Biology 1202

Introduction to Cell Biology

Laboratory manual
Spring 2024

Mount Royal University

Department of Biology

Written by Alexandria Farmer & Todd Nickle,

edited by Margot Williams, Jeffery Sheedy, Tatiana Rogasevskaia
In memoriam of our dear friend, Todd Nickle

“The obsession with grades really gets in the way….. I wish we could create
an environment where students could relax, and just learn…”
Don’t let grades get in the way. https://m.youtube.com/watch?v=CtPsOX6PA3M

Todd completed bachelor’s and master’s degrees at the University of Calgary, and earned his PhD
in Botany at Oklahoma State University. One year later (in 1999), he joined Mount Royal as a
faculty member. Todd noted, “I didn’t start out wanting to teach, but as I pursued my PhD and
master’s — there is working with students throughout that — I found that’s where I lit up. That’s
where I had opportunities to be creative and make analogies and be challenged and talk back and
forth. I kind of fell into it.” Of course, it was also something he excelled at. Todd was an
experienced and enthusiastic teacher, and he set aside remarkable amounts of time for his
students (office hours? “By appointment or just stop by!”). He brought science to life in inventive,
creative ways; he relished challenging his students and engaging them in authentic activities to
connect their studies to real life. For Todd, student learning was a journey of transformation.

In 2012, Todd was among the first group of faculty members to be promoted to full Professor, and
in 2015 he received the Distinguished Faculty Award, after being nominated by both students and
colleagues.

Everything about Todd's career reflected his passion for undergraduate teaching. He gathered a
group of biology educators from around the province and shepherded an official society: the
Alberta Introductory Biology Association (AIBA), which was later renamed the Undergraduate
Biology Educators of Alberta (UBEA). Todd was the inaugural president of AIBA, organized the
2014 AIBA conference (held at Mount Royal University), and continued to be involved with UBEA
through 2021. He was a textbook author, an early adopter of educational technology, and was
constantly innovating and experimenting with teaching techniques and assessment strategies.

More than all this, Todd was part of the Mount Royal family; he was a colleague and a friend.

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 Table of Contents
Page #
A little bit about Todd Nickle 1
Table of Contents 2
Preface 3-11

Lab 1 Microscopy I 12 – 26
Lab 2 Macromolecules 27 – 36
Lab 3 Instrumentation – Solution prep & Spectrophotometry 37 - 47
Lab 4 Microscopy II 48 – 65
Lab 5 Enzymes 66- 74
Lab 6 GI – studying how emulsifiers affect cellular membranes 75 - 80
Lab 7 GI II – performing the GI (YOU write this!) 81
Lab 8 Cell Respiration 82 – 87
Lab 9 Photosynthesis 88 - 96
Additional Literature cited 97

Appendices Contents list (Appendices are located on D2L lab site)

Appendix A: Using the Scientific Method in Laboratory Experimentation page 2
Appendix B: Good Laboratory Practice (GLP) page 4
Appendix C: Proofreading Tips page 7
Appendix D: Care and Use of Microscopes page 8
Appendix E: Determining Size and Scale page 11
Appendix F: Creating Solutions and Preparing Serial dilutions page 14
Appendix G: Using Micropipettes page 16
Appendix H: Using a Spectrophotometer page 17
Appendix I: Using a pH Meter page 18
Appendix J: Weighing substances on a desktop balance page 19
Appendix K: Questions to Ask while Peer Editing page 20

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 PREFACE TO LABORATORY

Required Materials for lab each week

Labs in binder with note paper or available on tablet/laptop
completed pre-lab questions (located at the end of each lab)
LAB ATTIRE: lab coat, close-toed shoes and full length pants (NO EXPOSED SKIN)
Following AHS and MRU current policies, a mask is required in all indoor spaces of MRU campus.

**at the start of each lab you will have completed prelab sheets prior to coming to lab to assure
familiarity with that day’s activities, and to be permitted access to lab. You will not be permitted to
access the lab if you fail to complete assignments or show up without proper attire.

Introduction to the Biology 1202 Laboratory

1 - Objectives of Laboratory Study

The aim of the Biology 1202 laboratory is to integrate knowledge presented in lectures with
practical aspects of biology. Concepts introduced in lectures will be reinforced and extended in
the laboratory to develop a more complete picture of biological principles. The objectives of the
Biology 1202 laboratory are:
1) to learn techniques in biological methods
2) to gain practical experience using laboratory equipment
3) to understand the principles of the scientific method and be able to use the scientific method
when doing experiments and writing laboratory reports.

2 - Approach to the laboratory

Although one can learn a great deal by simply reading, nothing beats direct experience for
helping us to understand and remember. In the study of biology, “direct experience”, means
hands-on work with living organisms and materials derived from them, as well as with
equipment and procedures used by biologists. Besides being interesting and enjoyable, such
work helps you gain insight into and experience in how biological questions are formulated, how
attempts to answer them are conducted, how data are collected and interpreted, and an
appreciation of the strengths and weaknesses of various scientific methods.
The student’s attitude to laboratory study is an important factor in determining the outcome of
the laboratory experience. The student must be willing to put in both time and effort to ensure a
pleasant and rewarding experience.
In Biology 1202 laboratories, a variety of organisms, materials and techniques will be used.
Some of these may be used for the first time by the student, so it is essential to be familiar with
appropriate terminology and techniques. This can best be accomplished by reading the laboratory
chapter and relevant textbook material

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*PRIOR* to the laboratory itself. It has been proven that prepared students learn and accomplish
a great deal more. In addition, “the real world” of science requires advanced preparation from
those in its employ. Charts, tables, and references are required of all scientific persons prior to
using valuable laboratory time. Reagents are often expensive and sometimes lab equipment is
available only during a narrow window of time.

3 - Student responsibilities
Students must:
i) attend every scheduled lab.
ii) read the lab manual prior to attending the lab.
iii) prepare your laboratory notebook with spaces for figures or data tables (as needed), and/or
notes PRIOR to each lab when required to do so.
iv) attend their regular section (unless explicitly making other arrangements which are agreed
upon IN ADVANCE by BOTH the normal and “new” instructors).
v) write lab quizzes and exams in their regular lab sections, and hand in assignments during the
lab section in which they are due.
vi) hand the lab reports in promptly on the day it is due. There is a 10% / day penalty for late lab
reports. For example, if you are registered lab is Monday 8:00 to 10:50, you must hand in the
report by 0830. Reports handed in at 8:31 on Monday will be penalized 10%. Reports handed in
after 8:30 on Tuesday will be penalized another 10%, after 8:30 on Wednesday another 10%,
etc..
vii) avoid plagiarism. Plagiarism is the submission of someone else’s work as if it were your
own. Lab manuscripts must be submitted electronically to “Turnitin” software located in
your D2L site.
So that all students are assured of a fair grade based on their own skills. In this course, it is
never acceptable to quote other sources directly: students are expected to practice
paraphrasing information. Even if you believe the original author stated the concept more clearly
than you could on your own efforts, you must ensure that you provide your own words in your
manuscript.
NOTE: Workload, computer, and computer-related problems will not be accepted as
reasons for handing in work late. See the Mount Royal Calendar for acceptable procedures for
getting work deferred or submitted late.

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The laboratory final exam will:
i) Include theory, procedures, materials, techniques, interpretation of results, etc. from all labs
covered (i.e. is cumulative).
ii) be closed-book. Notes you make during the regular laboratory sessions in your lab notebook
cannot be used in the exam. Your notes are strictly for study purposes.
iii) be scheduled and written during the students’ registered lab time (refer to syllabus for
responsibilities for missed exams).

4 General Instructions and Laboratory Procedures

At the beginning of the laboratory, the lab instructor will demonstrate techniques, indicate the
location of supplies, and give helpful hints. These introductory comments are meant as an
orientation to the laboratory and a quick review of salient features and concepts to be learned
during each laboratory period.
Proceed through laboratory exercises in sequence. Record observations and results whether working
alone or in a group. Answer all questions within each exercise and be sure to include any required
diagrams. This information may be essential for writing lab reports and preparing for examinations.
Coats, bags, briefcases, etc. are to be left in the designated area. To ensure an uncluttered work space,
take only the essentials to your laboratory bench. Do not work over your lab book; spills, particularly
of microorganisms or noxious chemicals may necessitate disposal of all pages that are contacted.
Laboratories start promptly on the hour and will proceed for 2 hours, 50 minutes. Before leaving the
laboratory, you must clean/disinfect and reset your area. Have the laboratory instructor sign your
laboratory results when required.
Attendance is taken in the lab and there will be no make-up laboratories for labs missed for
inexcusable reasons (slept in, work, etc.). With sufficient notification to both the regular and
receiving instructor, an occasional laboratory may be made up during another session offered on the
same week (case by case basis to be discussed with your lab instructor).

Laboratory Safety
1. All personal must receive training on the potential hazards associated with the work involved and
the necessary precautions to prevent exposure to infectious agents and release of contained material.
Personnel must show that they understand the training and documentation of the training is required.
2. Immediately notify the laboratory support centre and occupational health and safety officer in case
of spills, accident, exposure, injury or illness associated with laboratory activities or laboratory
preparation. Written records must be maintained.
3. Be aware that access to the laboratory is limited or restricted to authorized personnel when
experiments or work with cultures or specimens is in progress.
4. Doors to the laboratories must not be left open.
5. Open wounds, cuts, scratches and grazes should be covered with waterproof dressings.
6. Laboratories are kept clean and tidy.

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7. Wash hands after handling viable materials, after removing gloves and before leaving the
laboratory.
8. Do not eat, drink, chew gum, smoke, store food or personal belongings, apply cosmetics, handle
contact lenses or engage in horseplay in the laboratory.
9. Tie back long hair so it cannot come into contact with hands, specimens, containers or equipment.
10. Wear lab coats while in the lab including visitors, trainees, and others entering or working in the
laboratory. Remove and leave coats in the lab. Laboratory clothing must not be stored in contact with
street clothing.
11. If a known or suspected exposure occurs, contaminated clothing must be decontaminated before
laundering.
12. Suitable footwear with closed toes/heels and full length pants must be worn in all laboratory
areas.
13. Wear gloves when using infectious materials. Remove and change gloves when contaminated or
torn. Do not wear contaminated gloves outside of the lab. Do not wash or reuse disposable gloves.
14. Do not pipette anything by mouth. Mechanical pipetting devices are used.
15. Follow all safety procedures for handling sharps. Dispose of sharps in puncture resistant
container provided.
16. The use of needles, syringes and other sharp objects should be strictly limited.
17. All procedures are performed carefully to minimize the creation of splashes or aerosols.
18. Contaminated materials and equipment leaving the laboratory for servicing or disposal must be
appropriately decontaminated and labeled or tagged out as such.
19. Decontaminate work surfaces at completion of work, at the end of the day and following spills of
viable material with a suitable disinfectant. Any work surfaces which have become permeable to
biohazardous material must be replaced or repaired.
20. Disinfectants effective against the agents in use must be available at all times within the areas
where the biohazardous material is to be handled or stored.
21. All cultures, stocks and other regulated wastes are decontaminated before disposal by an
approved method such as autoclaving. The material must be contained in such a way as to prevent
the release of the contaminated contents during removal.
22. A biohazard sign is posted at the entrance to the laboratory and laboratory preparation area
whenever infectious agents are present.
23. Leak-proof containers are to be used for the transport of infectious materials within the facilities.

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WHMIS
All students and staff must have up to date WHMIS training by the institution (MRU).
WHMIS training is available online on your D2L site and must be completed to attend any wet labs.
Upon successfully passing the WHMIS quiz (80% or better), you will receive a WHMIS sticker on
your ID as proof of completion.
MSDS sheets are located in The Laboratory Support Centre in the MSDS yellow binders.

Know Your Chemicals

Whether you are doing biology or chemistry labs it is important to know your chemicals. Each
chemical has its own set of hazards. In some cases there can be more than one hazard associated with
a chemical. The hazards include the following:

Flammable and Combustible Material

Flammable material is material that will burn or catch fire easily at normal temperatures
(<37.8 degrees C or 100 degrees F). Combustible material is material that will catch fire if
heated (between 37.8 degrees C and 93.3 degrees F). Examples include propane, acetylene,
ethanol acetone, and toluene.

Oxidizing Material
Oxidizers are compounds which are capable of reacting with and oxidizing (i.e. giving off
oxygen) other materials. The primary hazard associated with this class of compounds lies in their
ability to act as an oxygen source, and thus readily stimulate the combustion of organic
materials. Common oxidizing groups include peroxides, nitrates, nitrites perchlorates, chlorates,
chlorites, hypochlorites, dichromates, permanganates and persulfates.

Poisonous and Infectious Material

Poisonous and Infectious material is material which can cause harm to your body. Some act
quickly and others require long term exposure. Examples are ethyl alcohol or cyanides.
Biohazardous Infectious Materials are organisms or the toxins they produce that can cause
diseases in people or animals. Examples are bacteria, viruses and fungi which can be found in
body tissues or fluids.

Corrosive Material (Acids and Bases)

Corrosive chemicals are those that react with human flesh, or with metals to form oxides. They
include both acids and bases. Examples of corrosive materials include hydrochloric acid and
sodium hydroxide.

Highly Reactive Material

It is defined as the tendency of a system to undergo violent or explosive decomposition under
appropriate conditions of reaction or initiation. Chemical names that include in part names such
per-, peroxy, azo- and cetylide should cause you to consider the possibility of fragile bonds of
peroxides, azides and acetylides. Examples of highly reactive material include picric acid, lead
azide, disodium acetylide and vinyl chloride.

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Low Hazard Materials
Materials which have no known harmful effects. Examples include glucose.

Chemical, Biological and Glass Waste

The Laboratory Support Centre is responsible for providing the proper containers for waste
disposal. Please ensure all containers are marked specifically for the type of waste. For chemical
waste the containers must include the information with the specific chemical. If you see an area
where we can minimize the waste please let us know. We have a specific waste policy which the
students and laboratory instructors must adhere to.
Chemical and Biological waste includes the following.
24. All waste from the experiments performed in the laboratories.
25. All leftover material in the laboratory experiments should be considered hazardous waste.

26. Only non-contaminated gloves and paper towel should be placed in the regular garbage waste
containers. Contaminated gloves and paper towels are considered chemical waste.
27. It is the responsibility of the instructor to instruct the students on how to dispose of the waste
properly in the labs.
28. Poisonous and reactive compounds should be disposed of in separate individual containers.
29. Separate glass containers will be available in the lab for glass waste.
The waste procedure in the laboratories is as follows:

Chemical Waste
Containers will be provided for all separate chemical waste. We will provide an assortment (1L,
4L, 10L and 20L) of specific containers for chemical waste in the laboratories. For most
chemical waste a specific type of container is required. If you do not have a container for a type
of chemical waste in the lab please call the Laboratory Support Centre general phone line and
someone will bring the container to you.
For contaminated gloves and paper towels there will be a separate container provided which will
be labelled.

Biohazard Waste
When required, there will be large yellow buckets labelled biohazardous waste available for all
biohazard waste in the labs including dissected specimens, contaminated paper towels and
gloves. There will also be smaller waste containers for things like slides, toothpicks etc. The
smaller containers will be either red or white and also have the biohazardous label on the outside.
For agar plates and cultures including stock cultures or any other regulated waste it must be
placed in an autoclave bag to be decontaminated before disposal.

Glass Waste
We have small and large cardboard boxes for glass waste which will be labeled. All glass waste
is disposed of as contaminated.

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Fill all the waste containers to maximum 90% full.

Accidents or Incidents
All campus accidents or incidents must be documented and reported. For accidents or incidents
which occur in the laboratory a Campus Accident / Incident Report Form must be filled out. The
forms will be found in the first aid kits in the laboratories. The original copy must be taken to the
Laboratory Support Centre which will keep a copy and forward the original report to the
Occupational Health & Safety Coordinator within 24 hours of the accident / incident occurring.
Examples of Accidents or Incidents
1. First Aid- basic first aid such as a band-aid for a cut or scratch.
2. Medical Aid- where the patient seeks medical aid. In the case of a chemical splash or burn it is
recommended the MSDS of the chemical goes with them for treatment.
3. Near Misses- did you almost have an accident?

If further follow-up or information is needed the Occupational Health and Safety Coordinator
will contact the instructor or student.
MSDS sheets are available on the Canadian Centre for Occupational Health & Safety website
and any mtroyal address can access information free of charge.

Emergency Procedures
If you witness a life-threatening situation such as an acute medical condition including
unconsciousness, profuse bleeding or chest pains, call 911 and Campus Security at 5900. For all
other emergencies including bomb threats, crimes in progress and suspicious activity, contact
Campus Security directly at 5900.
Fire alarm
Mount Royal College at Lincoln Park Campus has a two-stage fire alarm system in most areas
except for the East Arts Building I and II (EA and EB).
The first stage is a combination of “slow” horns at 20 blasts per minute with strobe lights. If
the first stage sounds, you should be alert and prepare to evacuate. Identify the safest exit, gather
your belongings and stand by for further instructions or the sounding of the second stage alarm.
The second stage horn produces a distinctive three-pulse universal “Temporal Pattern Fire
Alarm Evacuation Signal” and includes strobe lights. If the second stage sounds, you should take
the personal belongings in your immediate area only (especially your coat and keys) and leave
via the nearest safe exit. Move at least 300 feet (or 100 meters) from the buildings and follow
instructions from College representatives and/or emergency personnel.

In Case of FIRE
If you discover a fire
Activate the building fire alarm system by pulling the nearest fire alarm pull station

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 Shout “FIRE” to warn people in the area
Evacuate the area, closing all doors and windows
Assist any persons with disabilities, if requested. On a ground level exit, ensure they are away
from the building. On upper or basement levels assist persons with disabilities to an emergency
exit stairwell. Do not attempt to carry them up or down stairs. Advise Emergency Response
personnel or Security of the location of the person(s) with disabilities.
Telephone Security at 5900 or use any of the Help Phones, or the Fire Department at 9-9-1-1,
from a safe location, giving them your name, location and nature of the fire.
If trained, attempt to extinguish the fire by using the appropriate fire extinguisher.
DO NOT use the elevators.

When the fire alarm sounds, evacuate the building. You should be aware of the location of two
emergency exits, the fire alarm pull stations and the fire extinguishers.

Spill Response
Chemical Spills
Clean up incidental spills only. An incidental chemical spill is one staff can clean up without
putting themselves or others in danger. All spills should be cleaned up by trained personnel.
Pre-planning is required to respond safety to any chemical spill. Spill kits with absorbents and
protective equipment are available in each lab for chemistry and a portable spill kit is available in
the laboratory support centre for the biology labs to clean up incidental spills. All personnel
should know the location of the kits and how to use them.

Incidental Chemical Spill

Alert personnel in the immediate area. Avoid breathing vapours and try to determine what
spilled.
If someone has been splashed with chemical, immediately flush the affected area with copious
amounts of water for at least 15 minutes
Take the injured person to Health Services or telephone 5900 for security assistance
Notify the Laboratory Support Centre and the Occupational Health & Safety Coordinator
Personal must wear protective equipment including safety goggles, gloves, and long-sleeve lab
coat during cleanup
Confine the spill to a small area
Use the spill kit material or absorbent material from the laboratory to make a dyke around the
spilled materials.
There is absorbent material for acids, caustics, solvents and formaldehyde available.

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Major Chemical Spill
Avoid breathing vapours. Quickly identify the spilled material if it can be done safely
If the spill involves a flammable liquid, turn off all ignition sources if it can be done safely
Immediately evacuate the area, closing all doors
Laboratory personnel most knowledgeable about the spilled material should be available to
provide information to Emergency Response personnel

Notify security at 5900, the Laboratory Support Centre at 6677 and the Occupational Health &
Safety Coordinator 6038

If someone has been splashed with a chemical, immediately flush the affected area with
copious amounts of water for at least 15 minutes. Take the injured person to Health Services or
telephone 5900 for security assistance
Keep all personnel away from the spill area until Emergency Response personnel arrive to
evaluate and control the situation. Place a sign at all doors on the spill location advising
personnel to not enter the room

Biohazard Spill – inform your instructor and they will:

1. Alert all people working in the area and leave the area for 30 minutes for the aerosols to clear
2. Post signage to keep other people out
3. Inform the Laboratory Support Centre about the spill and make sure to fill out a incident report
form
4. Put on personal protective equipment and open the biohazard spill kit
5. Pour a disinfectant solution around and into the edges of the spill. A disinfectant will be
included in the kit
6. Leave the disinfectant for 30 minutes
7. Soak up the liquid with paper towel and place soiled paper into an autoclave bag
8. Make sure you disinfect you shoes as you are disinfecting the area and take off any clothing
that was contaminated
9. Apply disinfectant solution around and into the edges of the spill a second time and wait 10
minutes
10. Soak up the liquid with paper towel and again place it into an autoclave bag
11. Finally wash entire area with detergent and place all used material into the autoclave bag

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Microscopy I – An Introduction to the
use of microscopes
Required Readings: Fenton, et al. (2022), (microscopy as reviewed in chapter 1 & Purple pages),
this lab! Appendices D (care and use of microscopes) & E (determining size and scale).

Key terms: Light microscope, dissecting microscope, transmission electron microscope,

scanning electron microscope. Eyepiece (ocular lens), revolving nosepiece, objective
lenses (scanning, low power, high power, oil immersion), coarse focus knob, fine focus
knob, substage condenser, diaphragm, stage, mechanical stage, light source.
Parfocal, depth of focus, field of view, wet mount, slide, coverslip, simple stain,
methylene blue.

Objectives:
• Identify features of a compound and dissecting microscope and explain their functions.
• Define and explain magnification, working distance, illumination, resolving power and
focus.
• Understand how magnification affects resolution, and how to manipulate light.
• Measure and calculate field of view. Estimate the size of various samples.
• Describe the use of compound and dissecting microscopes, and key terminology (e.g.
resolution, magnification, parts of a microscope).
• microscope handling and care
• Define and explain: field of view; parfocal; real image; virtual image.
• Understand the differences between the dissecting and compound light - and electron-
microscopes (and between transmission and scanning electron microscopes)
• Proper slide preparation – wet mounts and simple staining.

INTRODUCTION
The ability to see cells, tissues and small organisms arose from the invention of microscopy. The
unaided eye cannot detect anything smaller than 0.1 mm (10 -4 meters) in diameter. With a light
microscope we can now identify objects as small as 0.2 micrometers (10 -7 meters) in diameter and with
an electron microscope we can now visualize objects as small as 1 nanometer (10-9 meters) (Figure 1).

There are four major types of microscopes we talk about in Biology 1202:
1) dissecting light microscopes
2) compound light microscopes
3) scanning electron microscopes
4) transmission electron microscopes

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 The compound and dissecting microscopes have more than one lens and the shape of the lens
determines the orientation of the object. Electron microscopes provide high-magnification views of
individual organelles but are not typically part of undergraduate instruction. Electron microscopes are
expensive and require full-time technicians and service contracts to remain operational because they
generate X-rays as they accelerate electrons to a high enough speed to penetrate tissue slices.
In compound light microscopes, a concave lens will impart a real image (which is also right side up),
while a convex lens will impart an inverted (upside down and backwards) virtual image. The
compound light microscope contains both concave ocular lens and convex objective lenses. The
dissecting microscope contains only concave ocular lens and concave objective lens rendering an
upright real image.

Figure 1. Relative sizes of biological structures. The grids represent logarithmic changes in
magnification. (Original artwork courtesy of Dr. David Bird).

Microscope Parts
Identify the following parts of the microscope (on Figure 2a and 2b, (use Appendix C, provided
bench materials, as well as your assigned microscope to help).
1. Eyepiece (ocular lens): Uppermost series of lenses through which a sample is viewed.
2. Arm: Provides support for upper components and serves as a handle.
3. Nosepiece: revolving component that holds the objective lenses.
4. Objectives (objective lenses): Convex lenses of various magnifications.
a. Scanning power objective (smallest): 4X lens used to view the whole slide.
b. Low-power objective (next largest): 10X lens used to view the object in detail.
c. High-power objective (next largest): 40X lens used to view the object in detail.
d. Oil immersion objective (largest): 100X lens and is used with immersion oil to give the
greatest magnification.

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5. Coarse-focus knob: Control used to bring object into coarse focus; used only with scanning power
objective.
6. Fine-focus knob: smaller control used to bring object into final focus when using low-power, high-
power, or oil immersion lens.
7. Substage Condenser: located immediately under the stage. Contains a system of lenses that
focuses light on the specimen.
8. Diaphragm (iris diaphragm): controls the amount of illumination used to view the object. It can be
opened or closed using the lever on the side of the condenser.
9. Stage: the platform on which a glass microscope slide is placed for viewing. A hole in the middle
of the stage allows light to pass up through the stage illuminating the specimen
10. Mechanical stage: a moveable stage that aids in positioning the slide. Stage clips hold
microscope slides and indicated knobs control both the forward/reverse and the right/left movement.
11. Light Source: an attached lamp that directs a beam of light up through an object. Light intensity
can be adjusted using a sliding dial or knob located on the side of the base next to the on/off switch
or on arm of newer microscopes.

Figure 2a: Dissection microscope (indicated parts highlighted in **paragraph below).

The dissecting microscope (Fig 2a) is used for viewing specimens – such as insects, eggs, large
protozoa, small plants or animals – at lower magnifications. The image seen through a dissecting light
microscope is not inverted but rather a real image (“right-side-up”). The major advantage of this type
of microscope is that specimens can be observed in three dimensions. Most dissecting microscopes
provide magnification in the range of 4X to 50X.

**The dissecting microscope has a fixed ocular objective (10X), two objective lenses (located on
a rotating drum), and two light sources (above and beneath the specimen). Coarse focus control
knobs, located on either side of the arm, raise and lower the ocular objectives. When the microscope
base is equipped with a transparent glass disk the lower light source tends to reveal internal structures.
If an opaque plate is inserted in the base the upper light source strikes the surface of the specimen
highlighting external features. As well as a large depth of view, which offers a 3 dimensional image,

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the dissecting scope has the advantage of a large working distance. This allows easy handling of the
specimen while it is being viewed.**

On a compound light microscope (Figure 2b) there is a fixed 10X ocular lens and four objective
lenses (scanning, low-power, high-power and oil immersion). The scanning objective lens is used for
viewing relatively large objects or for preliminary location of a specimen on a microscope slide. The
oil immersion objective is used for viewing small objects like bacteria, using immersion oil to enhance
resolution. NOTE: Regardless of the size of the specimen being observed, one ALWAYS starts
on scanning power.
(the ‘tab’ controls the)**

(the entire unit)

(the knob)

Figure 2b: Compound light microscope. Label the parts indicated with the arrows.

To accurately observe the specimen, the objective lens must be positioned at a specific distance from
the specimen (working distance) (figure 2c). When the objective lens is located at the proper working
distance the specimen will be in focus. The working distance of the lens is inversely proportional to
the magnification of the objective.

Magnification, Resolution, Illumination and Focus

The primary purpose of a microscope is to magnify (increase an object’s apparent size) the image of
an object. Total magnification (diameter units) is the product of the magnification of the objective
and ocular lenses:
For example: ocular lens (10X) X scanning objective (4X)= total magnification (40X)

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 (the working distance)

Figure 2c. Slide positioned over the stage/substage condenser showing working distance.

Therefore, the scanning objective has the greatest working distance and the oil immersion objective
has the smallest working distance.

Light microscopy is limited by resolving power (i.e. as the magnification increases, the resolution
(ability to distinguish between two points distinctly and clearly, decreases). The lens of the human
eye can resolve two points of an object that lie 0.10 mm (100 μm) apart. By convention, this is given
as a magnification of 1. The wavelength of visible light is the limiting factor in the use of glass lens
systems in high-magnification imaging of objects. The best magnification in a modern light
microscope is 1000 diameters (that is, a circle can be magnified in its diameter 1000 times), therefore
the best resolving ability is 100 μm/1000= 0.10 μm. As we increase magnification, we have a
number of strategies to improve resolution (e.g. including the use of light, high quality glass of the
slides which is also of the same refractive index as our lenses, and the immersion oil).

Using a blue light filter in a light microscope helps increase resolution. Blue light, has a shorter
wavelength of light than full spectrum white light so the filter makes more photons hit the specimen
which offers better visual acuity. The same premise applies to electron microscopy where electrons
have significantly shorter wavelengths allowing for incredible cellular detail to be observed (refer
back to figure 1). In the case of electron microscopy, the specimen is flooded with a concentrated
beam of electrons (Figure 3a-c). Because the wavelength of electrons is about 1/1000th that of visible
light a thousand fold increase in resolution is possible, thus increasing the useful magnification
1000X.

a b c

Figure 3a. This is a New Jeol JEM-1400Plus 120kV Transmission electron microscope and
resulting TEM image. c) Scanning electron microscope image (Images by kind permission of
Jeol USA INC.).

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EXERCISES

Exercise 1 – How to focus the compound light microscope.

See Appendix D for proper operation of the Compound Light Microscope.

Step 1 – Preparation of a mount of the letter ‘e’

1. Obtain a microscope slide from the side bench and a piece of paper with letter ‘ee’s on it.
2. Add a drop of water to your slide and place the paper into the water with the ‘e’s facing upward
and oriented as it would be if you were reading.
3. Place a coverslip over the ‘e’s (refer ahead to figure 8).
4. Return to your microscope.

Step 2 - Use of the compound light microscope – focusing on the LARGE ‘e’
1. Using the coarse-focus knob, lower (or raise) the stage all the way (which will
subsequently increase or decrease your working distance).

2. Turn the nosepiece so that the scanning objective lens (4X) is in alignment over the stage. You
should feel it ‘pop’ into place. (What is the total magnification at this power?)

3. Place your ‘e’s on the stage in the metal frame/pivoting arm as observed below (Figure 4).
Center the largest e using the stage control knobs on the right of the stage. Our microscopes
are par-centered so whatever is in the center of the field of view on this power will remain so as
you go up in power.

Figure 4. The placement of a slide on the stage of a compound light microscope. Observe the
placement of the arm holding the top corner of the slide and how it’s centered such that the beam of
light is penetrating the specimen.

4. On scanning power, turn the coarse focus knob while looking through the oculars until the ‘e’s
are in focus (you will be either moving the stage up, or down to focus the image and the slide will
typically be roughly only 3mm from the objective lens by the time you are done).

5. Adjust the iris diaphragm tab to give the maximum amount of light. (NOTE: you are maximizing
light due to the translucent/opaque properties of paper/ink. If you were looking at something more
transparent (usually types of cells/tissues and microscopic organisms, you would actually shrink
your iris diaphragm quite a bit to increase contrast and be able to see it (i.e. you would not be able
to observe a slide of red blood cells if your light was on maximum)). The iris diaphragm is the
best way to control light.

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6. Once focused, use the fine focus knob, being mindful to turn the knob in the same direction you
did using the coarse focus knob.
NOTE: Make a mental note of which way you turned the fine focus knob while looking through the
lens as this will be the same direction you turn it for focusing at higher powers (at which point you
will only use the fine focus knob, but the stage moves so minimally you will not be able to see it.
For example, if you are focusing objects by raising the slide towards the lens, you will be turning
the knobs away from you (counter clock-wise).

Light microscopes are parfocal (lenses with identical planes of focus but different working distances)
therefore, once the object is in focus with the lowest power, it should also be almost in focus with the
next highest power.

Prior to increasing magnification, draw what you observe in your field of view in the first circle
provided in Figure 5. Note the orientation of the letter e as you view it through the compound
microscope? Why does it look different from a letter e printed on this page?
________________________________________________________________________________
Move the mechanical stage and microscope slide slightly to the left. Which way do the letter ‘e’s seem
to move when looking through the oculars (left or right)? ___________________
Step 3 - Increasing the magnification:
7. Once the e’s are in focus with the scanning objective and you’ve drawn what you observed, move
next to the low power objective (10X) by turning the nosepiece counter-clockwise.

8. Use only the fine-focus knob for focusing this objective lens (and all higher magnification
lenses). Once the e is in focus, you may again need to adjust the amount of light by opening the
diaphragm (more light is filtered out as magnification increases). Draw your ‘e’ at 10X below.

9. Switch to the high power objective (40X) and repeat as above. Sketch your letter e at 100x below.

Scanning power (___X) Low power (____X) High dry ( X)

Figure 5. Sketches of largest letter e as observed with each field of view through a compound light
microscope.

10 (optional). You may want to try using the immersion oil lens to view your ‘e’s (or save this task
to do with a more ‘exciting’ specimen later).

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Using the oil immersion lens: once focused on high dry power (400X), move the lenses so they
straddle the slide (Figure 6 below), and place a SMALL amount of immersion oil directly onto the
cover slip of your slide. Next slide the 1000X lens into the oil.

Figure 6. Lens orientation when applying immersion oil.

When you are finished observing your inky paper fibers, FIRST change the lens back to scanning
power, THEN drop the stage and wash off your lens using lens paper and solution (as demonstrated
by your lab instructor).
Protocol Question: Why do you suppose you change the lens before attempting to drop the stage?

Exercise 2 – Measuring the Diameter of the Field of View (FOV)

Step 1 – Determining the Diameter of Field of View (FOV) at each magnification
It is important to know the scale of the objects you are looking at. The entire area you can see at one
time is called the field of view.

Measuring FOV (on scanning power)

1. To determine the diameter of the field of view under the scanning objective lens, place the inner
edge of a clear plastic ruler with the mm gradations in the field of view. Count the number of mm that
you see in the field (estimate to one decimal place i.e., 1/10th of a mm). Convert this figure to μm using
the following conversion factor:
(___)mm x (1000 μm/mm) = _____μm (hint: simply; 1mm = 1000 μm)

NOTE: you can only use the clear plastic ruler on scanning power.

Calculating FOV for the higher powers (above scanning power)

2. To calculate the diameter of the field of view for the next highest lens magnification use the formula:
FOVlower x Maglower = FOVhigher x Maghigher
where:
FOVlower= diameter (mm) for lower power (in this case the scanning power objective)
Maglower = magnification of lower power lens (again, scanning power in this case)
FOVhigher = the diameter of the higher powered lens we want to determine
Maghigher = magnification of this higher powered objective lens

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If your field of view at scanning power was 5mm, then to solve for the size of the field of view at low
power you would plug in all the values and solve for FOVhigher
5mm(40X)= FOVhigher(100X),

200/100 = FOVhigher
= 2mm
Therefore, if the field of view at scanning power (40X) is 5mm, on low power the field of view is
reduced to 2mm (or 2000μm).

3. Calculate the diameter of field of view for the remaining objective lenses.
Low-power diameter field of view:________ mm
High-power diameter field of view:________ μm
Oil-immersion diameter field of view:______ μm
(You may also refer to appendix E as well to calibrate the ocular scale properly. This gives readings
far more precise than the field of view estimate you just learned how to do.)

Fill in the table below (Table 1).

Table 1. Magnification for each objective power and their subsequent calculated FoV’s.
Objective Objective Occular TOTAL Diameter of Diameter of
lens magnification magnification magnification field of view field of view
(mm) in μm
Scanning 4X 10X
Low power
High dry
Immersion oil

Step 2 – Determining the size of your specimen based on FOV calculations

If you know the scale of the whole area you can see (i.e. FOV), you can estimate the sizes of the specimens
being observed. For example, if you know the field of view is 10mm in diameter and your specimen spans
1/5th of the field of view, then the specimen must be about 2mm in size.

10mm so this large is is ~2mm in size

5 cells
e
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4. Obtain your different-sized e’s slide again. Now that you know the size of the diameter of FOV for
each magnification, use this information to determine the size of each e.
5. Center and focus the largest e on your slide, then determine its size.
Record your calculations below.

Repeat for the small sized e, recording your calculations below.

Exercise 3. Effects of Depth of Focus on resolution observations

Depth of focus is the vertical distance that remains in focus at one time (Figure 7). If you chose to do
step 10 above, you also noted that on 1000X you could only get a small portion of your e into focus
while other portions were blurry. The depth of focus (the vertical distance that remains in focus at one
time) is shallow under higher powers than under low power.

It is possible to reconstruct a three-dimensional image from a series of near flat sections.

Figure 7: A demonstration of how a series of sections focused on at varying depths could produce a
3-dimensional structure. Each box to the right represents the view achieved at each plane intersecting
the cone at the left.

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Over-laying coloured cross-fibers slide
1) Obtain a microscope slide with coloured threads mounted on it. Using the low power objective, find
a point where the threads cross. Slowly focus up and down. Note that when one thread is in focus the
others seem blurred. As the stage moves upward, objects on top come into focus first.
Determine the order of the threads on the slide.

2) Switch to high power and observe again how only one coloured fiber can be focused at a time as
you change the plane of focus (i.e. move the lens up or down).
As magnification increases, depth of focus _____________(increases/decreases). Explain…
______________________________________________________________________________
____________________________________________________________________________

Exercise 4. Preparing wet mounts - Protozoa in suspension

1. Prepare a wet mount from one of the protista or pond water made available in the lab (which may
contain “Euglena”, “Paramecium”, or other “mixed cultures” of animals and algae). Do this by first
squeezing the air out of a disposable pipette or dropper then drawing up a small portion of culture from
the container (usually near the bottom of the container specimens are more concentrated). Place a dime
to nickel-sized drop onto the slide (Figure 8). You may also want to add a drop of methylcellulose
(Protoslo) to this slide to slow your critters down.
2. Next, cover your drop with a cover slip as shown below in Figure 8. Do NOT add any stain to this
slide or you will kill your specimens. Adjust your iris diaphragm to see these organisms well (almost
completely shutting it) will increase contrast.

cover slips

slide box dropper full of protozoan

suspension

Step 1. Obtain a clean slide Step 2. Place a dime sized drop of solution on the slide.
pipette

Step 3. Place cover slip on top of sample and observe using the microscope.
Figure 8. Wet Mount preparation. Notice in step 3 that you bring the coverslip towards the water at a
45 degree angle and once the water adheres to the glass you should be able to release it without air
bubbles.

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Use the bench materials provided (models and posters) to try and identify some of the organisms in
your samples and even some of their internal/external observable features.
Which organisms can you observe in your slide(s)?_________________________________

If you open the iris diaphragm to let the light flood in, what happens to the creatures you are
observing and why? ________________________________________________________________
_________________________________________________________________________________
Exercise 5. Preparing wet mounts of fresh plant tissue – Simple staining
Prepare a wet mount of a thin layer of cells from the white onion specimen provided by your lab
instructor.
1. First, add a drop of water to your slide and place it on the lab bench.
2. Next add a very small amount of methylene blue at the end of a dropper to this water
3. Carefully peel a thin section of the epidermis using a razor blade and forceps or your finger nail.
Note: this piece of onion is not going on a hot dog, it should be as thin as a layer of your own skin.
4. Place the thin section into the water on your slide, spread it out (remove wrinkles) as much as
possible, and place the coverslip on top (Figure 9). Observe your specimen.
At this point you should consider if your iris diaphragm should be open or closed (hint: this is
relatively transparent tissue).
Which features (organelles, membranes, cell wall), can you observe in your specimen?
_________________________________________________________________________________
_________________________________________________________________________________

Figure 9. Simple staining of translucent tissue. Take note of how little stain is needed to sufficiently
stain the tissue.

This type of staining which does not offer any diagnostic information but simply aids in visualization
is called simple staining. What do you suppose we mean by “diagnostic” information (i.e. how is it
different from simple staining)? ______________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

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Exercise 6. Dissecting microscope
1. Obtain a dissecting microscope. Select a specimen that is on display and examine it through the
microscope.
2. Adjust the distance between the eyepieces so that they are comfortable. You should be able to see
the specimen with both eyes as one three-dimensional structure.
3. Use the large focusing knob to bring the specimen into focus. Change the magnification by rotating
the nosepiece. Experiment with the various specimens at different magnifications until you are
comfortable with the binocular dissecting microscope.
Question: Why isn’t the image flipped and backwards as was observed in exercise 1? ____________
_________________________________________________________________________________
What is the advantage of the large working distance between the stage and the objective lens?
_________________________________________________________________________________
Flip between the 2 objective powers on your microscope to observe the difference in magnification.
What is the total power (magnification) of each lens? ______X and _____X respectively.

Exercise 7. Electron Microscopy (In-class Demonstration of electron microscope use).

If available, your lab instructor will demonstrate the use of the electron microscope we use at MRU;
the Hitachi TM-1000, Tabletop Scanning Electron Microscope (SEM).

Transmission electron microscopy (TEM) directs an electron beam at thin specimen sections
which are stained with metals to absorb electrons and enhance contrast. The specimen deflects
electrons away from the screen, and an image of the electron-opaque and electron-transmitting
sections from the specimen appear on a screen as shadows and light areas. Electrons transmitted
through the specimen are focused and the image of internal cellular ultrastructure is directed onto a
film.

Scanning electron microscopy (SEM) directs the electron beam over the surface of a pre-coated
specimen. Scanning and secondary electrons are excited, focused and collected onto a viewing screen
providing remarkable field depth and useful three-dimensional images. The SEM can provide
detailed information about chemical composition, electrical properties, and structural characteristics
of the surface of specimens. Due to the elaborate specimen preparation required for the SEM it is
possible to view only dead cells.

As mentioned, electron microscopy increases the magnification a thousand-fold over the light
microscopes. View the various pictures taken from the two types of electron microscopes. How do you
distinguish between the two types of electron microscopy.
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
Why do organisms have to be dead when being observed? __________________________________
_________________________________________________________________________________

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Additional Study Questions
1. Why is resolution a limiting factor to magnification? ______________________________
___________________________________________________________________________

2. Define parfocal and state why it is beneficial with the lens system of the compound light
microscope. _________________________________________________________________
___________________________________________________________________________

3. Why is immersion oil necessary when using the 100x objective? ______________________
____________________________________________________________________________

4. What is the function of the iris diaphragm? _______________________________________

____________________________________________________________________________

5. What is the function of the sub-stage condenser? __________________________________

____________________________________________________________________________

6. Describe some techniques or equipment we use to increase resolution. _________________

____________________________________________________________________________

7. What happens to depth of focus as magnification increases? __________________________

8. Which lens inverts the specimen image? Why? ____________________________________

____________________________________________________________________________

9. Which electron microscope produces a 3-D image? _____________________________________

10. Which electron microscope yields an image of internal cellular structures? __________________

11. What capabilities are similar between light and electron microscopes? How do they differ?
_________________________________________________________________________________
_________________________________________________________________________________

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 Microscopy I Prelab questions

1. The magnification of the eyepiece lens is ________ so, when the scanning objective lens

(magnification ____ ) is in place, the total magnification is _____ .

2. With the low power lens (magnification ______ ) in place the total magnification is_____ .

3. With the high power lens (magnification ______ ) in place the total magnification is _____ .

4. With the oil immersion lens (magnification ______ ) in place the total magnification is _____ .

5. Light microscopes use a beam of _____________ so we can see things in colour.

However, electron microscopes use a beam of _________________ so there is no

________________ .

6. The two kinds of electron microscope are ___________________ and __________________ .

7. Our light microscopes are parfocal. This means that _____________________________________

_________________________________________________________________________________

8. The whole area of the microscope slide that you can see through the microscope at any one time is

called the _______________________.

9. The formula used to calculate the diameter of the field of view is

_____________________________________________________________

10. Which is the only objective lens you can use a ruler on the stage to measure the FOV on?

_____________________________

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 Macromolecules
The major organic polymers
Required readings: Fenton, et al. (2022) “Purple pages”, p F2-F4, (Chapter 1) & look up amyloplasts!

Key Terms: Macromolecule, monomer, polymer, Carbohydrate, monosaccharide (glucose, fructose),

disaccharide (sucrose, lactose, maltose), polysaccharide (starch, cellulose,
glycogen).Benedict’s reagent (reducing sugar), precipitate,
Barfoed’s reagent (monosaccharide), iodine (starch, glycogen), amyloplast.
Lipid, glycerol, fatty acid, fat, oil. Grease spot, miscibility, polar, non-polar (lipid).
Protein, amino acid, peptide bond, polypeptide. Ninhydrin (amino acid), glycine, gelatin,
albumin, Biuret’s reagent (peptide bond), peptone.
Nucleic acid, DNA, deoxyribose sugar, RNA, ribose sugar, Dische diphenylamine.

Objectives:
• Know the building blocks of carbohydrates, proteins, lipids, and nucleic acids
• Know the reagents used to test for the presence of the above organic compounds
• Know the major classes of each macromolecule along with their monomer(s).
• Know some of the characteristics of each macromolecule.

INTRODUCTION
A polymer is a large molecule polymerized from few to many repeating subunits known as
monomers. Macromolecules are special types of polymers we often refer to as the building blocks
of life, and includes such groups as lipids, nucleic acids, proteins (polypeptides), and carbohydrates
(polysaccharides). Each group is made up of its own unique monomer. The combinations of
monomers coupled with how they are polymerized gives each of these macromolecules unique
properties which relate to their structure, aid in our ability to identify them, as well as provide them
with many specific functions within an organism. In this lab we shall investigate the chemical
composition of each macromolecule group, learn their monomers, and how to identify them using
a series of indicator reagents.

Carbohydrates
The wonderful thing about this macromolecule groups name “carb-o-hydrate” is that it conveniently
describes its molecular structure (as hydrated carbon; C, H, and 0, existing in a 1:2:1 ratio respectively).
Carbohydrates are a group of biomolecules consisting of sugars, and includes both simple sugars
(monomers) and polysaccharide chains (polymers). Carbohydrates function to provide an immediate
source of energy for the body, usually used as simple sugars (e.g. glucose) or stored as polymers (e.g.
glycogen or starch). Other functions of polysaccharides include structural purposes, such as cellulose
in plants or chitin in animal exoskeletons. Carbohydrates are divided into 3 groups based on how many
carbons are present in their structure. The 3 groups are:
i) Monosaccharides - or simple sugars, have 3-7 carbons in their structure. e.g. Glucose
ii) Disaccharides - made up of 2 monosaccharides, have 8-14 Carbons in their structure. e.g. Sucrose
iii) Polysaccharides - complex carbohydrates, important to add fiber to our diet. e.g. Starch, Glycogen,
Cellulose

27 | P a g e
Tests for Sugars
A simple and common test for carbohydrates is the Benedict’s Test. Benedict’s reagent is a chemical
that reacts with reducing sugars and changes colours. A reducing sugar is a carbohydrate with a free
or a potentially free aldehyde (-C =O-H) or ketone (-C=O) group. In a solution of sufficiently high pH,
reducing sugars can reduce weak oxidizing agents, such as cupric ions, to form a solid precipitate. For
example, Cu++ ions react with glucose to form a coloured precipitate of cuprous oxide. The colour of
the precipitate will range from green to reddish brown depending on the concentration of the reducing
sugar present.

(C6H12O6) (Glucose) +Cu (OH)2 Cu2O + H2O + oxidized glucose

heat (coloured precipitate)

a) Benedict’s Test
Benedict’s reagent contains sodium bicarbonate, sodium citrate and cupric sulfate. After heating with
a reducing sugar, the cupric ion of the cupric sulfate is reduced to the cuprous ion of cuprous oxide
that forms a solid precipitate ranging in colour from green to red-brown.

b) Barfoed’s test
Barfoed’s test is used to distinguish monosaccharides from disaccharides and polysaccharides. The
reagent is similar to Benedict’s reagent except that it is slightly acidic, having a pH of about 4.5. At
this pH, disaccharides and polysaccharides will not reduce Cu++ to Cu20, whereas monosaccharaides
will reduce when heated for exactly 2 minutes. Longer heating of disaccharides may lead to some
reduction because of the formation of monosaccharides by hydrolysis. Therefore is it essential that all
sugars be treated in exactly the same way and not boiled for longer than 2 minutes.

c) Iodine Test
The polymer we call amylose is more familiarly known as “starch”. Starch is important because most
plants store their carbohydrates as starch in specialized organelles called leucoplasts or amyloplasts
(we will learn about these in the cell structure lab), and cellulose is a major component of plant cell
walls. (Note: most animals store their carbohydrates as glycogen in the liver to be converted back to
simple sugars such as glucose during times of high energy demand).
Iodine molecules get trapped in the spiral strands of starch, which are simply lots of glucose molecules
all covalently joined. A solution of un-joined glucose will not darken because the iodine is not trapped
in the coils. Iodine turns deep blue if it is in a starch solution and red-brown in a glycogen solution.
Glycogen is similar to starch but is branched.

Exercise 1. Carbohydrate testing

1. Gather 8 test tubes. Try to use this same set of 8 test tubes to complete all 3 tests, rinsing into
supplied indicated waste bottles, and washing thoroughly between each reagent test.

Reducing sugars (Benedicts) test:

Place 0.5 ml of Benedict’s reagent in each test tube and add 1 ml of each of the provided 1%
carbohydrate solutions (as listed in table 1 below).
Place the test tubes in the boiling water bath for 1 minute (or until the tubes begin to change colour)
and allow to cool to room temperature.
Record your results in Table 1 noting the colour of the precipitate formed.

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Monosaccharides (Barfoeds) test:
Repeat above procedure only this time use 1 ml of Barfoed’s reagent in each test tube and again add 1
ml of each of the carbohydrate solutions.
Mix the solutions well and place the test tubes in a boiling water bath for exactly 2 minutes. A positive
test for monosaccharides is the appearance of a fine red precipitate of Cu 2O within 1 or 2 minutes. It
is easy to misinterpret this test as a negative result so hold each tube over a piece of white paper and
look to the bottom of the tube.
Record your results in Table 1.

Starch (Iodine) test:

Place a couple drops of the various 1% carbohydrate solutions in each of the wells of a spot plate. Add
1 drop of the IKI (Iodine) solution to each. A positive test for starch is the deep blue-black colour and
a positive test for glycogen is the red-brown colour. Record your results in table 1.

Table 1. Results for carbohydrate tests for reducing sugars, and complex carbohydrates.
Reducing sugar Monosaccharide Starch
Carbohydrates (state reagent/test
used:)->
water H2O
Glucose C6H12O6
Fructose C6H12O6
Lactose C12H22O11
Sucrose C12H22O11
Starch (C6H10O5)n
Glycogen (C6H10O5)n
Unknown A

Microscopy Break! Test for starch in potato tissue

Prepare a wet mount of a thin slice of potato tissue. Do this by using a razor blade to shave off a
thin slice of tissue, and place it into a tiny drop of water on a clean slide. Next add a drop of iodine
to the slide and place a cover slip on top. Observe your slide under the microscope. Starch is stored
in tiny plastids called amyloplasts. You make a sketch of your observations below.

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Study Questions
1. Why was the test tube of distilled H2O used and what does it show? _______________________
_____________________________________________________________________________

2. From your results, list the reducing sugars. ___________________________________________

3. From your results, list the disaccharides. _____________________________________________
4. For what type of carbohydrates is the iodine test suitable? _______________________________
5. Is the iodine test suitable for distinguishing between starch and glycogen? Explain. ___________
________________________________________________________________________________

Lipids
Lipids are substances that are insoluble in water (i.e. nonpolar) but soluble in fat solvents like acetone,
alcohol, chloroform, benzene and ether. Lipids are important because they provide us with long term
energy storage. Lipids provide insulation for our bodies and protection for the organs. Lipids include
fats, oils, waxes, phospholipids and steroids. Fats and oils are made up of 1 glycerol molecule and 3
fatty acid molecules joined together. We cannot over-simplify the monomers of a lipid however, as
some lipids, such as sterols, have a very different structure in the form of a cyclic ring structure.

Substances that contain a high percentage of fats can easily be identified by the familiar grease marks
they make on unglazed brown paper. However, if the concentration of fat is low, a more sensitive test
is required.

Exercise 2 – Lipid testing

Grease Spot Test

1. Lay out 3 small pieces of brown paper or paper towel.
2. Take 1 drop of distilled water and put it on one piece of paper.
3. Place 1 drop of vegetable oil on the second piece of paper.
4. Place 1 drop of Unknown B on the third piece of paper.
After the papers have dried for a few minutes, hold them up to a light and observe. You are looking
for the presence of a grease spot (translucent area) on the paper. Record your results into your
laboratory notebook. NOTE: WHY does it make a translucent spot if it is a lipid?

Layering/miscibility test
Given the fact that lipids are not water soluble, a simple test for the presence of a lipid is to try to
mix it with water. Recall the saying “like dissolves like”. If you mix a lipid with a non-polar
solvent it will readily dissolve (i.e. no layering will be observed after mixing).

1. Take 1 ml of distilled water and dispense it into a test tube.

2. Take 1 ml of vegetable oil and add this to the water and cover with parafilm.
3. Invert the tube several times, mixing the contents.
Did they mix? Yes/no (circle one)

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4. Now repeat this procedure using 2 ml of water with unknown B. Did they mix? Yes/no (try to
explain in your own words) ______________________________________________________
_____________________________________________________________________________

Questions:
1. Is unknown B a lipid? Explain how you got your answer. _____________________________
_____________________________________________________________________________

**All waste from the lipid tests may be disposed of in the garbage or rinsed down the sink.**

Proteins
Proteins are a very important part of our bodies involved in several structural and metabolic functions.
Proteins make up our hair, fingernails, connective tissue and muscles. There are proteins in our blood.
Some of our proteins are enzymes. Enzymes are biological catalysts, which aid the chemical reactions
in our bodies.

Proteins are composed of many monomers, amino acids, joined together by peptide bonds. A single
protein molecule is often referred to as a “polypeptide” – some biologists reserve the term “protein”
for one or more polypeptides that are capable of some kind of activity, such as enzymatic modification
of other molecules.

Each amino acid has an amino group (NH2) at one end and a carboxyl group (COOH) at the other end.
To test for a protein we can use a test that looks for the presence of the amino group and reacts if one
is present or we can use a test that reacts only if peptide bonds are present.

Ninhydrin Reaction test

Ninhydrin is a powerful oxidizing agent that removes the amino groups of amino acids, liberating
ammonia, carbon dioxide, the corresponding aldehyde and a reduced form of ninhydrin. The ammonia
then reacts with an additional molecule of ninhydrin and the reduced ninhydrin to form a purple
substance. Therefore, the formation of a purple colour indicates the presence of amino acids.

Exercise 3. Ninhydrin (amino acid) test

Obtain 5 clean test tubes. Add 2 ml of distilled water to test tube #1.
To test tube #2 add 2 ml of glycine (an amino acid).
To test tube #3 add 2 ml of gelatin solution (collagen – peptide & protein).
To test tube #4 add 2 ml of egg albumin (egg white).
To test tube #5 add 2 ml of Unknown C.

Add 1 ml of Ninhydrin reagent to each of the 5 test tubes. Mix the tubes carefully so that you do not
get any Ninhydrin reagent on your hands. (Ninhydrin is believed to be a carcinogen). Carefully
place the test tubes in a beaker of hot but NOT boiling water that has been removed from the hot
plate once steaming. Results can be recorded in Table 2.

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 !! SAFETY NOTE: DO NOT BOIL NINHYDRIN!!
Table 2. Results for protein identification using ninhydrin
Test tube # Contents Colour explanation
1 Distilled water
2 Glycine
3 Gelatin
4 Albumin
5 Unknown C

WHEN YOU ARE DONE, RINSE TUBES INTO LABELED WASTE BOTTLES.

Biuret Test
Biuret Reagent is a solution of sodium or potassium hydroxide (NaOH or KOH) mixed with dilute
copper sulfate. It reacts specifically with the peptide bonds formed between amino acid groups.
Biuret changes from blue to violet in the presence of a protein (long chains of amino acids joined by
peptide bonds) and from blue to light purple in the presence of peptides (short chains of amino acids
joined by peptide bonds). The reaction is based on the formation of a purple-coloured complex between
cupric ions and two or more peptide bonds. Proteins give a particularly strong biuret reaction because
they contain large numbers of peptide bonds.

The biuret reaction may be used to quantify the concentration of proteins because peptide bonds occur
with approximately the same frequency per gram of material for most proteins. One could then
determine the concentration of an unknown protein solution by measuring colourimetrically the
intensity of their colour production in the biuret reaction as compared with the colour produced by a
known concentration.

Exercise 4. Biurets (peptide bonds) test

Obtain 5 clean test tubes and add the solutions as shown in Table 3. Add Biuret Reagent last and mix
thoroughly by rotating the tubes between the palms of your hands. The colour will remain stable for
about 1 hour. Grade colour intensity for each tube by comparing to the control (tube #1). Use the scale
(0, +, ++, +++) for colour intensity. Record your results in Table 3.

Table 3. Tube contents and results for Biurets test

Tube # Vol. of sample Vol dH20 Vol. Biurets Observations
1 0 ml 1 ml 1 ml
2 0.5 ml peptone 0.5 ml 1 ml
3 1 ml peptone 0 ml 1 ml
4 1 ml gelatin 0 ml 1 ml
5 1.0 ml unknown D 0 ml 1 ml

WHEN YOU ARE DONE, DISPOSE OF WASTE IN LABELED WASTE BOTTLES.

Questions:
1. According to your Ninhydrin test results, which tube has the highest concentration of protein
(amino acids) in it and how do you know?_________________________________________

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2. According to your Biuret test results, which tube has the highest concentration of protein (peptide
bonds) in it and how do you know?_______________________________________________

3. Do Unknown C and D contain protein?_____________________________________________

Nucleic Acids
Nucleic acids are made up of monomers called nucleotides. A nucleotide is a three part structure
composed of a sugar, a phosphate group and a nitrogenous base. The bases include; adenine, cytosine,
guanine, thymine and uracil. Some examples of nucleic acids are DNA, RNA and ATP. In this lab we
will study the differences between DNA and RNA. Chemically DNA differs from RNA in that DNA
has deoxyribose sugar and RNA has ribose sugar. DNA and RNA also have a base difference.

The reagent Dische diphenylamine reacts with deoxyribose sugar to form a blue complex. The
intensity of the colour corresponds to the concentration of the DNA. Dische diphenylamine does not
react with ribose sugar.

Exercise 5. Dische diphenylamine test for DNA

1. Obtain 4 clean test tubes and label them 1-4.
2. In test tube #1 place 1 ml of distilled water.
3. In test tube #2 place 1 ml DNA solution.
4. In test tube #3 place 0.5 ml DNA and 0.5 ml distilled water.
5. In test tube #4 place 1 ml RNA solution.
6. Add 1 ml of Dische diphenylamine reagent to each tube. Mix thoroughly.
7. Place the tubes in hot steaming water bath for up to 10 minutes (have tubes pointing away from
you).
8. Then let cool. Record your results in table 4.

Table 4. Results of testing molecules for the presence of nucleic acid using Dische diphenylamine.
Test tube # Contents Reaction with Dische diphenylamine (explanation)
1 dH20
2 1 ml DNA
3 0.5 ml DNA
& H20
4 1 ml RNA
WHEN YOU ARE DONE, DISPOSE OF WASTE IN LABELED WASTE BOTTLES.

**Be prepared to be able to identify the macromolecular constituents in a sample (which may contain
more than one macromolecule group) during your laboratory final exam. **

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 Macromolecules prelab questions

In this weeks’ lab you are asked to work with a number of reagents. For each of the indicated
reagents below, determine what it is. Gelatin, for example, is collagen; peptides and protein.
Knowing this will help you decipher if the test results you get in lab, are appropriate, or if you
have false positive/negative result.

A – Fructose is a ________________________, and as such should yield a positive result for the
___________________ test, which turns _________ in colour.

B – Glycine is a ________________________, and as such should yield a positive result from the
____________________ test, which turns _________ in colour.

C - Peptone is a _______________________, and as such should yield a positive result from the
___________________ test, which turns _________ in colour.

Which test is specifically for looking for the presence of starch? ____________ a positive result
is ______________ in colour.

The only macromolecule group test results which can go down the sink this week are for the
_______ tests.

Do lipids mix with water? Why/why not?

From your research on each of these reagents, and reading your lab, fill in the following
tables with your predicted outcomes for each macromolecule test you will be performing.

Table 1. PREDICTED results for tests for monosaccharides, reducing sugars & complex
carbohydrates (note as + or – and the resulting colour).
Reducing sugar Monosaccharide Starch
Carbohydrates
Reagent used for testing: (example below)
Glucose C6H12O6 + orange
Fructose C6H12O6
Lactose C12H22O11
Sucrose C12H22O11
Starch (C6H10O5)n
Glycogen (C6H10O5)n

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Table 2. PREDICTED results for protein identification using ninhydrin
Test tube # Contents Colour Why? (explanation – what is each substrate?...)
1 Distilled water clear because H20 has no amino acids in it (negative control)
2 Glycine
3 Gelatin
4 Albumin
5 Unknown C

Table 3. Predicted results for Biurets test and why.

Tube # Vol. of sample Vol dH20 Vol. Predicted outcome and why
Biurets
1 0 ml 1 ml 1 ml Negative control
2 0.5 ml peptone 0.5 ml 1 ml
3 1 ml peptone 0 ml 1 ml
4 1 ml gelatin 0 ml 1 ml
5 1.0 ml unknown D 0 ml 1 ml

Table 4. Predicted results of testing molecules for the presence of DNA.

Test tube # Contents Reaction with Dische diphenylamine (why? explanation)
1 dH20
2 1 ml DNA
3 0.5 ml DNA
& H20
4 1 ml RNA

Blank macromolecules chart next page to be filled in for study purposes.

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36 | P a g e
 Instrumentation;
Solution Preparation & Spectrophotometry;
Generating Standard Curves
Required Readings:
- Appendix F: Creating solutions and Preparing serial dilutions
- Appendix G: Using Micropipettes
- Appendix H: Using a spectrophotometer
- Appendix I: Using a pH meter (if one is provided on the side bench check it out)
- From the GI guidelines: Graphing and statistical analyses using Excel
Key terms: Micropipette, microlitre (µl). Spectrophotometer, absorbance, blank, wavelength,
standard curve, independent variable, dependent variable, negative control, serial dilutions.
pH meter, calibrating buffers.

Objectives: -to learn appropriate laboratory techniques (pipetting, measuring pH, and
spectrophotometer use)
- solution preparation and associated calculations
- to use absorbance data from a spectrophotometer to generate standard curves, and to
determine the concentration of an unknown solution from graphs and from the slope
of a line formula
- how to prepare serial dilutions
- learn how to graph data using Excel

INTRODUCTION
One of the earliest instruments used in identifying and the subsequent study of cells was the light
microscope. Over the years, improvements in the manufacture of microscopes have resulted in these
instruments remaining one of the primary tools of cell biologists today. As our knowledge of the cell
expanded and as scientists moved from the observation of cells, cell structure and sub-cellular
organelles, to the study of the functions of these sub-cellular fractions and their biochemistry, new
tools were required. In this laboratory you will be introduced to three important instruments commonly
employed in modern chemistry and biology labs. These are: micropipettes, spectrophotometers, and
pH meters. In this lab you will gain familiarity with the application and proper use of each of these
items.

Micropipettes
Pipettes are devices used to measure volumes of up to 25.0 ml accurately. For measuring extremely
small volumes (i.e. in the μl range), a specialized pipette called a micropipette is used. 1μl is the
equivalent of 1/1000th of a ml, or conversely, 1000 μl = 1 ml.
In our labs, we have three kinds of micropipette available; each is distinguished by the maximum
volume it is intended to measure: P20 = up to 20 μl, P200 = up to 200 μl and P1000 = up to 1000μl.
You can identify each by the number on the top of the round plunger (Figure 1a).

RULE OF THUMB: To achieve the greatest accuracy,

always select the smallest pipette size that will handle the required volume.
Accuracy decreases as you use larger pipettes for small volumes
(i,e., you would not use a P1000 to measure out 10µl).
37 | P a g e
How to Read the Volume on the Micropipette
Look at the front face of the pipette and you will see a window with three digits inside (Figure 1b). Do
not exceed the maximum value for your pipette (i.e. do not exceed a setting of 1000 for your P1000
pipette or 200 for your P 200 pipette). To exceed these values will put the pipette out of calibration.
Re-calibration is expensive. Note that only three digits are found in the window. This means that the
20 μl micropipette can be set to 10 ’s of a μl (the bottom digit is red) and the 1000 μl micropipette
th

measures in 10’s of μl (the top digit is red).

Changing Load Volume

Change the load volume by turning the ridged plastic knob at the top of the hand grip (Figure 1d). Be
sure you do not take the numbers higher than the maximum load or lower than the minimum
load. When in doubt, ask for help.

Technique
Hold the pipette with your thumb on the plunger, “like a knife in a horror film”. Load a sterile tip (blue
for P1000; yellow for all others) and then push the plunger down slowly to the point of first
resistance: this is the load volume. Put the tip into the solution so that it is immersed just enough to
cover the end. Slowly release the plunger to draw up the liquid. To deliver the volume, place the tip
into the receiving vessel and slowly press the plunger all the way to the bottom - this expels all the
liquid and gives a little extra volume to force ALL the fluid out. WITHOUT RELEASING THE
PLUNGER, withdraw the tip from the receiving vessel.

Note regarding small volumes:

\With small volumes (particularly the 1 - 10 μl range used for molecular biology), you need to know
where the drop of fluid was expelled from the pipette so that you do not inadvertently remove it as you
withdraw the tip. Expel the liquid as a drop on the inside wall of the receiving vessel (tube) so that you
can see it. If adding the fluid to a larger volume of solvent fluid, flush the tip with the solvent liquid
after expelling the droplet to make certain you get all the delivered liquid into your larger volume.
Practice this with small volumes of food colouring water until you are comfortable with the
mechanism. Figure 1 shows you the micropipette you will most commonly use in this lab, the P1000.
The volume being pipetted is displayed in the window and can be changed by turning the black knobs
observable in behind in Figure 1b and c.
Knob for adjusting
Volume dispensing
the volume being
knob pipetted.
Release
button for
disposable
micropipette
tips

a. b. c.
Figure 1. Images of the P1000 (100/1000) which will be commonly used when measuring volumes
between 100 and 1000µl. The volume shown in (b) is 500µl (0.5ml) and in (c) is 1ml.

38 | P a g e
**NOTE: Micropipettes are delicate instruments. Handle them with care and when not in use
PLEASE ensure that they are stored upright in their racks. DO NOT lay micropipettes on their sides
on your bench top. Fluid may leak into the barrel of the pipette and clog the plunger mechanism.**
Be certain to make notes on the volumes indicated in the windows for each pipette.
Example:
P10 (or 2/20) P100 (or 20/200) P1000

= ________ml = _______ml = _______ml

or ________µl or ________µl or ________µl
Other notes:

Spectrophotometers
In nature, all atoms and molecules have the capacity to both absorb and reflect light. Pigment molecules
reflect light of only certain wavelengths and absorb light of other wavelengths. For instance, we see
the leaves of trees as being predominantly green during the summer months. This is because the leaves
contain high concentrations of the photopigment chlorophyll which reflects green light and absorbs
light from the blue, yellow, and red portions of the visible spectrum. Knowing that molecules will
absorb specific portions of the visible spectrum, scientists have developed mechanisms to measure this
absorbance. The instrument used to measure the absorbance of light by substances in solution is called
a spectrophotometer (Figure 2). The data generated by spectrophotometers can be presented
graphically, and used in quantitative analyses.

Screen
displays
the spec
is set at
546nm
toand has
increase
orandecrease
nmabsorban
ce
used to
reading
“zero or
of 0.000
blank”
the spec.

Figure 2. The (Thermos Genesys 20 Vis)- spectrophotometer used in lab.

39 | P a g e
Spectrophotometers are able to measure both how much light is absorbed by a solution containing
a dissolved solute, as well as measure how much light is reflected (transmitted) through the
solution. Within the spectrophotometer, white light derived from an internal light source is
separated into light of different wavelengths (and hence different colours) by passing the white
light through a prism. The spectrophotometer can be programmed or set to select a particular colour
of this separated light (a specific wavelength of light energy) to illuminate a cuvette (or spec-
appropriate test tube) that is best for the solution of interest. When light of the selected wavelength
is passed through a cuvette (or test tube) containing a sample of your solution of interest, some of
this incident light will be absorbed by the dissolved solute while the rest of the light will be
transmitted through it. The more diffused solute present in your solution (i.e. the higher the
concentration of dissolved solute), the greater the absorbance of the selected wavelength will be.
Light that is transmitted through the sample cuvette (or tube) is detected by a detector and
converted to an electrical signal. The strength of this signal can be measured and displayed on a
meter. The ratio of transmitted light to incident light is referred to as transmittance (T) and is
expressed as a percentage. The negative log of T is referred to as absorbance (A) (Figure 3). Your
spectrophotometer can be set to read either T or A. For the purposes of our labs, we are concerned
with absorbance values as they allow us to quantify molecular responses in the biological processes
we will be examining in several labs.

cuv
ett
Light moving through solution e
Light that is transmitted through
being “absorbed” the solution (Transmittance)

Absorbance of light

Figure 3. Schematic to illustrate principles of spectrophotometry and the difference between

absorbance and transmittance.

Every molecule absorbs light of certain wavelengths, so for each solute of interest we choose the
wavelength that will be absorbed more strongly than others in our quantitative analyses. For example,
you will use absorbance values in the cell membrane permeability lab to assess cell membrane
disturbance by quantifying the leakage of the purple pigment betacyanin (the absorbance of light
caused by betacyanin suspended in solution), from beet tissue. For the examination of factors affecting
enzyme activity, you will be able to quantify amounts of evolved oxygen, an end product in the enzyme
catalyzed reaction using peroxidase.

In order to ensure accuracy when using the spectrophotometer you must allow for the possibility that
molecules other than the ones of interest may be present in your solution and that these molecules may
absorb light of your selected wavelength. To overcome this problem, a blank containing your solvent
(water, buffer, etc.), and absent of the molecules of interest, are used to “zero” the spectrophotometer.

40 | P a g e
Recordings of absorbance are expressed as A (wavelength used) = x. If for example, a wavelength
of 530 nm (1 nm = 10-9 meters) was used, it would be expressed as A530 = x. Note: absorbance
has no units, it is merely expressed as a number (see below for further explanation).

A linear relationship exists between absorbance and concentration for dilute solutions at a specific
wavelength. This relationship is known as Beer's Law and, while you need not concern yourself
with this (i.e. no, it won’t be on your test!), it is given by the equation:

A = εbc, where:

A = the absorbance (measured)

ε= the absorptivity constant (unique for each substance)
b = the thickness of the sample (determined by the width of the sample cuvette and usually 1 cm)
C = the concentration of the absorbing substance.
[With respect to units: A = εbc = (M-1·cm-1)(cm)(M) therefore, A is unit-less]

In today’s exercise we will be using food colouring to prepare solutions of known concentrations
(you will calculate these), to determine the optimum wavelength for absorbance readings. We
will then collect absorbance data to generate a standard curve; a graph which displays the
relationship between two variables, or, quantities (Figure 4). A standard curve uses a set of
known values or standards (eg. concentrations) to determine a concentration of an unknown.
Such a curve can be used to determine the concentration of an unknown solution. Standard curves
can also be used to calibrate instruments when known concentrations have responding readings of
previously accepted values.

1.2
1.1
1
0.9
0.8
Absorbance

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0 ?
0 0.2 0.4 0.6 0.8 1 1.2
Concentration (mg/ml)

Figure 4. Example of a standard curve generated by measuring the resulting absorbance values from
glucose solutions of known concentrations.

41 | P a g e
Application: Using the above figure, roughly determine the concentration of an unknown solution that
yields an absorbance value of 0.6. Concentration =___________ (don’t forget to include units)

Exercise 1. Generating standard curves using known concentrations (to determine the
concentration of an unknown)

1. Turn on your spectrophotometer and set it to the appropriate wavelength for your chosen colour
of solution. **DO NOT OPEN CHAMBER WHILE SPEC IS WARMING UP **

OPTIMAL WAVELENGTH FOR ABSORBANCE for:

yellow: 420nm blue: 630nm
red: 521nm green: 415 nm
Obtain 15-20 mL of one of the stock dye solutions in a graduated cylinder or beaker.

2. Acquire 5 small, clean, test tubes and label them 1 to 5. Add the indicated amounts of stock
(i.e. 100%) food colouring solution and distilled water as indicated in Table 1 to each tube. First
you will have to calculate the volumes (a sample calculation is below).

Sample preparation
In preparing our solutions, we want to keep the total volume constant at 5 ml. For your first calculation,
your initial ‘concentration’ is 100%, or C1= 100. C2 is the concentration you need to make (i.e.
90%) and your final volume will be 5ml. So you need to solve for V1 (to determine how much of the
stock solution do you need to add, and how much distilled water).
Example: C1V1 = C2V2
100(x) = 90(5)
x = (90(5))/100
x = 4.5 ml
So you would add 4.5ml of stock solution and 0.5 ml of distilled water to make 90% solution.

Table 1. Table for % solution preparation using the formula C1V1=C2V2. All tubes contain the
same final volume of 5 ml.
distilled
Test stock dye water Concentration (expressed
tube (ml) (ml) Absorbance as %)
1 0 5 (0%) Blank (use to zero spec)
2 25%
3 50%
4 75%
5 5 0 100%

42 | P a g e
3. Use the appropriate micropipette (the P1000), to carefully measure the determined volume of
each stock solution and water. After adding, mix the contents to prevent layering.
4. Wipe tube 1 with a Kimwipe to ensure it is clean of finger prints, etc. and place it in the
spectrophotometer. Zero the spectrophotometer. Remove your blank.

*PLEASE NOTE: A BLANK IS NOT THE SAME AS A NEGATIVE CONTROL!*

5. Place Tube 2 in the spectrophotometer and record the resulting absorbance and repeat with each
subsequent tube. You may choose to blank the spectrophotometer between each reading.

6. Use the data you have collected in Table 1 to make a standard curve on the graph paper provided
(do not forget an appropriate, inclusive, figure-heading).

Using the Standard Curve to determine the concentration of an UNKNOWN solution.

7. Now determine the absorbance of your unknown (pour ~5 mls of ‘unknown’ of the same colour
you used to make your standard curve, into a test tube and record the absorbance).
**Use the standard curve to determine what % solution it is.**

Figure ________________________________________________________________________
________________________________________________________________________
NOTE: label your figure appropriately. REMEMBER our spectrophotometers only read up to a maximum of 2 (and
2 is not a valid measure), so you may spread your y-axis up to this value (if necessary). Your DEPENDENT variable
goes on the y axis, and your INDEPENDENT variable goes on the x axis

Now to throw you a “Curve ball” (pun totally intended); determine the concentration of
your unknown WITHOUT using your figure! Rather, just using the formula for the slope

43 | P a g e
of a line! y= mx+b. You know that absorbance is your (y) value, and you want to determine
the concentration of the "U" (x). If we assume that b = 0, because the tube with 0%
concentration was used as "blank, then the equation will be y = mx. To solve for x (concentration
of U) you must first calculate the slope "m" in x=y/m. m= rise/run, or more appropriately:
m= y2-y1
x2-x1

Determine the slope of the line using your data, then calculate the concentration of the
unknown (solve for x). Does it match what you extrapolated from your figure?

Serial dilutions
Serial dilutions are dilutions which greatly amplify the dilution factor between each step. Each
dilution prepared comes from diluted material of the previous solution. An application of this
technique is used in microbiology. Microbiologists often need to see colony characteristics to
identify a bacteria. A culture of bacterial cells growing in a nutrient broth may contain millions
of cells and as such, plating of such a concentrated solution would not allow such a visualization.
By removing 1 ml of this dense nutrient stock and placing it in 9 ml of sterile water, a 1:10
dilution factor has been produced. The next step would be to take 1 ml from the 1:10 dilution
stock and add that to a new sterile tube of 9 ml water, creating a 1:100 dilution. Repeating this
again would yield a 1:1000 dilution and this may then be plated to see individual colony growth
(Figure 2).

1ml from stock 1ml from stock 1ml from stock

broth in (a) broth in (b) broth in (c)
pipetted to 9ml pipetted to 9ml pipetted to 9ml
of sterile water of sterile water of sterile water
in tube (b). in tube (c). in tube (d).

a. stock b. 1:10 dilution c. 1:100 dilution d. 1:1000 dilution

Observable
Undifferentiated colony growth
bacterial growth from 1:1000
plated from dilution of
undiluted stock original stock
broth. broth.

Figure 2. Application of serial dilution techniques in microbiology.

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Exercise 2. – preparing serial dilutions
Prepare a series of serial dilutions using the same food colouring stock you chose for the previous
exercise (to compare the huge difference in dilution factors).
Procedure (summarized in Table 2):
1. Obtain 5 clean test tubes. Place 5 ml of stock solution (100%) in a test tube labelled 1.

2. Micropipette 0.5 ml of stock solution from tube 1 and place it into 4.5 ml of distilled water.
Agitate the tube to mix the contents completely. This is tube 2.

3. Micropipette out 0.5 ml of the contents of Tube 2 and place it into Tube 3 and add 4.5 ml
of water.
4. Repeat the above procedure for one more dilution into Tube 4 (Tube 5 is just water (why?)).

5. Set your spectrophotometer to the wavelength appropriate for your chosen colour.

6. Place tube 5 in the spectrophotometer and use it to zero/blank the spectrophotometer.

7. Wipe tube 1 with a kimwipe and place it in the spectrophotometer and record absorbance.

8. Repeat step 8 for the subsequent serial dilutions (tubes 2 through 4). You may choose to
blank between each reading. List the dilutions you have prepared.

Tube 1 - (100%) (concentrated stock)

Tube 2 – 1:10 dilution or % (how might you determine this?)
Tube 3 - 1:100_______________
Tube 4 - ____________________
Tube 5 – 0% (blank)
Table 2. Preparation of serial dilution summary table with each tube at a final volume of 5ml.

Test Concentration distilled water

Dilution Stock (ml) Absorbance
tube (expressed as %) (ml)

1 1:____ 100 0 Stock dye: 5

2 1:____ 4.5 Tube 1: 0.5
3 1:____ 4.5 Tube 2: 0.5
4 1:____ 4.5 Tube 3: 0.5
5 1:____ 5 0

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Graph your results (from lowest concentration to highest) on the graph paper provided. Remember
to label your axes and give your figure an appropriate figure heading.

Figure ________________________________________________________________________
________________________________________________________________________

Curve ball! Once again place the provided unknown in your spectrophotometer and
record the absorbance. Determine the dilution of the unknown using the slope
formula. Place your work below.

Now determine it through extrapolation on your figure. The values should match.

46 | P a g e
 Instrumentation Prelab

1. Micropipettes typically measure very small volumes measured in units called

___________________

2. There are _________ ml in 1 l (litre). There are __________ μl in 1 ml.

3. A solution is comprised of two parts: __________ and ____________.

4. In all our spectrophotometer exercises in today’s lab, we will use a cuvette or test tube
containing only water. The contents of this tube is referred to as the __________. This is used to
make sure the spectrophotometer is only measuring the absorbance of _______________.

5. If 1 ml = 1000 μl, how many ml is 456 μl? ______ ml.

37 μl = ________ ml. 9 μl = _________ml. 1927 μl = ______ml.

3.241 ml = ______ μl. 0.647 ml = ______ μl. 0.012 ml = ________ μl.

6. What is a standard curve?

7. When graphing standard curves today, your dependent variable (i.e. absorbance) goes on the
_______ axis, while your independent variable (i.e. concentration) goes on the ______ axis.

8. A set of dilutions prepared from the diluted material of the previous dilution are called
_____________ dilutions.

9. Would you use a P1000 to pipette 73 μl of solution? _____ If not, what would you use instead?

10. What is the maximum value our spectrophotometers are accurate to for measuring absorbance
(i.e. values above _____ are not recorded).

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 Microscopy II – Cell Structure and
Function of prokaryotic and eukaryotic
cells
Required Readings: (Fenton et al., 2022), (Ch 2: pp 31-46; 50-56 & look up gram-staining bacteria
as well!), Appendix D/E.

Key Terms: Prokaryote, eukaryote, cell membrane/plasma membrane, cell wall, cytoplasm, nucleus,
nucleolus, nuclear envelope, mitochondrion, chloroplast, amyloplast, ribosome, endoplasmic
reticulum (ER), rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi
apparatus, centriole, centrosome, cytoskeleton, lysosome, vacuole, vesicle, cilia, flagellum.
selectively permeable, passive transport, Brownian movement, diffusion, osmosis, solute,
solvent. Petri dish, agar, potassium permanganate, methylene blue, molecular weight. Thistle
tube. Tonicity, hypotonic, hypertonic, isotonic, turgor pressure, lysis, hemolysis, plasmolysis,
crenated, turgid, flaccid. Cytology, histology

Objectives:
• Explain the significance of the cell as the fundamental unit of life.
• Identify eukaryotic cell (plant and animal) organelles and describe their function.
• Identify prokaryotic cells and be able to differentiate gram positive from gram negative cells
• distinguish between prokaryotic and eukaryotic cells
• Define and describe the processes that mediate molecular exchange across a membrane.
• Understand the effect of molecular size, and other variables, on diffusion rates.
• Define and explain the effects of isotonic, hypertonic and hypotonic solutions with respect to
red blood cells and plant cells, cytology versus histology
• Identify red blood cells in an isotonic, hypertonic and hypotonic solution.
• determining the appropriate magnification power for viewing cells and tissue
• draw appropriate diagrams from a slide, including proper labeling of scale and size
• prepare legends for all figures that you will be producing in this lab

PART A – CELLS – Cell structure and function

INTRODUCTION
To refresh your memory of the taxonomy hierarchy you can review the figure below (Figure 1).
Within the 3 main Domains (the classification category above Kingdom) there are two main cell
types: i) prokaryotic (which are found in Archaea and Bacteria) and ii) eukaryotic (within
Eukarota). Prokaryotic cells evolved before eukaryotic cells and lack membrane-bound organelles
(‘pro’= before; ‘karyon’= kernel or nucleus). Prokaryotic DNA is concentrated in a region within the
cell called the nucleoid region. Prokaryotic cells lack most types of organelles that are found in
eukaryotic cells (Table 1). This does not mean that the cells do not carry out the functions performed
by the eukaryotic organelles, but rather the function simply occurs within the cytoplasm or cell
membrane. For example, prokaryotes have genetic material but it is not packaged within a nuclear

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membrane. Some prokaryotes have respiratory enzymes but they are not assembled into
mitochondria. Some prokaryotes have chlorophyll but it is found in the folds of the cell membrane
rather than in the membrane of the chloroplast. Prokaryotes exhibit a distinct lack of
compartmentalization both structurally and functionally.

Figure 1. Taxonomy hierarchy. The ‘example’ shows the classification information for the
Paramecium you will be observing in lab.

Eukaryotic cells (‘eu’=true; ‘karyon’= kernel or nucleus) have membrane-bound organelles, and
specifically a nucleus. The region between the nucleus and the cell membrane, the cytoplasm, consists
of a semi-fluid medium called the cytosol, in which organelles (tiny structures of special form and
function) are suspended. Tables 1 lists the differences between eukaryotic and prokaryotic cells and
differences between plant and animal cells.

The cell is considered the most basic unit of life and cell structure is similar throughout the eukaryotic
kingdoms. Eukaryotic organisms differ first in the number of structures in the cells, and also in the
kinds of structures present in cells, as well as from the differences in the number and type of cells
making up the organism. The theoretical implications of this fact is that all organisms are related
through evolution from a common ancestor. The practical implications are that facts about the structure
and function of typical cells usually apply to all organisms. Each organism is made up of cells
displaying a remarkable diversity of forms. They can live as single cells or as more complex organisms,
such as plants and animals. Their bodies are a cooperative of many specialized cells that could not
survive for long on their own. However, even when they are arranged into higher levels of organization,
such as tissues and organs, cells can be singled out as the organism’s basic units of structure and
function. You can use your textbook, the models (and their keys) in the lab, and the table below to
review the various organelle functions then use Table 2 to summarize what you have learned.

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Table 1. Comparison of organelle and cell component structure in animal and plant cells.
Structure Composition Primary Function
Cell membrane Phospholipid bilayer with Passage of molecules in and out
embedded proteins of cell
Cell wall (plant) Contains cellulose fibrils Support and protection
Centriole (animal) 9+0 microtubule pattern Forms basal body
Chloroplast (plant) Grana within inner and outer Photosynthesis
membrane
Cilia and flagella (animal) 9+2 microtubule pattern Cell movement
Cytoskeleton Microtubules/microfilaments and Cell shape and movement of its
intermediate filaments parts
Endoplasmic reticulum Membranous flattened channels Synthesis of proteins and other
(ER) and tubular canals substances, and transport by
vesicle formation
Rough (ER) Studded with ribosomes Protein synthesis
Smooth (ER) Ribosomes absent Lipid synthesis, drug and poison
detoxification
Golgi apparatus Stack of membranous saccules Processing, packaging, secretion
of proteins, and carbohydrate
synthesis
Lysosome (animal) Membranous vesicles housing Intracellular digestion
digestive enzymes
Mitochondrion Inner cristae with outer membrane Cellular respiration
Nucleolus Area of chromatin, RNA and Ribosome formation
proteins
Nucleus Nuclear envelope housing the Cellular reproduction and protein
nucleoplasm, chromosomes, and synthesis
nucleoli
Ribosomes Protein and RNA in 2 sub-units Protein synthesis
Vacuole and vesicle Membranous sac Storage of substance

Table 2. Comparison of prokaryote and eukaryote (plant and animal) cell composition.

Structure/Organelles Prokaryotic Eukaryotic

Animal Plant
Cell membrane
Cell wall
Centrioles
Chloroplast
Cytoskeleton
Endoplasmic Reticulum (ER)
Lysosomes
Mitochondrion
Nuclear envelope
Ribosomes
Vacuoles

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 Plasma membranes – structure and function (transport mechanisms)
A cell is dependent on its environment. It is continually exchanging gases, nutrients, and wastes
between intracellular and extracellular compartments. Every cell is surrounded by a selective
boundary (plasma membrane) that lets certain ions and molecules enter the cell while keeping
others out. Inside the eukaryotic cell, most organelles are surrounded by a membrane that is
composed of the same constituents as the plasma membrane. This allows for compartmentalization of
the cell with each organelle having a specific function and a specific environment in order to carry
out its task. Because a membrane is selectively permeable, it regulates and maintains internal cell
conditions. Therefore, what can be exchanged and how the exchange occurs is largely determined by
properties of the plasma membrane and the structures involved.

Exchange across a membrane can occur by several methods: i) simple diffusion - movement of
substances without energy expenditure, ii) carrier-mediated transport - movement of substances by
specialized proteins (note that this may be passive or active transport, but in both circumstances a
trans-membrane protein is involved), and iii) bulk transport - movement of very large substances
which involves interaction of the cell membrane and vesicles. This lab focuses on the passive
transport mechanisms of diffusion and osmosis (use your textbook to learn about the latter two on
your own).

Passive transport
All molecules and ions are in constant motion. The constant random high-speed bouncing of individual
molecules in solution resulting from kinetic (heat) energy is called Brownian movement. Motion is
directly related to temperature and inversely related to particle size. Thus the higher the temperature
and the smaller the molecule, the more rapid the molecular motion. At absolute zero molecular motion
is believed to stop altogether.

Diffusion is the net movement of molecules and ions with the concentration gradient (i.e. from
regions of higher concentration to regions of lower concentrations). Diffusion is the result of the
constant random motion of all molecules and is one way that substances can move across membranes.
It occurs rapidly in gases, more slowly in liquids, and extremely slowly in solids. Diffusion is
temperature dependent, occurring more rapidly as temperature increases. It is also dependent on
molecular size. Small molecules or ions diffuse more rapidly than larger ones.

Osmosis is a special case of diffusion of a solvent (usually water in living systems) in response to a
non-penetrating solute. The solvent diffuses across a selectively permeable membrane from a dilute
solution (high water concentration) to a more concentrated solution (low water concentration).
Osmotic pressure is the amount of force it takes to oppose osmosis and can be related directly to the
concentration of the non-penetrating solute. The greater the concentration of non-penetrating solute
the lower the concentration of solvent (water) – the greater the movement of water by osmosis from
dilute solution to concentrated solution – the greater the opposing pressure to stop the osmotic flow.
Therefore, a solution with a high solute concentration has a greater osmotic pressure than a solution
with a low solute concentration. The selectively permeable membrane allowing only certain substances
to cross is essential for osmosis to occur.

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Diffusion
Simple diffusion is the result of the constant random motion of all molecules. It occurs rapidly in
gases, more slowly in liquids and extremely slowly in solids. Diffusion is also dependent on
temperature and the size of the molecule.
Many factors can affect rates of diffusion. Think for example, about how quickly the sugar dissolves
in your hot tea or coffee, however, on a hot summer day, when you try to stir sugar in to your iced tea
it takes much longer and requires mechanical energy (stirring).

Exercise 1 – Class demonstration -Effect of Molecular Size on Rates of Diffusion.

Compare the rates of diffusion of two ionic substances with different molecular weights through an
agar medium.
1. On one petri dish containing agar, drop a tiny crystal of potassium permanganate (or use KMnO4
solution if provided in liquid form).
2. On another petri dish containing agar, drop a tiny crystal of methylene blue (or drop of solution).
3. After 1 hour observe the diameter of the diffusion ring in each plate.

How does molecular weight affect diffusion rate?

_________________________________________________________________________
_________________________________________________________________________
Sketch differences in observations as a visual reminder of the experiment performed.

Potassium permanganate methylene blue

Exercise 2 – Class demonstration - Osmosis – Thistle tube experiment –

NOTE – This activity does not appear in your virtual lab. But will be set up for the in-
person lab.

To demonstrate osmosis, the bulb of a thistle tube is filled with sugar solution (or molasses).
The lower opening of the thistle tube (or other tubing) is then covered with a selectively permeable
membrane (dialysis tubing). At the beginning of the lab, the tube will be lowered into a beaker of
distilled water so that the membrane is submerged. Mark the height of the molasses/sugar solution in
the column with a grease marker.
After 45 minutes examine which way the solution height in the column has moved.
Did the column height increase or decrease? Why? Briefly explain what caused the column of sugar to
change height. (Space to sketch thistle tube set up on the next page).
_________________________________________________________________________
_________________________________________________________________________
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_________________________________________________________________________
_________________________________________________________________________
Space to sketch thistle tube set up

Tonicity
Osmosis is affected by tonicity and we will study its effects on osmosis using red blood cells and
red onion cells. Tonicity refers to the concentration of solutes on one side of a membrane relative to
the other side of the membrane (for the purposes of this lab, the concentration of solutes in a cell
relative to that found in the solution the cells are placed in). Red blood cells, like other body cells, are
in osmotic equilibrium with the blood plasma. That is, the solute concentration inside the cells is about
the same as the solute concentration outside and is therefore said to be isotonic. If a cell is placed in a
hypotonic solution (less solute than found in the cells) such as distilled water, water enters red blood
cells by osmosis and causes them to swell and burst, or undergo lysis (hemolysis if referring to blood
cells). A plant cell on the other hand has a cell wall therefore it reaches a state of maximum turgor
pressure. Conversely, if a red blood cell is placed in a hypertonic medium (more solutes in the solution
than found in the cells), it shrinks (water is drawn out by osmosis) and the cell margins appear scalloped
(crenated). Plants cells in this situation have decreased turgor pressure (or become flaccid).

Physiological saline used intravenously, for example, is 0.9% NaCl because this is isotonic to human
red blood cells. If a person were to receive an intravenous solution of 10.0% NaCl however, this would
have a drastic effect on cells. A salt solution greater than 0.9% salt would be hypertonic to the cells,
causing them to lose water, (crenate). A solution of distilled water (100% water) would be hypotonic
to animal cells and cause cells to undergo lysis (if blood cells: hemolysis). Animal cells are typically
isotonic with their environment, or have developed elaborate mechanisms to maintain homeostasis
with their environment (paramecium for example, have contractile vacuoles to expel excess water
which moves into their bodies which are living in fresh water environments).

Exercise 3. Effect of osmoticity on red blood cells (sheep blood)

Animal red blood cells are in osmotic equilibrium with blood plasma. Therefore, the solute
concentration inside the cells is about the same as the solute concentration outside. Animal body
fluids have a salt (NaCl) concentration of 0.9%.

This initial set up will be done by your lab instructor:

1. Obtain 3 small test tubes labelled; dH20, 0.9%, and 10%. A drop of blood will be placed in the
bottom of each tube, followed by 3 ml of the indicated distilled water or NaCl solutions.
YOU will:
2. Hold the prepared test tubes to the light or over the writing on your page and observe whether they appear
transparent (like cranberry juice), or opaque (like tomato juice). Give explanations for the appearance of
each (use appropriate terminology).

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dH20_________________________________________________________________________________

_____________________________________________________________________________________

0.9%NaCl____________________________________________________________________________
_____________________________________________________________________________________

10%NaCl ____________________________________________________________________________
_____________________________________________________________________________________

Exercise 4. Microscopic view of the effects of tonicity on red blood cells

Prepare a wet mount for each tonicity of solution.

1. Start with 0.9% NaCl and place 1 drop onto a slide (this will allow you to observe cells under
“normal” conditions). Next, just touch the dropper from the red blood cells bottle to the slide beside
your water drop (do not squeeze a drop out - less is much better), and do not touch the dropper
to your water drop as this will contaminate the dropper (figure 1). Alternatively you could add a
small amount of blood to the slide using a toothpick. As you lower your coverslip over the water at
a 45 degree angle, the water will move over the blood. Release the coverslip and it should land with
minimal air bubbles (though air bubbles sometimes offer a nice point of reference for focusing).
0.9% NaCl blood

a. how to place drops b. lower cover slip to draw solution over the blood
Figure 1. Schematic of how to set up your slides. In (a) the blue dot is your salt solution, red is blood.

2. Examine the cells under the compound microscope and draw what you observe.
3. Repeat this procedure with 10% NaCl and a fresh slide (or, try adding 10% NaCl to the edge of the
original slide and see if you can observe the crenation of those cells). Below, sketch the appearance
of the cells and give the correct scientific term that describes the appearance. Be prepared to be able to
replicate and/or interpret such an experiment on your lab final.

Tonicity:
ISOTONIC __HYPERTONIC__

________________________________ ____________________________
________________________________ ____________________________

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4. Why didn’t we ask you to draw cells placed in dH2O? ___________________________________
_________________________________________________________________________________
Exercise 5. Plasmolysis in a red onion
Plant cells store secondary metabolites within their cells interior (often within the central vacuole)
making the interior hypertonic relative to their environment, which causes water to enter their
cytoplasm. When plant cells receive adequate water, (the cell wall restricts excess water from entering),
it moves in to the central vacuole and cells are considered turgid. Consider the spray systems in
grocery stores, which constantly spray freshwater on produce to create a hypotonic external
environment which keeps plant tissue looking supple and fresh as cells reach their maximum turgor
pressure. Plant cells that are isotonic to their environment tend to be limp. Sprinkling salt on unwanted
“weeds” is an effective weed control because in this hypertonic environment the plasma membrane
collapses from the cell wall and the cells shrivel as water is pulled from the central vacuole; this is
called plasmolysis or decreased turgor pressure.

1. Prepare an epidermal peel (as you did in lab 1), by first placing a drop of distilled water on a
slide.
2. Carefully slice red onion tissue with a razor blade and gently (twisting) while you peel back
the tissue with forceps or your finger nail to obtain a thin layer (single cell layer) of the
epidermis (IMPORTANT: make sure to get the red pigment containing cells).
3. Add the cover slip at a 45 º angle (at this point you may need to add a little more water to the
side of the cover slip and let it diffuse in).
4. Place the slide on the microscope (be sure to close the iris diaphragm) and focus your slide.

Describe what you see (what is the appearance of the central vacuoles/distribution of red pigment
within the plant cells)? _______________________________________________________
_________________________________________________________________________
_________________________________________________________________________
Include a rough sketch below.

***The next slide you prepare is to be drawn with attention to detail and scale as part
of your histology drawings ( space for drawing is at end of lab on page 38).***

5. Place drops of 10% NaCl on the same slide you used previously beside/touching the edge of
the coverslip and allow it to diffuse across the slide by drawing the salt solution with a tiny piece
of paper towel touching the opposite edge of the coverslip. Observe the cells again. Describe
what is happening using proper terminology. Draw the cells after NaCl has been added on pg 38.

Describe what you see (what is the appearance of the central vacuoles/distribution of red pigment
within the plant cells)? _______________________________________________________
_________________________________________________________________________
_________________________________________________________________________

Exercise 6. Class demonstration - Effect of osmoticity of potato strips

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Your lab instructor will:
1. Cut two strips of potato using a cork-borer and remove the outer epidermis from each end.
2. Mark two test tubes 1 and 2.
3. Place one potato strip in each test tube.
4. Fill tube 1 with enough distilled water to cover the potato strip.
5. Fill tube 2 with enough 10% NaCl solution to cover the potato strip.
6. After 30-45 minutes examine both strips. Record and explain your resulting observations using
appropriate terminology.
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
Sketch results below- draw your potato strips appearance in the test tube images below.

Tube 1 Tube 2

PART B – Cytology & drawing cells

INTRODUCTION
Cytology is the study of both the form (i.e. anatomy), and the function of plant and animal cells.
Interestingly, the term cell, which means ‘tiny room’ itself dates back to the 1600’s, when Robert
Hooke used it to describe plant and animal cells, looking similar to the tiny rooms or cellula that monks
lived in, or the cells of a honeycomb. The importance of cytology in medicine and pathology has been
paramount to the understanding and diagnoses of cancers through viewing abnormalities in cells.
Similarly, histology, the study of tissue, is equally important in diagnosing many different disease, as
cells seldom exist or function alone. Both cytologists and histologists are trained to be able to identify
microscopic views of plant and animal cells/tissue, in a normal and healthy states as well as in an
abnormal state.

Prokaryotic cells
Domain Bacteria are divided based on whether they are Gram-positive, or Gram-negative. This
is an example of diagnostic staining. A Gram stain (named after the techniques developer Hans
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Christian Gram) indicates something about the cell’s molecular composition. If you reviewed your
text book you know that prokaryotic cells can have very different cell wall compositions and as
this wall is its means of protection, this composition can determine how virulent a bacteria may
be. Many bacteria have capsules comprised of polysaccharides which creates a slime-layer around
the cell wall, allowing them to stick to substrates or protects them from a hosts’ immune response.
Gram stains specifically look for the cell wall component peptidoglycan and differentiates Gram-
positive bacteria from Gram-negative bacteria. Gram-positive bacteria have a thick mesh-like
layer of this polysaccharide peptidoglycan. Gram-negative bacteria have a thick lipid-layer
external to a much thinner peptidoglycan layer. The medical significance of this is that this lipid
layer often protects the bacteria from host immune response, making them more virulent.

Exercise 1. Examining prepared slides Using the microscope to study prokaryotic cells such as
bacteria will require even more fine-tuned microscopy skills and we will again being using the
immersion oil. You may want to refer back to Lab 1 (or your appendices) on the proper technique
for using this oil so that you do not damage the microscopes. Remember that ONLY the
Immersion oil lens (1000X) can be used with oil as none of the other lenses are sealed.
Further, use of this lens without the oil will likely damage the lens.
If the circle below represents your field of view, how big would a single cell look within it?
State the Genus species (i.e. Latin name) of a slide you determined to be gram positive:
______________________________________
State the Genus species (i.e. Latin name) of a slide you determined to be gram negative:
______________________________________

Draw just a few to scale as best as you can in the circle below, then move on to the next
activity.

Drawing cells (with a focus on eukaryotic cells)

The larger focus of this portion of the lab is on preparing adequate drawings of microscopically viewed
specimens. No, this is not an art class, but being able to produce detailed and meticulous, hand-drawn
diagrams of specimens that are to scale, will help solidify your understanding of the material being
presented today, aiding in future study both within this course, and in future courses (i.e. organelles
within plant versus animal cells, microbiology, anatomy and physiology, and histology to name but a
few). Do take your time and pay attention to detail (scale, visible cell features, etc.)!

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At an individual cell level, the shape of the cell tends to match its function. Today one of the slides
you are making is an epithelial cell suspension slide. Epithelial cells are simple squamous epithelial
cells. These cells are found lining lumen throughout your body and comprise the respiratory
membranes of your lungs. The best way to imagine them is as microscopic fried eggs! They are 1/10th
the thickness of a piece of tissue paper. What processes might being so thin facilitate?
_________________________________________________________________________________

Other epithelial cells, such as simple cuboidal cells, are quite supple-looking, and often line excretory
vessels (think of them as the ‘squirtie’ cells). Muscle cells are long and packed with cytoskeleton.
Think on why this might be.

The figure below illustrates how much smaller prokaryotic bacterial cells are than eukaryotic cells
(Figure 2a & b).

a. b.
Figure 2. a. 2 human cheek cells (at 1000X -each cell is roughly 60µm in size). b. staphylococcus
bacteria (1000X- each cell is roughly 1-2 µm in size). Photo credits to Carolyn Ly

When doing histology drawings from microscopes:

- DO give a title of what the drawing is of, and state total magnification
- DO include scale bars in field of view diagram (i.e. “1cm=_______ μm” or mm)
- DO NOT use arrow heads to label your drawings, a simple line will suffice and labels/descriptions
of indicated features should be outside the drawing field of view (the circle).
- If you use a stain (methylene blue for example), state that. (“Methylene blue stained onion cells…”)
- For each cell type you will be calculating the size of a single cell. Show your work/how you
determined it.
-delineate cellular boundaries distinctly, ideally with a pencil/ coloured pencil crayon and
highlight cell characteristics that are distinguishing to the cell (not ‘grunge’ or anomalies such as
remnants of the lettuce you had in your sandwich). Again, this can be done in colour or grayscale
depending on what is appropriate.

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Sample calculations for Field of view and scale calculations.

Below are human red blood cells observed in the field of view for a compound light microscope at
1000X (total magnification) (Figure 3).

Figure 3. Microscopic view of red blood cells at 1000X on a compound light microscope.

1. First we need to determine the size of the field of view at 1000X.

To do this you must get a ruler and determine the field of view at scanning power (40X-total
magnification). Remember you can only use the ruler this way on 40X.

In this microscope the field of view is determined to be 7.2mm (far larger than what your own field of
view will be).
If we are calculating for calculating for oil immersion as indicated above, we use 1000X as our Maghi.
So remember: FOVlow X Maglow = FOVhi X Maghi
We fill in and solve: (7.2mm)(40X) = x (1000X)
=0.288mm field of view (FOV) at 1000X, or, more appropriately, 288µm.

2. Now to determine the size of one cell you need to determine roughly how many cells across fill
your field of view. For something as small as above (or bacteria), you may only want to count for ½
the field of view, or even only 1/3 or ¼ depending on how small the cells are.
For the cells above, I would roughly say 17 red blood cells across ½ the FOV.

288 µm /17 cells =16.94 *0.5 = 8.47 µm is our calculated estimate for 1 red blood cell

The known red blood cell size is actually known to be ~ 9µm so we are ‘close’.

NOTE: You will find your calculated sizes may vary known sizes (example: for cheek cells you should
be ‘near’ 60 µm), and may vary from person to person depending on the plant type you use, or how
wrinkled your cells are.

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Calculating scale bars for your pictures
(Sample calculations continued)

NOTE: You do NOT use your calculated scale bar to determine your cell size, you compare
your scale bar, to your drawing, to determine if your drawing is to scale. You calculate cell size
as above (determining number of cells across your field of view).

You draw the blood cells (I will only draw a portion here because I am lazy (you can’t be!)☺).

Magnification
1000X

From the previous calculations we

determined the FOV at 1000X = 288
µm,

The distance in cm, across the drawn circle is 9cm SO: 288 µm /9cm = 32 µm (per
cm).

Below the 1cm bar you

then put =32 µm

From this I can see that if 1cm = 32 µm, then I should be able to fit nearly 4 of my little red blood
cells across that bar (if I’ve drawn it to scale properly). In this instance I would say it has been
drawn to scale.

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Exercise 2. Cytology drawings
Wet mount preparation for provided mammalian epithelial cells
1. Place a drop of the 0.9% NaCl solution onto the center of a slide.
2. Obtain a toothpick and using the wide end, gently scrape any dryer portion of your skin tissue
to obtain some epidermal cells (the skin on your arm usually works well). Discard toothpicks
(and later, slides) in the biohazard buckets provided (these will be pointed out in lab).
3. Smear the scraped cells on your toothpick end in the water on your slide and stain with
methylene blue (directly beside the water so not to contaminate the dropper).
4. Place the cover slip on the two separate drops which will cause them to merge.
5. Diagram TO SCALE, a few of the best looking (i.e. shows cellular characteristics most
clearly) observed cells at immersion oil (1000X).
6. Include a title, scale bar, the magnification, and label the nucleus, cytoplasm and cell
membrane.
7. Calculate the size of a single cell in µm. Show your calculations at the bottom of the page.

Epithelial cell slide

Total magnification __________
(include scale)

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Exercise 3. Red onion epithelial cells in 10% NaCl solution. (Use the slide you prepared in Part A
of exercise 5).

Sketch a single cell layer with attention to scale, what you see at 400X magnification, labeling your
drawing with as much detail as possible (you should delineate cell wall, and the central vacuole at
minimum).
Calculate the width of a single onion cell in µm. Show your calculations at the bottom of the page.

Total magnification __________

(include scale)

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 Microscopy II Prelab

1. Molecules that are large/heavy will diffuse ____________ (faster/slower) than lighter ones.
2. In this lab, a thistle tube is to be filled with molasses, covered with dialysis tubing, and
inverted into a beaker of water. This experiment is studying the process of
____________________.

3. A solution is said to be isotonic to red blood cells if it contains ______________ % solutes.

4. Would a solution that is isotonic to blood cells necessarily be isotonic to plant cells as well?
Why/why not?
______________________________________________________________________________

5. If blood is added to a test tube containing 10% NaCl solution, the cells would
______________ (crenate/hemolyze) due to the solution being ______________ tonicity to
the cells.

6. The cells of potato strips placed in 10% salt will undergo ____________, causing the strips to
appear ‘shriveled’.

7. Histology is the study of _________________ while cytology is the study of

_________________.

8. If the FOV at 400X works out to be 0.4mm, and 1 plant cell takes up 1/4th the field of view at
this power, how big is one cell (in µm)? (Use the sample calculations in the lab and show your
work below).

9. If you drew what you were viewing in question 8 onto paper in a 10cm circle, your 1 cm scale
bar would represent _____ µm. (Use the sample calculations in the lab and show your work
below).

10. Prokaryotic cells that have been gram stained represent a _________________ stain, where
cells containing large amounts of peptidoglycan in their cell wall will stain __________ (colour),
and are therefore gram ______ (+ or -). Conversely, high levels of mycolic acid produce a
gram____ (+ or - ) result.

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 Animal and plant cell model images to be labeled and used for study purposes.

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65 | P a g e
 Enzymes - assessing the enzymatic
activity of peroxidase
This lab procedure was modified from pages 109-113 of: Pitkin, R. B. 1992. Enzyme
investigations for introductory courses. Pages 107-188, in Tested studies for laboratory teaching. Volume
13. (C. A. Goldman, Editor). Proceedings of the 13th Workshop/Conference of the Association for
Biology Laboratory Education (ABLE), 191 pages.

Required Reading: (Fenton et al., 2022), (Chapter 3 – 59-82)

Key Terms: Enzyme, catalyst, substrate, enzyme-substrate complex, active site, product, hydrolysis,
dehydration synthesis. Peroxidase, rutabaga, hydrogen peroxide, dye-coupled reaction, guaiacol,
mortar, pestle, buffer, centrifuge.

Objectives: -understand the structure and function of enzymes and related terminology (e.g.
activation energy, catalyze, anabolic, catabolic, substrate, end-product)
-to understand the peroxidase-specific reaction
-use standard graphing methods to determine relationships between dependent and
independent variables and determining rates of reactions.

INTRODUCTION
Many biochemical reactions occur within living cells. Almost all of the biochemical reactions are
mediated by cellular enzymes. In most cases enzymes are proteins which act as biocatalysts for
metabolic processes in living systems. Enzymes catalyze (accelerate) reactions by lowering the energy
of activation needed for that specific reaction to occur.

The 3-dimensional structure of an enzyme determines its ability to catalyze a reaction. An enzyme will
bind to the reacting molecule (called the substrate) to form an enzyme-substrate complex. The area
of the enzyme where the binding occurs is called the active site. The enzyme-substrate complex exists
for only a few milliseconds, during which the covalent bonds of the substrate either come under stress
or are oriented in a manner that allows them to be attacked by other molecules. The energy that is
required for this step is called the energy of activation, which is lowered by the enzyme. Reactions
can include splitting substrate molecules into smaller product molecules by the addition of water, or,
hydrolysis (“water splitting”), or piecing substrate molecules together to form a larger product upon
the removal of a water molecule (dehydration synthesis). At product formation, the enzyme is
released and is free to combine again with more substrate molecules.

Often an enzyme will need the assistance of a helper molecule to ensure catalytic enzyme activity.
These molecules may bind tightly to the active site or loosely with the substrate. They are referred to
as cofactors if they are inorganic (e.g., metallic ions Fe3+, Mg2+, Ca2+, or Mn2+), and coenzymes if they
are organic. Typically, vitamins are coenzymes or coenzyme precursors, and like cofactors, function
in a variety of ways to facilitate enzyme catalysis.

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Peroxidase
For today’s lab you will investigate the function of the enzyme peroxidase extracted from rutabaga
tissue using the provided protocol and studying its roll in the catabolism (breakdown) of the substrate
hydrogen peroxide (Figure 1). It is worth mentioning that many enzyme names end with the suffix
“ase” or “in” and are often named for the substrate which they bind with. “Lipase” for example, is an
enzyme that breaks down lipids.

Peroxidase is a large protein containing several hundred amino acids. An iron ion is located in the
active site, which allows conversion of toxic hydrogen peroxide (H2O2) into water (H2O) and oxygen
(O2).

peroxidase
2 H2O2 --------------------> 2 H2O + O2
(substrate) (enzyme) (end products)

Figure 1. The catalytic activity of the enzyme peroxidase. The above equation represents how
enzyme-catayzed reactions are written out (minus the sub-headings below).

The oxygen often reacts with other compounds in the cell, forming secondary products. We can follow
the peroxidase reaction by following the formation of oxygen. We could trap the oxygen in a closed
system that measures the volume of accumulated oxygen, or we could look for evidence of oxygen
chemical activities. We will use the latter method. To do this, a dye-coupled reaction will be used, in
which a dye; guaiacol, that is initially colourless becomes coloured (orangish-brown) as oxygen, an
end-product, reacts with it (Figure 2). Peroxidase is not affected by the dye, but the products of the
peroxidase reactions can be followed by coupling it with this dye (hence the name).

peroxidase
2 H2O2 --------------------> 2 H2O + O2
(substrate) (enzyme) (end products)
clear guaiacol dye orange/brown

Figure 2. The catalytic breakdown of hydrogen peroxide can be monitored using guaiacol dye in
a dye-couple reaction which turns from clear to brown when oxidized (as oxygen is evolved).

To quantitatively measure the peroxidase activity, we need to be able to ascertain the emergence of the
brown colour. To do this, we must mix the enzyme, substrate, and dye in a tube and immediately place
it in a spectrophotometer to precisely measure the amount of light that can is being absorbed as the
substrate breaks down. As the guaiacol darkens, light (with a wavelength of 500 nm) gets absorbed by
the oxidized dye. The rate at which the solution darkens is proportional to the enzyme activity (i.e.
reaction rate).

Effects of concentration on enzyme activity

In a biological reaction, enzyme activity is affected by the concentration of the enzyme relative to its
substrate. If there are many more substrate molecules than enzyme molecules in the reaction, then the
reaction rate is low, since it takes the few enzyme molecules longer to convert the higher number of
substrate molecules to product. In this situation, increasing the concentration of enzyme increases the
reaction rate, since there are now more enzyme molecules to react with the same number of substrate
molecules. However, there is a limit to an increasing reaction rate with an increasing enzyme

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concentration. At some point, all the enzyme molecules are engaged with substrate molecules and
adding more enzymes will not increase the reaction rate.

Cells must regulate the rate at which they carry out most of their reactions. They have many
mechanisms by which they control enzyme activity, but one common way is to control the amount of
enzyme they have in their cells. Cells in different organisms and in different tissues of the same
organism can use different enzymes or even different amounts of the same enzyme, depending on their
metabolic needs.

Normally when running an experiment using biologically active tissue we would need to standardize
for the activity of a given tissue source (i.e. test several concentrations to best characterize activity)
because every preparation of a biological substance results in products of varying quality and purity.
However for this labs purposes (we are only running the experiment for one day) this will not be
necessary. Rather in this experiment we will examine the effect increasing concentrations of enzyme
have on resulting enzyme activity (i.e. reaction rates).

Using rates to illustrate trends

In science, data are often plotted graphically to enhance visualization of trends. The convention is to
plot the independent variable (i.e. what you manipulate) on the horizontal (X-axis) and the dependent
or responding variable (i.e. what you are influencing) on the vertical (Y-axis).

Note that in this experiment, you will be making many measurements for each trial. That is, each trial
consists of a series of absorbance measurements over time (i.e. absorbance change/time = rate). This
will let you see the progress of each trial over time, and that a constant and steady increase often occurs
for each trial which will vary with the concentration of enzyme present. Within each trial, time is the
independent variable and will be used to determine the reaction rate (Figure 3). If a plateau in the
values occurs, you can assume you are no longer watching the reaction progress (for instance, if all the
guaiacol is used up). Data that do not lie along a straight line are not useful for making a general
statement about how “efficiently” the enzyme does its job, and can be legitimately excluded from
further analysis.

The most important part of this study is not the individual time points rather you are going to study a
series of different enzyme concentrations and calculate the rate of enzyme reaction (Figure 3a,b). You
will ultimately be able to plot the reaction rates (calculated from the slopes of the lines you generate
plotting absorbance vs time) on the Y-axis (as your dependent variable) using your independent
variable (the treatment that changed between trials) on the X-axis.

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 0.0025

Reaction rate (abs/time)

0.002
0.0015
0.001
0.0005
0
0 0.5 1 1.5 2
concentration

a. b.
Figure 3.a Using the absorbance versus time data to calculate the reaction rate (i.e. slope of the line)
for the enzyme peroxidase, at three concentrations yields slopes equal to 0.0005, 0.001, and 0.002
which are plotted in b. b. Resulting graph of reaction rates (generated from slope calculations) for the
3 shown concentrations of enzyme extract.
You will orient yourselves to the system for analyzing enzyme activity. You will make extracts from
plant tissue (rutabaga) and practice using the spectrophotometer to follow the progress of the reaction.
The mass of tissue from which the enzyme will be extracted will be 0.5 g. If time allows, the whole
class could have a discussion about what kinds of other variables you could study in this system and
hypotheses/predictions on how it might alter observed reaction rates.

Exercise - Enzyme Extraction

1. Weigh 0.5 g peeled rutabaga on a balance and place this in a cold mortar (ceramic bowl). (Note: other
plant materials that could be used include (but are not limited to; rutabaga root, horseradish root, or potato
tuber).

2. Obtain 50 ml of cold phosphate buffer using the autopipette-topped bottles. A little at a time, add
10 ml aliquots of the cold pH 7 phosphate buffer (0.1 M), and a little sand, grinding tissue/buffer
suspension with a cold pestle. Add subsequent aliquots of the buffer until the full volume has been
mixed into a homogenous solution and the rutabaga sufficiently macerated.

3. Place the slurry into a 50 ml centrifuge tube, and spin at 1500x rpm for 2-3 min (this should all be
preset on the centrifuge). When complete, the fluid at the top contains the plant extract (the pellet
remains behind at the bottom of the tube and is discarded). The extract will last for at least 4 hours
without degrading if refrigerated. Immediately place this tube into a beaker of crushed ice. This crude
extract will contain peroxidase as well as hundreds of other enzymes.

You will prepare two test tubes for each enzyme concentration (i.e. reaction rate) you will study.
You will fill each with different parts of the total reaction mixture careful to keep the final volumes
constant as your double the enzyme concentration each time (Table 1 and Figure 4).

When you are ready, you will pour the two test tubes together and quickly mix them to initiate the
reaction (time = 0 when you first combine them). Refer to Figure 4 on the next page. Note that if

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you mistakingly added each component in order of listing in table 1 below to a single test tube, the
reaction would begin too early for you to be able to reliably measure the rate. By splitting the reactions
into two tubes, you can set them up beforehand and mix them as needed. Note that substrate (H 2O2)
and dye are the same in each sample but that the amount of extract and buffer changes. Note, however,
that the total volume is also the same between samples (the volume of buffer is subtracted to
accommodate additional extract volume (i.e. increase enzyme concentration).

Experimental Set-up to study effects of doubling enzyme concentrations:

1. Collect and label seven test tubes as shown in Table 1.

2. Materials to be measured out (the rutabaga/peroxidase enzyme extract, pH 5 buffer, H2O2 stock,
and guaiacol dye), are all in bottles which have auto-pipettes on top to make dispensing both easy and
accurate (and AVOID senseless waste of materials- ONLY PUMP OUT the indicated volume).
Ensure the volume is set properly (manipulated with knob as needed) and lift the pump, releasing
slowly.
The tube referred to as “Blank” contains the substrate, buffer, and dye, but does not contain
peroxidase, therefore will not turn brown. We use this to blank (i.e. zero) the spectrophotometer
because we are only interested the absorbance that is a result of oxygen evolution (i.e. indicating
peroxidase activity), therefore we must blank for all other molecules, or control for their presence.
Again, Do NOT confuse this with a negative control (e.g. if you had designed an experiment to
examine the effects of the addition of a vitamin enhancer to the system, the negative control would
contain all reagents listed below INCLUDING the enzyme, but not the vitamins).

Table 1: Contents of the test tubes prior to mixing. Note that enzyme is separated from the substrate so that the
reaction commences only after mixing (time 0). Test tube pairs for A, B, and C represent dilute, medium, and
concentrated enzyme extracts.
ID Tube Tube set pH 5 H 2O 2 Guaiacol Enzyme extract
# contents buffer (ml) (ml) dye (ml) (ml)

blank Blank no enzyme 5.0 2.0 1.0 0.0

Set A Ai Low enzyme 2.0 1.0
Aii concentration 4.5 0.5
Set B Bi Med. enzyme 2.0 1.0
Bii concentration 4.0 1.0
Set C Ci High enzyme 2.0 1.0
Cii concentration 3.0 2.0

3. Make sure the spectrophotometer is on, warmed up, and set to a wavelength of 500 nm.

4. Choose one person to be the timer/recorder, another to be the “spec” reader, (depending on group
size - a third person, if necessary, could also be the recorder). Note the time to the nearest second and
then mix the first pair of tubes (A-i and A-ii) together. Pour them back and forth twice (quickly, but
don’t spill any!) and enter them into the spec. after wiping the tube’s outside (BEGIN TIMING UPON

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MIXING and have them in the spec prior to the first 20 second reading) (see figure 4 below). Consider
the absorbance to be equal to that of the blank at time 0 (i.e. absorbance =0 at 0 seconds).

Mix back and Record

forth, wipe with absorbance
kimwipe, place every 20 seconds
IMMEDIATELY or until spec.
in to spec. reaches 2.0
SET
Ai + Aii mixing = time 0 sec

Figure 4. Diagram demonstrating the experimental protocol to be used for standardization of enzyme
activity of peroxidase extracted from a rutabaga. Absorbance readings are a response to the oxidation
of guaiacol dye changing from clear to brown/orange as the substrate H2O2 is catabolized.

5. Collect an absorbance reading every 20 seconds over the full two-minute trial or until the spec
“maxes out” (i.e. reaches 2). Record your readings in table 2 below.

Table 2. Raw data table for examining the effects of increasing enzyme volumes on reaction rates.
Enzyme Absorbance readings at each time interval (seconds)
concentration
Time (seconds) 0 20 40 60 80 100 120
A (low) 0
B (medium) 0
C (High) 0

6. Plot the values on graph paper with “time” on the X-axis. Determine the rate of enzyme activity
from the slopes on that graph. Re-graph the results with “concentration” along the X-axis and the
rates on the Y-axis. You’ve now plotted the independent variable for standardization
(concentration) where it belongs; on the X-axis. The most effective rate is easily seen from this
plotting.

7. Graph your results. Note that when you present your final figure, you’ll be using a rate. Rates
represent change over time. DO NOT PLOT YOUR FINAL RESULTS WITH “TIME” AS THE
INDEPENDENT VARIABLE, that is just a step towards your final figure so just RAW DATA
(i.e. plot the results against time to visualize the slope; “rise over run” (that is, the rate) and plot
those rates with your independent variable on the abscissa in your final figure.

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Graph paper for raw data (plot of absorbance vs time) to determine slopes of each line (reaction rate for each enzyme volume)

Figure ___________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

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Graph paper for slopes in response to doubling enzyme volume (resulting reaction rates for each enzyme volume)

Figure ___________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

*This lab was adapted from: Pitkin, R. B. 1992. Enzyme investigations for introductory courses. Pages
107-188, in Tested studies for laboratory teaching. Volume 13. (C. A. Goldman, Editor). Proceedings of
the 13th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 191 pages.

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 Enzyme Function Prelab

1. The enzyme which we will use is called ______________. The substrate for this enzyme is
______________ (which is harmful to the plant) and the products of that reaction are
______________ and ______________ (which are harmless).

2. We are able to measure the reaction rate by recording the amount of ______________ which
is produced, by using a dye called ______________, which changes colour from
______________ to ______________ when exposed to oxygen.

3. The equipment we use to measure the colour change is called a ______________ and we set it
to a wavelength of ______________.

4. We will get the enzymes by crushing up a small piece of the root of the RUTABAGA plant
(find the genus and species using Google. Capitalize the genus but not the species, and be sure to
underline or italicize both genus and species!). ______________ ___________________

5. To crush the root, we will use a bowl called a ______________ and a hand held device called
a ______________.

6. We will separate the buffer containing the enzymes from the unwanted plant material using a
______________.

7. We will test three volumes of “enzyme extract”. They are _____ ml, ____ ml, and ____ ml.

8. Once we have our results, we will draw two graphs. The first will be an _______________
versus________________ graph. From the slopes of these lines, we will calculate the reaction
rates of the three different volumes of extract.

9. We will use these values to draw a ______________ versus ______________ graph.

10. When investigating the effect of temperature on enzyme activity, the independent variable is
______________ (temp/activity) and the dependent variable is ______________ (temp/activity).

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 Cell membrane permeability
Using Spectrophotometry to quantity membrane disturbance
in beet tissue (Beta vulgaris)
(Guided Inquiry topic)

Required Readings: Fenton et al. (2022), (Chapter 4: pp 83-100; 104-108 as necessary)

- Appendix G: Using Micropipettes
- Appendix H: Using a spectrophotometer
- http://www2.mtroyal.ca/~tnickle/stylesguide (for demonstrations of how to display data).

Key Terms: Beetroot (Beta vulgaris) Cell wall, cell membrane, cytoplasm, tonoplast, central
vacuole, betacyanin. Cork borer, beet puck, blank, negative control, treatment tube,
extraction tube.
Objectives
- Use an experiment to test the “fluid mosaic” model of membrane structure.
- To investigate, using proper experimentation, some of the influences upon membrane
integrity in a plant system.
- review how to graph data using Excel
- modify this labs protocol/study system to investigate your own hypothesis

INTRODUCTION
Cell membranes function to separate the contents of the cell from its external environment and to
organize the cellular compartments (this applies to eukaryotes only; remember prokaryotes typically
only have a plasma membrane). Membranes allow the passage of certain molecules and ions into and
out of the cell and partition them within appropriate intracellular compartments.

In general, the lipid bilayer of a biological membrane is impermeable to most polar molecules
because of its hydrophobic interior. This prevents the water-soluble contents of the cell from escaping.
Synthetic protein-free lipid bilayers have been used to study the permeability of membranes. In vitro
experiments show that almost any molecule will diffuse across these bilayers given enough time. The
rate of diffusion depends upon the size of the molecule and its relative solubility in oil. Molecules that
are smaller and more hydrophobic or nonpolar dissolve more rapidly across them. Large, hydrophilic
molecules move diffuse across more slowly.

Study system
In this exercise you will use living beet cells to relate the structure of a membrane to its function.
Beet cells contain a red pigment called betacyanin. This pigment is located in the large central
vacuole of the cell, which is located entirely within the cytoplasm of plant cells and surrounded by a
membrane called the tonoplast (tonoplast is the proper term for the membrane surrounding the
central vacuole). The cell itself is surrounded by another phospholipid bilayer, the plasma membrane.
As long as both these membranes remain intact, the betacyanin will remain within the cell. If the
membrane permeability is increased, however, the betacyanin will leak out and cause the fluid
around the cells to become red (Figure 1).

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 In this exercise, you will subject discs of beet root to temperature stresses and assess the amount of
damage to the cells by measuring the leakage of betacyanin into the environment. The amount of
betacyanin leaked from the cell can be easily measured using a spectrophotometer. Betacyanin
absorbs light maximally at 525 nm, and so as more betacyanin is leaked from the cell, there is an
increase in absorbance (also known as the optical density - OD). You will be measuring the OD 525

using a spectrophotometer which will be demonstrated by your instructor. The instructions for this
piece of equipment can be found in Appendix H if you need to review.

Test tube with beet

Nucleus tissue samples in it.
Mechanical damage to
Cell wall
tissue shows up in
Plasma membrane solution as leaked
pigment through the
Tonoplast surrounding
membranes.
central vacuole
containing betacyanin
a. b. *note the striations c.

Figure 1. Visualization of the location of betacyanin within the tonoplast of the beet (a), cross-section
of beet to show striations in betacyanin content (b), and (c) shows our study system to manipulate.

Exercise - Procedure for temperature treatment experiment using beet tissue

1. Select a large, fresh beet root located on side bench or in the fridge.

2. Using a cork borer, obtain a long cylinder of beet root. Use a razor blade and a ruler to cut roughly
8 disks that are 5 mm long (pay attention to the colour of your pieces as some will be lighter and
some will be darker due to the naturally occurring striations of betacyanin. Try to choose one or
the other for all your disks). This will give you more than enough disks for the experiment. Cut the
epidermis away at each end of the cylinder and avoid including any tissue within 5 mm of the
epidermis.

3. Rinse all disks in a beaker of cool tap water for about 10 minutes to remove pigment from damaged
cells. The disks may be left in a small amount of water until you are ready to use them. While they
are rinsing carry on with step 4 below paying attention to **highlighted note**.

4. Obtain 11 test tubes; 5 will be for the 1 minute treatment, 5 will be for the 30 minute extraction,
and 1 will be for a ‘blank’ to zero your spectrophotometer. Label 5 of the test tubes for the
temperature treatments (0, 20, 40, 60, and 80ºC) and fill each of these test tubes with 5 ml of water.
**Immediately place these tubes into their corresponding temperature treatment baths and
carry on preparing your beet tissue.** Label the corresponding extraction tubes with the same
temperatures and fill with 5 ml of water.

**Your temperature experiment is written out on the next page in the form of a flow diagram to
help clarify the procedure (Figure 2).**

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 cylinder of beet root

-“Stab” the beet with cork-borer Slice with razor blade in to 5 mm pieces (‘pucks’)
(pull out ‘core’ of beet tissue)

-(Rinse pucks in tap water for ~10 minutes)

-PLACE TUBES CONTAINING DISTILLED WATER IN EACH OF 5 TEMPERATURE

TREATMENT WATERBATHS TO REACH INDICATED TEMPERATURE
When tubes are at appropriate temperature, Place a ‘puck’ into each of 5 test tubes containing 5 mls
distilled water. TREAT FOR ONE MINUTE.

THESE ARE YOUR TREATMENT

TUBES AND PUCKS ONLY
REMAIN AT TEMPERATURE FOR
1 MINUTE

-POUR OFF WATER (tip pucks on to separate pieces of kimwipe or a tea

sieve) (keeping your treatment ‘pucks’ separate)!

Gently, place each puck into clean test tubes of new clean water all at room temperature and extract for 30
minutes.

THESE ARE YOUR

EXTRACTION TUBES AND
PUCKS REMAIN IN THESE FOR
30 MINUTES.

After 30 minutes, agitate one more time and once the puck has settled, place tubes in the spec and record
resulting absorbance readings (after zeroing the spec with water)).

Figure 2. Flow diagram of a membrane permeability experiment using beet tissue (Beta vulgaris)
subjected to increasing temperature treatments, followed by extraction of betacyanin in tap water
quantified via a spectrophotometer measuring absorbance (of light).
(Once the water in the tubes is at the same temperature as the water bath):

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6. Take one of the beet disks and immerse it into the treatment tube you labeled as 0ºC that is in the
ice water bath and let it sit for exactly one minute.

7. After one minute, carefully remove the disk by draining the water into the sink and tapping the disk
onto a kimwipe or into a small sieve. Immediately place it into the extraction tube of water labeled
0ºC. Leave the tube at room temperature for 30 minutes (in the room temperature bath) to
allow extraction of the betacyanin from the disk in response to having been exposed to 0 ºC. Record
the exact time you start the extraction to ensure you are precise with when you read the absorbance.

8. Repeat steps 2-7 for the remaining temperature treatments. Once all 30 minute temperature response
extractions have begun, turn the spectrophotometer on and set the wavelength to 525 nm.

9. During the extraction period, agitate the beet root disk by gently vortexing tubes every 10 minutes.

10. After 30 min, vortex the tube one last time. Blank your spectrophotometer first with your test
tube of water then place each tube of betacyanin extract into the spectrophotometer and record the
absorbance. You may want to blank repeatedly between absorbance readings to prevent ‘drift’.

11. Once all the data is recorded beet disks can be discarded in the garbage.

12. Collect results from other groups in the class. Determine the average absorbance at each
temperature and standard deviation. (Report the actual temperatures your baths are at).

Table 1. Absorbance caused by betacyanin leakage from beet tissue samples in response to 1 minute
temperature treatment.
Absorbance readings at each temperature
Trials 0ºC 20ºC 40ºC 60ºC 80ºC
1
2
3
4
5
6
7
8
9
Average
Standard
deviation

10. Graph your results complete with concise and all-inclusive figure heading (graph paper provided
on the next page).

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Figure .________________________________________________________________
_________________________________________________________________________________

With the time remaining, refer to your GI guidelines and planning forms. You MUST have the
planning forms completed and submitted prior to the GI lab, to perform your upcoming GI
experiment (see syllabus for due date).

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 Prelab
1. How will you quantify the amount of damage to the cell membranes of your beet tissues?
_______________________________________________________________________________
2. What is the name of the pigment which will leak out of the beet with disturbance and where is it
located within the cell? ______________________________________________________________

3. In your temperature vs. absorbance figure, temperature goes on the ____ axis, because it is the
____________________________ variable, while Absorbance (no units), goes on the ___ axis,
because it is the ____________________ variable.

4. What is a reasonable hypothesis for todays lab looking at the effect of temperature on cell membrane
permeability? (Remember a hypothesis is a prediction complete with a rationale).
_________________________________________________________________________________
_________________________________________________________________________________
5. If you were to design an experiment similar to the one written out here, only instead you decide to
look at the effect detergent has on the beet cell membranes permeability, what do you predict the
mechanism of disturbance would be? (i.e. you predict increased membrane permeability due to soap
affecting what aspect of the membrane structure)? ________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
6. In a sample protocol for looking at the effect of methanol on membrane permeability, Tommy
prepares 6 test tubes containing the following:
Tube 1 – 5 ml water (0% methanol)
Tube 2 – 5 ml 5% methanol
Tube 3 – 5 ml 10% methanol
Tube 4 – 5ml 15% methanol
Tube 5 – 5ml 20% methanol
Tube 6 – 5ml water (0% methanol)
Tommy puts a beet ‘puck’ into each of the first 5 test tubes for 1 minute, while tube 6 is left with
none. Explain this as it relates to preparing a blank versus a negative control.
_________________________________________________________________________________
_________________________________________________________________________________

7. Why did you remove the epidermis of the beet sample?

8. Why do you put your room temperature tubes in a water bath?

9. You were very careful to calibrate your spectrophotometers, measure volumes, etc. yet you likely
noticed in some cases, large variation in absorbance readings within treatments. What are some
reasons for this variation?

10. Are there other changes to methodology you might include based on your answer to 9?

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 Guided Inquiry Lab – part II
Performing your membrane permeability experiment -
using an emulsifier as your independent variable
Required readings:

You must have your G.I. Planning forms completed to

attend todays lab! Your Lab instructor will come around and make sure all criteria have
been met before you can begin your G.I. experiment and data collection/analysis.

The guidelines for this is in your Guided Inquiry Folder. Refer to:

1. Guided Inquiry Guidelines (pages 2-4 in particular for today)

2. The Guided Inquiry Assignment Template

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 Cellular Respiration
Required Readings: (Fenton et al, 2022), (Ch 5: pp 101-124)

Key Terms: Aerobic cellular respiration, mitochondrion, anaerobic cellular respiration,

fermentation, glycolysis, cytoplasm, sodium bicarbonate (NaHCO3). Carbonic acid (H2CO3),
phenol red. Durham tube, methyl red. Yeast, fermentation tube.

Objectives:
- understand the process of cellular respiration
- learn the balanced formula, steps, and evolved by-products and end-products (including energy
in the form of ATP)
- know where the most ATP is produced and through which processes
- differentiate between aerobic cellular respiration and anaerobic respiration (and the different
end products)
- compare and contrast cellular respiration to photosynthesis
- understand how substrate and temperature can affect rates of respiration in yeast

INTRODUCTION
Cellular respiration is the metabolic process of generating ATP (adenosine triphosphate), the bodies
utilizable form of energy, from the biochemical energy we acquire through the ingestion of
macromolecules. Imagine if you will, going to a gum ball machine for a gum ball but all you have is
a hundred dollar bill. That hundred dollar bill will not stuff into the coin slot no matter how small you
fold it. The point is, you have the funds for the gum ball, but it’s the wrong currency. We ingest
carbohydrates, proteins, and lipids, all of which feed into cellular respiration at some point in the
process to generate varying amounts of ATP. Recall from the previous enzyme lab our discussion on
how enzymes regulate all the metabolic processes occurring within the body. It is this ATP that works
as a coenzyme, providing energy for our cellular processes, such as active transport, (for example,
generating the polarity of a resting membrane potentials across in a neuron).

Aerobic cellular respiration

The balanced general equation for aerobic cellular respiration is as follows:

C6H12O6 + 6 O2 6 CO2 + 6 H2O + ATP (32-38 give or take)

The site of aerobic cellular respiration is the mitochondria. Glucose (6 carbons in length) conversion
first occurs in the cytosol which yields pyruvate (2 - 3 carbon molecules per glucose molecule), as well
as ATP (through substrate-level phosphorylation and NADH2 (the reducing agent; a hydrogen-
carrying molecule which transfers these hydrogens to the electron transport chain). Upon entering the
mitochondria the pyruvate molecules undergo further catabolism as they are converted to acetyl groups
(only 2 carbon-atoms in length releasing the other two cleaved carbons as carbon dioxide) moving into
the Kreb’s cycle (citric acid cycle) which occurs in the matrix, with the assistance of coenzyme A
(acetyl coA formation).

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The Krebs cycle will “turn” twice to completely oxidize one molecule of glucose and again it will yield
ATP but only minimal amounts (1 ATP per turn), as it also occurs via substrate-level phosphorylation.
During this cycle however, 6 molecules of NADH2 as well as 2 molecules of FADH are produced, and
the energy stored in these bonds will be fed to the inner membrane to carry out oxidative
phosphorylation via two components which we will discuss next: electron transport chain, and
chemiosmosis. This final stage will produce the bulk of the ATP produced through aerobic cellular
respiration. At this point glucose has been completely oxidized with the remaining 4 carbon atoms
released in the form of carbon dioxide.

The NADH2 and FADH (the electron acceptor/hydrogen carrying molecules) have been feeding their
hydrogens into the electron transport chain which resides in the cristae of the mitochondria (allow
your mind to wonder to the typical membrane structure model- the phospholipid bilayer with proteins
embedded in it). In this case the hydrogen atoms are passed to electron acceptors but they do not
‘accept’ the protons of the hydrogen so these get moved to the inter-membrane space and will act as
part of the proton gradient, necessary to driving ATP synthesis. As electrons get passed along the
electron transport chain, they ‘fall’ in free-energy, as energy is used up at the site of ATP synthase
channels and in the presence of ATP synthase, ADP and H, to produce ATP (chemiosmosis). So, to
summarize; in the electron transport chain, electrons are passed from one molecule to
another, and energy released in these electron transfers is used to form an electrochemical
gradient. In chemiosmosis, the energy stored in the gradient is used to make ATP.

For every one molecule of NADH2 we generate 3 ATP, and for every FADH, we generate 2 ATP. The
final electron acceptor in this chain is oxygen and the bi-product of this is water production (& roughly
30 ATP per glucose molecule).

Ultimately this reaction is both exergonic and catabolic (breakdown of sugar and subsequent storage
of energy in high-energy bonds of ATP. This is the opposite of photosynthesis which is endergonic
and anabolic. For now, compare the formula above with the formula below for photosynthesis
(Table 1).
Recall, in Photosynthesis …

(photon) energy + CO2 + H2O -----------------------> (CH2O)n + O2

carbohydrate
(e.g. glucose C6H12O6)
Note: While ATP and NADPH are both produced during the light reactions of photosynthesis but
immediately utilized in the Calvin cycle (where carbon is fixed and sugars are made).

Aerobic cellular respiration takes place within the mitochondria. The initial stage, glycolysis,
however, occurs in the cytosol. This is the only stage that will occur in the absence of oxygen and in
this case, we refer to it as anaerobic respiration or fermentation (depending on the circumstances
and/or organism). In this case glycolysis results in the formation of lactic acid in animals, or in the
case of plants and yeast, alcohol (ethanol) and carbon dioxide are end products.

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Table 1. Comparison of the features of cell respiration and photosynthesis (fill in more).
Aerobic cellular respiration compared to photosynthesis

aerobic cellular respiration photosynthesis

mitochondrion chloroplast

oxidation reduction

releases energy requires energy

requires O2 releases O2

releases CO2 requires CO2

CO2 fixation and Oxygen production in plants

Animals are dependent on plants for a supply of oxygen (O2) produced during the light reactions of
photosynthesis. Equally important are levels of atmospheric carbon dioxide (CO2) which is required
for the second stage of photosynthesis; carbon fixation. Carbon dioxide is taken up by the plant and
reduced to a carbohydrate. Therefore remember that plants still must also rely on cellular respiration
for the formation of ATP to carry on cellular processes so this will also contributes some measure of
CO2 as well as consumes O2 (in other words, chloroplasts and mitochondria are separate organelles,
alas, no teamwork is occurring).

CO2 produced during cellular respiration can combine with water to form carbonic acid (H 2CO3) as
follows:

CO2 + H2O H2CO3

(carbonic acid)
pH neutral  → (pH drops)
This provides another way to observe cellular respiration through this acid production coupled with
the use of an indicator dye. pH is a measure of the acidic or basic properties of a solution. Solutions
having a pH = 7 are neutral, solutions having a pH < 7 are acidic, and solutions having a pH > 7 are
basic. We will use methyl red to detect changes in pH resulting from CO2 production during cellular
respiration. Methyl red is a pH indicator that turns from yellow in a base, to red in an acid.

Aerobic respiration uses oxygen as the terminal electron acceptor in the electron transport chain. During
respiration, CO2 is produced while O2 is consumed. The light reactions of photosynthesis do not occur in
the dark because there is no energy, photons of light, to run the system. Therefore, plants can only respire
in the dark. However, in the presence of light plants can respire and carry-on photosynthesis at the same
time.

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Exercise 1. Respiration in dry vs germinating green peas
Seeds represent a dormant stage of a plants life cycle. Germinating peas, much like animal embryos,
are going through massive amounts of cell division and therefore require huge amounts of energy to
develop and grow. A germination pea should exhibit notable amounts of cellular respiration which we
can observe again using a pH indicator, methyl red.
Obtain four large test tubes and label them 1 through 4. Place the following in each:
1. two-thirds full of germinating green peas (GREEN)
2. two-thirds full of germinating chick peas (NOT green)
3. two-thirds full of germinating peas
4. two-thirds full of dry heat-killed peas (to stop any minute amounts of respiration that may occur)
5. two-thirds full of plastic beads (why?)

Into each place a small test tube containing methyl red, an indicator which turns from yellow to red
in the presence of acid. Place parafilm over the lids of all 4 tubes and wrap tube 2 in aluminum foil so
no light may enter. Let sit for 45 minutes and record any colour change observed. Record your results
in Table 2 below.

Table 2. Results for observing cellular respiration in germinating peas.

Tube Germinating green Germinating Germinating Dead peas Plastic
contents: peas in the light chick peas in the peas in the dark in the light beads
light
Colour of
methyl red
Why/what
process
occurred

Anaerobic Respiration/fermentation in yeast

We will investigate anaerobic respiration using a yeast (Saccharomyces cerevisiae). Yeast are a
eukaryotic single-celled organism from the Fungus Kingdom (Division Ascomycota). Yeast are non-
photosynthetic, rather they are chemo-organotrophs that acquire energy through converting
carbohydrates such as sugars to carbon dioxide and alcohol. Yeast are facultative anaerobes which
means that they can respire in the presence of oxygen but will switch to fermentation in its absence.

Exercise 2. Determining optimum substrates for metabolism by yeast

For this experiment we will look at how sugar source affects fermentation using yeast in fermentation
tubes. You will prepare a yeast suspension in each of the provided substrates as listed below. In each
case use 25-30 mls of metabolite and stir in ½ teaspoon of yeast using a stir stick.

Obtain 5 fermentation tubes and label them for the following treatments/contents;
Tube 1: yeast with 25-30ml 100% distilled water only
Tube 2: yeast with 25-30ml 1% glucose solution
Tube 3: yeast with 25-30ml 1% sucrose solution
Tube 4: yeast with 25-30ml 1% splenda (sucralose) solution
Tube 5: yeast with 25-30ml 1% flour solution
-Tip each tube backwards such that there is no air in the elongated end (Figure 2), and place a tiny
piece of parafilm over the opening with a pin-prick hole.

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Figure 2. The diagram on left shows how to tip your fermentation tube back to fill the chamber,
while the image on right shows a fermentation tube collecting carbon dioxide gas in the chamber.

Place these tubes in the 37ºC incubator for 10-15 minutes (but check frequently after maybe 3 minutes
in case some tubes start bubbling over) and record observed results. Draw a table of your results in the
space below. Again, be able to explain the results in each tube.
Tube Substrate Results/explanation (they can or cannot metabolise…)
Tube 1 water (why?)
Tube 2 Glucose
Tube 3 Sucrose
Tube 4 Splenda
Tube 5 flour

Exercise 3 – microscopic view of yeast (Saccharomyces cerevisiae)

Depending on what is provided in the lab, either use a prepared slide of yeast, or prepare a wet mount
of yeast using the materials provided. Draw what you observe at 400X.

Is it prokaryotic or eukaryotic? To which kingdom does it belong? What type of asexual reproduction
does it use? _______________________________________________________________________
_________________________________________________________________________________

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 Cellular Respiration Prelab
1. Write out the (general) balanced equation for aerobic cellular respiration, then rewrite the
formula for photosynthesis (to review and help gain familiarity):
A- cell resp:
B-photosynthesis:
2. What are the end products of aerobic cellular respiration?__________, ___________ and
_______ATP.
3. Most of the ATP generated in aerobic cellular respiration is made through the process of
____________________________________ which occurs where? ________________
4. The most obvious control in an experiment is one where the experimental variable has been removed.
How does this differ from a ‘blank’?

5. Cellular respiration is broken down into stages, only _____________________can occur in

the absence of oxygen. This process occurs in the ____________________ (where in the cell),
while all other stages occur inside the ________________________.

6. When yeast cells undergo fermentation, what are the end products? ______________ and
_______________________ and only ____ ATP.

7. What do you expect to see in your fermentation tubes when yeast is provided with optimal
conditions (utilizable substrate and a warm environment)?

8. In this lab we will use methyl red which is __________(colour) in a basic pH, and _________
(colour) at an acidic pH. We are using this dye to look for the evidence of production of which
gas? ______________ The formation of ________________ causes pH change which is
indicative of the process of ________________________.

9. Why might there be a colour difference in your methyl red in the germinating pea experiment between
the live green peas placed in the light, and the live green peas placed in the dark?

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 Photosynthesis
(**Exercise 1 is adapted from an original experiment written by Dr Debbie Eldridge of King Ecgbert School in Sheffield, UK).

Required Readings: (Fenton et al., 2022) Ch 6: pp 135-160. Appendix H

Key Terms: Photosynthesis, chloroplast, grana, stroma, pigment, chlorophyll a, chlorophyll b,

carotenoids, carotene, anthocyanin, xanthophyll, chromatography, origin, solvent front,
Rfvalue, chromatogram, oxidation/reduction, epidermis, stomata, guard cells,
evapotranspiration. Absorption spectrum, pH indicators, carbonic acid, carbon dioxide.

Objectives: - explain how pigments give rise to the colour we perceive in objects
- separate and identify pigments present in leaves
- determine the absorption spectra of plant materials
- examine leaf tissue surface for stomata and guard cells
- quantify photosynthetic activity through qualitative & quantitative means

INTRODUCTION
Photosynthesis is the major process by which external energy (derived from the sun) is made available
to the living world. Light energy striking pigments in the chloroplast is transformed first to electrical
energy (excited electrons) and then to chemical energy bonds in the molecules ATP and NADPH.
Some of these bonds are subsequently broken down, and in the process, energy is released which is
used to drive the enzymatic reactions which change atmospheric inorganic carbon dioxide, a low
energy molecule, into organic molecules in the form of sugars. Although photosynthesis is restricted
to only a few types of organisms (plants, some protists, and some bacteria), the sugars they produce
can be used by all living organisms, via glycolysis and respiration, to provide chemical energy for
living processes. Based on your background reading and from your lecture material, you should be
aware that chloroplasts contain a variety of different pigments. Chlorophyll a, chlorophyll b, and the
carotenoids are the major pigments associated with photosynthesis. Each pigment is responsible for
capturing light in the range of specific wavelengths and using it to power further photosynthetic
reactions.

The overall photosynthetic reaction is:

LIGHT REACTIONS

CALVIN CYCLE

(photon) + 12 H2O + photosynthetic + 6 CO2 (CH2O)n + 6 O2 + 6 H2O

energy pigments carbohydrate
(E.g., glucose: C6H12O6)

When we look at a leaf, our eyes can distinguish only a general green colour. However, by using
the process of chromatography, we can separate the pigments using filter paper (chromatography
paper) and a solvent. More on this later! We can also use various means to observe photosynthetic
activity, such as by using pH indicators as carbon dioxide is used, or produced in an aqueous
solution.

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Exercise 1. Quantifying photosynthetic activity using Algae beads (adapted from original experiment written
by Dr Debbie Eldridge of King Ecgbert School in Sheffield, UK).

Students will work in groups for this exercise to ensure there is enough time and materials. Where
materials are limited, groups may be assigned just 1 or 2, of the 3 protocols, with the expectation that each
group will share the results of their experiment. Students will be tested on results from all 3 experiments
regardless of the one your group performed.

This experiment which uses algae beads, requires 1 – 2 hours for measurable change, so we will
set this up first! The algae ‘beads’ you are using for this experiment, are made up of jelly-like
balls consisting of hundreds of immobilized microscopic algae (a single-celled Protist) (Figure 2).
They are prepared by taking dense algae suspensions and mixing them with sodium alginate
solution. Note that the sodium alginate is not harmful to the algae, and makes it much easier to
standardize the quantity of plant material found in each ‘ball’ to approximately the same volume.

Figure 2. Image of algae beads (right) prepared from an algae suspension (left).
Photo credit: https://knowledge.carolina.com/

Carbonic acid (H2CO3) indicator is very sensitive to changes in carbon dioxide levels. When you place
the algal balls in carbonic acid indicator solution and illuminate them, the colour of the pH indicator changes
from orange through red to purple. As the algae use carbon dioxide in photosynthesis, the concentration of
carbon dioxide in the solution falls leading to a colour change (Figure 3).

Increasing CO2 Decreasing CO2

in indicator in indicator

pH 7.6 --> 9.2 in 0.2 increments

0.03% CO2
Atmospheric air

Figure 3. The indicator is orange/red in colour when equilibrated with atmospheric air. Carbonic
acid indicator changes to yellow when more carbon dioxide is added and changes through red to a
deep purple colour when carbon dioxide is removed as shown above. (Image/information: taken from
original source write up)

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Investigating factors that affect photosynthesis – groups will each be assigned one of the protocols
outlined below. The set up in each case will result in something similar to what is seen in the figure below;
showing colour changes in response to algae beads suspended in H2CO3 and placed at different distances from
a light source (figure 4).

Figure 4. Sample results for todays study system. (Image taken from original source write up)

1. Light intensity – this can be achieved in two ways:

-either vary the distance from the lamp, or using different light sources. We will use distance.
Increasing distance from a light source:
1. Obtain 5 small test tubes and add ~5 algae beads to each test tube, along with 3ml H2CO3 .
2. Seal each tube with either a cork/stopper, parafilm (*note the cover needs to be air-tight), or
screw tops if provided.
3. Place tube 1* 10cm from an elevated light source. Place tube 2 30cm from tube 1, and
subsequent tubes at 30cm distances from each other. NOTE: All tubes are to be placed on their
sides!
4. Now wait for colour changes to occur as the algae undergo photosynthesis and absorb the CO2.
*This will take 1 – 2 hours.
5. Record the colours and corresponding pH’s using the charts provided, and graph your results.
*Have one tube completely covered with foil to prevent any light from penetrating. (why?)
WHEN EXPERIMENT IS COMPLETE:
6. Pour the H2CO3 from each test tube for each distance through a tea strainer into the
indicated ‘spent’ container. * Place algae beads in indicated ‘spent’ beaker to rest.

2. Wavelength of light - using coloured filters (cellophane)

1. Obtain 5 small test tubes and add ~5 algae beads to each test tube, along with 3ml H2CO3 .
2. Seal each tube with either a cork/stopper, parafilm (*note the cover needs to be air-tight), or
screw tops if provided.
3. Place each tube 10cm from 1 of 4 different coloured light sources (using either cellophane or
coloured light bulbs). *Use the information from tube 1 -Light intensity above as your white light
value. *Have the 5th tube completely covered with foil to prevent any light from penetrating.
NOTE: All tubes are to be placed on their sides!
4. Now wait for colour changes to occur as the algae undergo photosynthesis and absorb the CO2.
*This will take 1 – 2 hours.
5. Record the colours and corresponding pH’s using the charts provided, and graph your results.
*Have one tube completely covered with foil to prevent any light from penetrating. (why?)
WHEN EXPERIMENT IS COMPLETE:
6. Pour the H2CO3 from each test tube for each distance through a tea strainer into the
indicated ‘spent’ container. * Place algae beads in indicated ‘spent’ beaker to rest.

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3. Concentrations of algal cells
1. Obtain 5 test tubes and add along with 3ml H2CO3 to each tube. Add 2, then 4, then 6, then 8 beads
to tubes 2-5, and leave tube 1 with no beads.
2. Seal each tube with either a cork/stopper, or parafilm. (note the cover needs to be air-tight)
3. Place in front of light source.
4. Now wait for colour changes to occur as the algae undergo photosynthesis and absorb the CO2.
*This will take 1 – 2 hours.
5. Record the colours and corresponding pH’s using the charts provided, and graph your results.
*Have one tube completely covered with foil to prevent any light from penetrating. (why?)
WHEN EXPERIMENT IS COMPLETE:
6. Pour the H2CO3 from each test tube for each distance through a tea strainer into the
indicated ‘spent’ container. * Place algae beads in indicated ‘spent’ beaker to rest.

Exercise 2. Chromatography
Chromatography is a way to separate molecules from a complex mixture. The pigments involved in
photosynthesis need to be bound to the thylakoid membrane and are therefore hydrophobic. Because
of their complexity, the degree to which they are hydrophobic is slightly different. We can take
advantage of the chemical differences of pigments to separate them. The structural differences of the
pigments make them capable of harnessing different qualities (wavelengths) of light (and therefore
they also appear to be of different colours).

By spotting a mixture of pigments onto chromatography paper (this position is called the “origin”)
and then allowing a solvent to flow over the spot, the pigments will drift up in the direction the leading
edge of the solvent travels (also known as the “solvent front”). The varied chemical characteristics of
the pigments mean they spend characteristic amounts of time either dissolved in the solution or
absorbed to the paper. A strongly nonpolar chemical will spend more time in the liquid, and thus will
be carried upward close to the solvent front. A more polar chemical will spend more time bound to the
paper and thus will not move as quickly. The rate of solvent flow can change substantially between
different separations because of humidity, changes in solvent composition, volume of solvent, size of
paper, and so on. So that different chromatography runs can be compared, an “index” that allows them
to be compared can be generated.

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This index is called the Rf and describes the distance moved by a pigment to the distance moved by
the solvent front as a ratio (and should always be a number less than 1).

Rf= Distance moved by the pigment

Distance from the pigment origin to the solvent front

chromatography paper
Suspend the
chromatography paper with
chloroplast sample inside a
chromatography chamber
capillary tube (containing a small amount
containing of chromatography solvent
chloroplast/solvent - below origin)
suspension added
drop-wise repeatedly
Keep this apparatus in the
to same spot at fumehood and check it
‘origin’ frequently to prevent the
solvent front from coming
off the page.
origin

Figure 5. Schematic demonstrating the procedure outlined in exercise 1 for paper chromatography
(separating plant pigments based on their polarities).

Chromatography using spinach extract **(Extract preparation will be created once by your
instructor for the whole classes use).**
Extract preparation (all text shown in orange= done by lab instructor)
The pigments mixed in a plant extract (obtained by grinding photosynthetic tissue in an organic solvent
-acetone) can be separated according to the following protocol.

1. Obtain 10 g of spinach (or other provided plant material) for the extract. Remove the leaf petioles
and cut up as much as possible before placing in a cold mortar.
2. Add 10 ml of acetone and a small pinch of sand.
3. Grind this tissue with a pestle until it’s as homogeneous as possible, pouring off the resulting green
chloroplast suspension into a test tube and allow the more solid material to settle.
Preparation of Chromatogram
4. Cut chromatography paper as shown in Figure 5 above, careful not to touch the surface of the paper
but gently hold by the edges as oils from your fingers can obscure results and interfere with the
movement of the solvent and pigments. Trim the bottom into a point as shown above and mark it with
a pencil line about 2 cm from the pointed tip. This is the “bottom” of your strip and is called the
chromatogram’s origin.

5. Apply some extract at the mark, and gently blow on the extract until the acetone has evaporated
and dot is dry. Repeat this at least 10 times or until a visible quantity of greenish pigment remains.

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6. IN THE FUMEHOOD - Add 1-2 ml of chromatography solvent (9 parts petroleum ether: 1 part
acetone) to your chromatography chamber (~1cm solution in bottom of large tube) Find a black stopper
with a paperclip hook anchored to it that will seal the test tube. Fasten the top of the paper to the hook
and lower it into the test tube such that the pointed end is just moistened by the solvent.
Be sure only the pointed end of the paper is immersed in the solvent and the origin is above the
solvent reservoir. YOU MUST KEEP THESE IN THE FUMEHOOD METAL TUBE RACK!!
7. Let the test tube stand vertically in a rack for 10-15 minutes (this time is very approximate). Monitor
Frequently and stop the separation when the solvent front (the leading “wet edge”) is approximately
5-10 mm from the top of the paper. Make sure you Mark the location of the solvent front right
away with a pencil (this will become invisible when the paper is dry).
8. Observe the bands of colour. Measure the distance from the origin to the solvent front (this is the Rf
function’s denominator) and from the origin to each of the pigment bands (there should be 4;
chlorophyll a, chlorophyll b, carotene, and xanthophyll).
Calculate the Rf value for each pigment, and compare to those obtained by other classmates.
Holding the filter paper up to the light will aid you in discriminating among the pigments; you may wish
to outline the bands of pigment using a pencil. Identify the pigments by their colours (Chlorophyll a -
blue-green, Chlorophyll b - grass-green, Carotene - yellow, and Anthocyanins - purple (this
pigment does not dissolve in the solvent and so will not move from its original spot).
Record your results here complete with Rf calculations.

Questions:
What is the order of pigments on the chromatography paper (based on your results drawn above)?
_________________________________________________________________________________
What does this indicate about their relative solubility in this solvent? What are the roles of the
pigments in
photosynthesis?____________________________________________________________________

Exercise 3. Observing the absorption spectrum for spinach extract in sucrose buffer
We will use the spectrophotometer to examine the response in absorbance for spinach chloroplast
suspension to different wavelengths of light. By now in lecture, you have studied both action and
absorption spectrums (and should be able to describe the relationship between the two(?)). As was
mentioned earlier in the lab, different pigments absorb light optimally at different wavelengths and
from reviewing your chromatography results we know we have several pigments in suspension so we
will not be able to isolate each pigment, however, we can get an overall absorption spectrum for our
specific plant tissue sampling of spinach.

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1. Obtain 1 ml of the Concentrated spinach in sucrose buffer solution concentrate your lab instructor
will have created at the start of the lab. This will be found in a clearly labeled large centrifuge tube
that has been wrapped in foil and placed on ice, on the side bench.
2. Add 24ml sucrose buffer to your 1ml . You will use this to generate your action spectrum.
3. Use a second tube containing 4-5ml of just sucrose buffer as your blank.
2. Set the spectrophotometer to each of the wavelengths listed below, blank using the buffer, and then
take an absorbance reading of your spinach extract and record it in Table 2 below.
**NOTE: You must blank every time you change the wavelength of your spectrophotometer.**

Table 2. Absorbance readings measured with a spectrophotometer for spinach extract in 0.05M sucrose
solution.

*Place a star beside your Amax (optimum wavelength for light absorbance).*
Next, plot your absorbance values (responding variable) vs wavelength (independent variable) on the
graph paper provided and compare your results with your textbook and materials provided at the side
benches.

Figure__ ________________________________________________________________________

Exercise 4. Microscopic observations of plant epithelium

We will revisit microscopic observation of plant tissue in this next exercise using fava bean plants
(Vicia faba). Factors that influence photosynthetic activity include light, and carbon dioxide. The
stomata are openings in the surface of leaf epidermis and they serve to regulate evapotranspiration,

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or the movement of water and gases O2 and CO2. Stomata largely impact photosynthetic activity by
regulating the amount of carbon dioxide available for photosynthesis. The opening or closing of stoma
is in turn regulated by the surrounding guard cells. When stomata are open, carbon dioxide may enter
the leaves and be captured in the calvin cycle. Stomata may close at night or when water stress is
occurring to prevent water loss (on hot days for example). The location and arrangement of stomata on
a leaf surface will vary according to the type of plant (monocot vs dicot – topics to be re-addressed in
biology 1204), and orientation of the leaves to maximize CO2 uptake while minimizing water loss.

Exercise 3. Observing the stomata (stomata) in fava bean epithelium.

1. Prepare a wet mount as you have in previous labs by first applying a drop of water to a clean slide
placed on the bench.
2. Carefully remove a cell-layer thin strip of epithelial tissue from the provided fava bean plant leaves
and place it into your drop of water. Try to smooth out any wrinkles in your tissue using tweezers as
needed.
3. (optional) – you may choose to add a very small amount of methylene blue to your slide to enhance
your visualization of details. Conversely, closing the iris diaphragm should have a similar effect. Either
way, close the iris diaphragm to enhance observations of cellular details.
4. Look for stomata on the leaves of your tissue sample and determine if they are open or closed. Try
to draw a stomate in each state (both open and closed).
(Draw and label your slide observations in the space provided (include guard cells, stomata, cell walls,
chloroplasts, etc.).

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 Photosynthesis Prelab
1. Write out the balanced equation for photosynthesis. CLEARLY indicate which components of
the reaction are part of the light reactions, and which are part of the Calvin cycle.

2. What happens to the ATP and NADPH that is generated in photosynthesis?

3. Look at the question highlighted in yellow on page 90 of this lab. What is missing in these
experimental protocols (Hint: think of how you set up your macromolecules tests, and your beet
membrane GI).

4. What happens to the colour of carbonic acid indicator as photosynthesis is occurring? Why?

5. The coloured bands we observed from doing TLC separated based on their
_______________,(molecular property) with the _________(most/least) of this property moving
the farthest distance up the paper.

6. What is the function of a stomate? What surrounds the stomate?

7. 4 different factors are explored in Exercise 1 of this lab. List them and predict how each factor
might affect photosynthesis.
Factor Predicted outcomes/colour changes (why)

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 Additional Literature cited

Pitkin, R. B. (1992). Enzyme investigations for introductory courses. Pages 107-188, in Tested studies for
laboratory teaching. Volume 13. (C. A. Goldman, Editor). Proceedings of the 13th
Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 191 pages.

Russell, P.J., Hertz, P.E., McMillan, B., Fenton, M.B., Addy, H., Maxwell, D., Haffie, T., and B. Milson,
(2015). Biology: Exploring the Diversity of Life, 3rd Canadian Ed. Nelson Education, Toronto,
Ontario.

Scott, N. and B. Greenberg (1995). Measurement of Photosynthetic Activity in Plant Cell Fractions.
pages 71-80, in Tested Studies for laboratory teaching, Volume 16 (C. A.Goldman, Editor).
Proceedings of the 16th Workshop/Conference of the Association for Biology Laboratory
Education (ABLE), 273 pages.

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