organisms living in the leaf litter.
3. Be prepared to discuss the answers to questions 1 and 2 with the class.

Activity 1: Collect Leaf Litter and Set-up Berlese Funnel Traps

Safety Precautions
● Use caution when collecting leaf litter and other environmental samples. Watch out for
sharp objects, harmful plants, and dangerous animals that may sting or bite. Wash your
hands as soon as possible after collecting the samples.
● Do not touch the light bulbs on the Berlese funnel apparatus, as they are on for long
periods during this lab and will likely become hot enough to cause burns.
● Do not ingest the alcohol in the collection beaker, as it is not edible and will cause
extreme sickness if ingested.

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 ● Wear safety goggles throughout the laboratory activity.
● It is highly recommended that you wear rubber gloves and a lab apron when handling
any chemicals.
● Immediately inform your teachers of spills.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.
● Clean up any spilled water or other fluids to prevent slippage.
● Immediately wash your hands if they directly come in contact with any chemicals. It is
important to wash your hands after the laboratory activity to avoid any possible
contamination of other surfaces with chemical residues that might be on your hands.
For this activity, you will need the following:
● Plastic sandwich bags
● Notebook or index cards
● Pencil
● Ruler (cm)
● Waterproof marker
● Thermometer
● The Leaf Litter Sample Information Form
● Two-liter plastic soda bottle
● One-quart jar or 500-mL beaker
● Mesh screen
● Moveable neck lamp with a 60- or 100-watt incandescent bulb
● Scissors
● Tape
● Ethanol (70–95 percent) or isopropanol (70 percent)

For this activity, you will work in pairs or groups.

Activity 1 Instructor Preparation and Teaching Tips

The following are recommendations for the Structured Inquiry for Activity 1:
● Remind students days in advance that they are going outside for this lab and should
wear appropriate clothing as well as shoes that they don’t mind getting muddy.
● Students working in pairs may require a bucket or other carrying container for their
equipment. If taking additional environmental data (see next bullet), then groups of at
least three students may be best, given the greater amount of time required to take
additional environmental data.

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 ● The environmental data that students take at their sites can be expanded to include (1)
soil moisture using a simple soil moisture meter sold at garden supply stores, (2) relative
humidity taken with sling psychrometer (much teach how to calculate humidity from this
as well), as well as soil nitrogen and phosphorus levels using simple test kits available at
garden stores, and (3) precipitation levels using a rain gauge that students set up during
a previous lab.
● Select naturally forested sites for student sampling, if possible. Disturbed soil or those
near development or walkways may have a depressed leaf litter community.
● In constructing the Berlese funnel, use a mesh that has no greater than 1-cm2 openings.
This will prevent excessive debris from falling into the collection beaker. Water can be
substituted for alcohol in the beaker; however, some organisms may be able to escape
from the surface of the water if left for too long. Avoid filling the container more than
halfway to the top if using water.

Structured Inquiry
Part 1: Collecting the Leaf Litter Samples:
Note—You may be instructed to collect two litter samples for this activity. It is very important to
pay attention to the environmental conditions of each area where you collect a sample. You will
be recording this information in your notebook. It is also important to get a complete
representation of the leaf litter. The sample bags should include the full depth of leaf litter.
Step 1: Hypothesize/Predict: Describe the environmental conditions for the leaf sites that you
sampled. Explain how the differences in the two sites may influence the number of species in
each sample. How would the biodiversity of predators in the leaf litter community affect their
prey and vice versa? Write your ideas in your notebook.
Step 2: Obtain the plastic bags, index cards or notebook, pencil, ruler, waterproof markers, and
thermometer from your teacher.
Step 3: Go to one leaf litter sample area selected by your teacher.
Step 4: Use the ruler to mark off a 15-cm by 15-cm square of ground to sample.
Step 5: Student-Led Planning: Discuss with your partner how you will record the following
information in your notebook or on the Leaf Litter Sample Information Form (Figure 21.1).
● Date and time of day
● Location
● Type of ecosystem
● Temperature
● Sky conditions
● Atmospheric conditions
● Ground conditions
Also, make a quick drawing or take a photograph of the area that you will be sampling.

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 Table 21.1: Leaf Litter Sample Information Form
Your Name (s): Sample #
Date: Time: Location:
Circle Type of Ecosystem: (Forest) (Grassy field) (Swamp) (River bank) (Beach) (Desert)
(Other)*
Temperature Sky: Check one: (Full sun) (Partly cloudy) (Overcast) (Night sky)
Circle Atmospheric Conditions: (No rain) (Drizzly) (Slight rain) (Moderate rain) (Heavy rain)
(Snow) (No wind) (Slight breeze) (Moderate wind) (Heavy wind) (Dry) (Slightly humid) (Humid)
Ground conditions: (Full shade) (Partial shade) (No shade)

*Explain other:

Step 6: Collect leaf litter from the 15-cm by 15-cm area. Remember to include the uppermost
layer of the soil. Place the sample in the plastic bag. Use the waterproof marker to write your
name or names on the bag and provide a sample number. For example, if collecting multiple
samples, label the first bag Site 1 and the second bag Site 2.
Step 7: Critical analysis: Explain why it is important to record the date and time of day for your
sample. Describe how temperature, atmospheric conditions, and ground conditions could affect
the organisms found in your sample. Record your answers in your lab book.

Constructing the Berlese funnel:

Step 8: Use the scissors to cut off the bottom of a 2-liter plastic soda bottle.
Step 9: Cut and bend the mesh screen so that it fits snugly inside the upside down two-liter
plastic soda bottle. Make sure the mesh screen is completely in contact with the plastic walls of
the bottle. This will prevent leaves from going past the mesh screen.
Step 10: Pour approximately 50 mL of the ethanol or isopropanol solution into the 1-quart jar or
500-mL beaker. This is your collecting container.
Step 11: Place the upside down two-liter plastic soda bottle into the mouth of a one-quart jar or
500-mL beaker. It should look like the Berlese funnel image in Figure 21.1. Use the tape and the
ruler to secure the two-liter plastic soda bottle and hold it in place.
Step 12: Carefully carry your Berlese funnel to a location designated by your teacher. It may
need readjustment to make sure that it is secure. Place the leaf litter from one of the samples
into the 2-liter plastic soda bottle.

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Step 13: Place the moveable neck lamp near your Berlese funnel. Adjust the light so that it
directly shines on the leaf litter sample in the funnel. The light should be about 20 cm above the
top of the Berlese funnel. It will take about two days of incubation for a majority of leaf litter
organisms to enter the collecting container.
Step 14: Clean up:
● Clean and return any of the supplies that you do not need.
● Pour any excess ethanol or propanol down a sink unless your teacher instructs
otherwise. Run water into the sink until the smell of the ethanol or propanol is
not evident.
Step 15: Critical Analysis: Use the Internet to research the types of organisms that live in leaf
litter. Discuss with your partner what you would expect to find in the leaf litter sample you
collected. Why might your sample be lacking in some of the leaf litter organisms you researched
on the Internet? Discuss with your partner why your sample may differ from samples collected
by other students. How could the size of the openings in the mesh screen affect the results of
an analysis using a Berlese funnel? Write your answers in your lab notebook.

Assessments
1. A student read that oak leaves release harmful substances upon decaying. The student
wants to determine whether these substances from the oak leaves reduce the
biodiversity of leaf litter organisms in a forest. How would you design an experiment to
test this hypothesis? [SP 3, Questions and Methods]
2. Based on the experimental protocol you followed using the Berlese funnel, what
environmental conditions are likely preferred by leaf litter organisms? How do you
know? [SP 4, Representing and Describing Data]
3. What adaptation of leaf litter organisms is exploited by the Berlese funnel? [SP 1,
Concept Explanation]
4. Your teacher asks you to investigate whether heat or light is the major factor in
separating leaf litter organisms in a Berlese funnel. Explain how you would design an
experiment to test this. [SP 3, Questions and Methods]
5. Pretend you are working as a scientist in a botanical garden. The director asks you to test
the effects of fertilizer on the biodiversity of leaf litter organisms in the botanical garden.
Explain how you would design an experiment to test this. Write your answers in your
notebook. [SP 3, Questions and Methods]
[Answer Key]
1. The most appropriate design would include a long-term field experiment with two
sample plots in an area that has a variety of trees, including oak. One plot would be the
control, an area that is undisturbed. In the other plot, the student would regularly
remove the oak leaves. The student would then perform the analysis using a Berlese
funnel. The student could also design an experiment in a terrarium containing leaf litter
organisms. In this setup, oak leaves and other types of leaves can be tested separately.

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 2. The Berlese funnel dries and heats the leaves, which repels the leaf litter organisms. This
suggests that the leaf litter organisms are adapted to cooler and more moist conditions.
3. The leaf litter organisms have evolved behavioral mechanisms to avoid unfavorable
conditions. They move away from areas that are hot and dry, preferring to stay cool and
moist. This behavior allows the Berlese funnel to trap leaf litter organisms.
4. The student would have to set up two different Berlese funnels. One would use a cool
light source, and another would have a non-illuminating heat source above the leaf
litter.
5. The student would first set up several plots having the same environmental conditions in
a particular region of the botanical garden. The student would then apply different
amounts of fertilizer to the plots. One plot must be kept as a control with no fertilizer.
Then the student would use a Berlese funnel setup to determine whether biodiversity
differences exist.

Activity 2: Pre-Assessment
1. Answer the following question in your notebook: Explain how the temperature and
rainfall of an ecosystem could affect the biodiversity of leaf litter.
2. Answer the following question in your notebook: Describe how the type of vegetation
in an area can affect the biodiversity of leaf litter organisms.
3. Be prepared to discuss the answers to questions 1 and 2 with the class.

Activity 2: Estimate of the Index of Biodiversity and Independent

Project
As in any environment, the biodiversity of the leaf litter habitat is affected by
interactions between each organism and between the organisms and the physical features of
the environment. In this activity, you will compare leaf litter from different areas to determine
whether location affects the biodiversity of organisms living in the leaf litter. You will collect the
organisms separated by the Berlese funnel and calculate the overall biodiversity of your sample.
Biodiversity will be measured using a mathematical tool called the Simpson Diversity Index. It is
one of many ways of representing biodiversity. Calculating the Simpson Diversity Index requires
you to keep track of the number of different groups of organisms collected and the abundance
of each. The Simpson Diversity Index provides a number scaled from zero to one, with zero
being the lowest biodiversity and one being the highest. In this activity, you will characterize
and count the leaf litter organisms in order to calculate the Simpson Diversity Index.
Safety Precautions
● Wear safety goggles throughout the laboratory activity.
● It is highly recommended that you wear rubber gloves and a lab apron when handling
any chemicals.
● Immediately inform your teachers of spills.
● Inform your teacher immediately of any broken glassware, as it could cause injuries.

310
 ● Clean up any spilled water or other fluids to prevent slippage.
● Immediately wash your hands if they directly come in contact with any chemicals. It is
important to wash your hands after the laboratory activity to avoid any possible
contamination of other surfaces with chemical residues that might be on your hands.
For this activity, you will need the following:
● Berlese funnel with leaf litter sample from Activity 1
● Several Petri dishes per group
● Dissecting microscope or hand lenses
● Forceps or tweezers
● Dissecting pins
● Pipette or eyedropper
● Small jar with a screw top
● Blank piece of paper
● Ethanol (70–95 percent) or isopropanol (70 percent)

Supplemental Information
● Soil or leaf litter organism keys, charts, and information:
[Link]
[Link]
[Link]
[Link]
[Link]
● Internet access to Simpson's Diversity Index website (optional):
[Link]
● Internet access to Biodiversity Calculator:
[Link]
For this activity, you will work alone, in pairs or in groups.

Activity 2 Instructor Preparation and Teaching Notes

The following are recommendations for the Structured Inquiry for Activity 2:
● It will likely be difficult for students to identify many of the leaf litter organisms down to
the species level, and they will need help with the characteristics in question. Review
your chosen key(s) carefully and investigate some organisms under the microscope
before lab so you can help students with this step.
● To clarify relative abundance to students, perhaps using the number of girls versus boys
in your class as a quick example.

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 ● Emphasize that a Simpson’s Diversity Index value close to zero means very low
biodiversity, while a value close to one is a very high biodiversity.
The following are recommendations for the Structured Inquiry for the Guided Inquiry for
Activity 2:
● Students could remain in groups while doing the Guided Inquiry, but you may wish to
have each student write their own paper or have each student graded individually on
their contributions during a presentation. This is to ensure that each student contributes
equally to a group project.
● You will want to check each student’s hypothesis before he or she proceeds with
research to ensure that it is testable, given the available equipment and time. The
student will also likely need advice on site selection.
● Students often will not include all vital components of a research paper or presentation
due to their inexperience. Providing a grading rubric for papers or presentations can
guide students to include the proper analyses and critical thinking in their
papers/presentation.
● In Step 6, clarify to students the difference between error analysis (conditions in the
experiment they just performed that could have skewed the results) compared to
creating new questions. Students will often come up with new research questions that
simply involve re-running their experiment with the errors corrected. Tell students that
they should come up with a completely new hypothesis based on the results of their first
study.

Structured Inquiry
Now that you have collected your leaf litter organisms, it is time to identify the organisms in
your sample. This involves categorizing the organisms into groups at the species or genus level.
It is important to record your information in a data table to help organize the data for
biodiversity calculations. Additionally, you should keep your sampled organisms in a container in
case you need to confirm your data later. It also helps to have your sample in case you made an
error identifying an organism. You can calculate the Simpson Diversity Index by either using the
mathematical formula or entering the data in an online diversity index calculator.
Step 1: Draw a data table in your lab notebook like that shown in Table 21.2. Make enough rows
to account for up to 20 different organisms that you may collect from the leaf litter.

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Table 21.2
Species Absolute Abundance (n) Relative Abundance

Species 1
Species 2
Species 3
Species 4
Total Total number of organisms collected Should total roughly 1.00
number of (N) =
different
species =

Step 2: Move your incubated Berlese funnel to your workstation.

Step 3: Carefully disassemble the unit so that you can remove samples from the collection
container.
Step 4: Add about 20 mL of ethanol (70–95 percent) or isopropanol (70 percent) to the screw
top container.
Step 5: Use forceps or a pipette to place small samples of organisms from the collecting
container into a Petri dish.
Step 6: Place the Petri dish under the dissecting microscope.
Step 7: Use the suggested online identification charts or keys to categorize the organisms in the
leaf litter sample. You should do your best to identify the organisms down to the species or
genus levels.
Step 8: Record the information in your data table. Carry out the calculations on your data as
shown in Table 21.3.

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Table 21.3
Species Absolute Abundance (n) Relative Abundance
Species 1 36 36/87 = 0.413
Species 2 27 27/87 = 0.310
Species 3 10 10/87 = 0.115
Species 4 14 14/87 = 0.162
Total number of different Total number of organisms Should total roughly 1.00
species = 4 collected (N) = 87

Step 9: Pour the contents of the Petri dish into the screw top jar and repeat. Continue until you
have collected all the organisms from your collecting container.
Step 10: Clean up:
● Dispose of the used leaf litter in a garbage basket.
● Clean the disassembled Berlese funnel and return any reusable components.
● Clean and return any other supplies.
● Pour any leftover alcohol solutions in a sink and flush the sink with water until the
alcohol smell is not evident.
● Store your screw top container in a designated area as instructed by your instructor.
When directed by your instructor, the organisms should be poured into a sink and
washed down the drain.
Step 11: Using the information in your data table, calculate the Simpson’s Diversity Index value
for your sample. This can be done in one of the following ways:
● Mathematically: Read the information on the Simpson’s Diversity Index website at
[Link] Then use the following formula to
determine the Simpson’s Diversity Index value (n = total number of organisms of a
particular species, N= the total number of organisms of all species).

Simpson’s Diversity Index = 1 – [Sum of n (n–1) / N(N–1)]. For example, using Table 21.2,
the calculation would be:
1. Simpson’s Diversity Index = 1 – [(36 × 35) + (27 × 26) + (10 × 9) + (14 × 13) / (87 ×
86)]
2. Simpson’s Diversity Index = 1 – (2,234 / 7,482)
3. Simpson’s Diversity Index = 1 – 0.30
4. Simpson’s Diversity Index = 0.70

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 ● Online Simpson’s Diversity Index Calculator:
1. Go to the online calculator at [Link]
2. Use the New Input area to enter the number of categories of organisms in the
designated box. For the sample in Table 21.2, you would enter 4 and press the
Submit button for the New Input area.
3. In the data input chart, enter the name of the category (species or genus) and
the number of organisms in the category (n). For our example, we would enter
the information as shown in Table 21.4.
Table 21.4
No. Category Value
0 Species 1 36
1 Species 2 27
2 Species 3 10
3 Species 4 14

4. When done entering your data, press Submit in the Submit Values area.
5. Subtract the Simpson Dominance (R1) value from 1 to calculate the Simpson’s
Diversity Index. For our example, the Simpson Diversity Index is 0.3017. So, the
Simpson’s Diversity Index = 1 – 0.30 which is 0.70.

A Simpson’s Diversity Index value close to zero means very low biodiversity, while a value
close to one is a very high biodiversity.
Step 12: Record the Simpson’s Diversity Index value for your sample (or samples, if you analyzed
multiple samples).
Step 13: Critical Analysis: Compare the biodiversity of your sample to the biodiversity observed
in samples collected by other students in class. Use the information from each sample’s Leaf
Litter Sample Information Form to explain any similarities or differences between your sample
and the other samples. Discuss with your partner how differences in the environment may
explain the variations in the biodiversity data from different samples collected by other
students.

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Guided Inquiry
Step 1: With your teacher’s direction, you will now perform an independent research project
comparing the biodiversity of leaf litter organisms among different leaf litter sites using the
Berlese funnel and Simpson’s Diversity Index. Based on the results of your Structured Inquiry,
was any particular organism predominant in the leaf litter? What could be a reason for one
particular organism dominating the leaf litter community? Using the Internet, search for
resources describing the diets of leaf litter organisms. Compared to the population of organisms
that feed on decaying leaves, would you expect the predator population to be larger or smaller?
Also, research how the physical conditions of the leaf litter, such as temperature and moisture,
may affect leaf litter biodiversity. Take notes in your notebook and print off your resources as
needed.
Step 2: Hypothesize/Predict: Based on your research, think of a question you wish to explore
about the leaf litter community, such as how does the diversity of leaf litter species change in
sites that have different moisture levels. Write your question in your notebook. Then create a
clear and specific hypothesis that you will test for your project. Present both your question and
your hypothesis to your teacher for approval.
Step 3: Student-Led Planning: Discuss with other students the best way to test your hypothesis.
Try to think of ways to control for external variables, especially when determining where and
how you will collect your leaf litter. Your teacher may have you write and submit an
experimental plan for review.
Step 4: Collect leaf litter according to your experimental plan. Then, run your leaf litter through
one or more Berlese funnel setups as described by your experimental plan. Set up your funnel
as described in Activity 1. Remember to set up each funnel in the same way for each leaf litter
sample and run each for the same amount of time. Then, determine the diversity of leaf litter
organisms as described above. Calculate the Simpson’s Diversity Index for your samples.
Step 5: Combine and analyze your data as necessary, based on your hypothesis. You may be
presenting your data to the class or writing it up as a lab report as instructed by your teacher.
Create graphs, if possible, or use other methods to neatly and efficiently present your results.
Step 6: Critical Analysis: Was your hypothesis supported or unsupported by your results? What
do your results indicate about the diversity of leaf litter organisms under different
environmental conditions? How could you improve your experiment and what question could
you investigate next? Include answers to these questions in your presentation or lab report, as
directed by your teacher.

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Assessments
1. Describe an experiment to determine whether evolution is occurring in the leaf litter for
an environment in which the type of vegetation dominating the area is changing. [SP 3,
Questions and Methods]
2. Describe an experiment to determine how the nutrient levels of leaves affect the
biodiversity of leaf litter. [SP 3, Questions and Methods]
3. Describe an experiment to determine the effect of moisture levels on the types of
insects and spiders in leaf litter. [SP 3, Questions and Methods]
4. Explain why the leaf litter you analyzed produced the Simpson’s Diversity Index value
that it did. [SP 1, Concept Explanation; SP 5, Statistical Tests and Data Analysis]
[Answer Key]
1. An appropriate design would include a long-term biodiversity study to determine
whether one or more organisms are changing in some type of characteristic that
correlates to environmental change. The phenotypic change would then require further
study.
2. An appropriate design would include the collection of many leaf litter samples in the
same region, and measurement of the leaves for their nutrient contents. A biodiversity
study could then be performed to determine whether variations in biodiversity correlate
to leaf litter nutrient levels, as measured by the amount of carbohydrates, lipids, or
proteins in the leaves.
3. An appropriate design would include a series of studies on the populations of insects
and spiders in leaf packets having different moisture levels. The relative dominance of
the insects and spiders could then be measured to determine whether the populations
respond to the different moisture levels.
4. Answers will vary. Environmental factors can affect one or more types of organisms in
the leaf litter, based on the organisms’ adaptations to a particular environmental
condition. For example, a lack of moisture might decrease the earthworm population
but not the spider population. Earthworms are much more sensitive to drying than
spiders. Also, the decrease in one species may affect the populations of organisms that
interact with it.

Extension Activities:
1. Design a study to show how pesticide applications could affect the arthropod
biodiversity of soils. Explain why it is important to demonstrate this using a controlled
experiment and explain the accurate procedures for sampling soil arthropods.
2. Design a study to show how the biodiversity of plants in a vacant lot varies over a
five-year period. Use that information to explain how local animal biodiversity may be
affected by changes in the vacant lot’s plant biodiversity.

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