Laboratory 1

INTRODUCTION AND SAFETY PROCEDURES

LAB REPORTS
For the scientific enterprise to be successful, scientists must communicate their work. Major
scientific findings are never kept secret. Instead, scientists share their ideas and results with other
scientists, encouraging critical review and alternative interpretations from colleagues and other
scientific community. Communication both verbal and written occurs at every step along the
research path. While working on projects, scientists present their preliminary results for
comments from their coworkers at laboratory group meetings and in written research reports. At
a later stage, scientists report the results of their research activities as a poster or oral presentation
at a scientific meeting. Then the final report is prepared in a rather scientific paper format and
submitted for publication in an appropriate scientific journal. At each stage in this process
scientists encourage and require critical review of their work and ideas by their peers. The final
publication in a peer-reviewed journal generally promotes additional research and establishes
this contribution to current knowledge.
One of the objectives of every lab topic in this manual is to develop your writing skills. You
will generate and write hypotheses, results, observations, answers to questions, and more, as
one way of learning biology. Also, you will practice writing in a scientific paper format and
style to communicate the results of your investigations. By the time you have completed the
lab topics in this manual, you will have written the equivalent at least two complete scientific
papers.
As you investigate the different lab topics, you will make observations, ask questions, and
propose hypotheses. You will design and conduct experiments using procedures of your own
design or fallowing procedures in the manual. You will record results, designing tables and
graphs to present your data in a logical and organized format. You will then interpret results and
come to conclusions based on your hypotheses. This process is reflected in the design of
scientific paper and format you will use for your laboratory papers. Each paper will be divided
into sections that reflect these activities.
A scientific paper usually includes the following parts: a Title (statement of the question or
problem), an Abstract (a short summary of the paper), an introduction (background and
significance of the problem), a Materials and Methods section (report of exactly what you did
), a Result section (presentation of data), a Discussion section (interpretation and discussion of
results), and References Cited (books and periodicals used), a Conclusion (restatement of
conclusions) and Acknowledgments (recognition of assistance) may also be included. Below
you will find a format, follow the given information in the writing of your lab reports.
Title of Experiment
e.g. An Introduction to the Laboratory
• Name and Surname:
• Student ID#:
• Department:
• Date of experiment:
Abstract
Summarize in a paragraph the purpose of the experiment, data presented and major
conclusions in about 100-200 words.
Introduction
This section presents background information about the subject of the experiment. A well-
written introduction should be through, but only include information that is directly relevant to
the experiment. Background information can be obtained from lab manuals, textbooks, and other
sources. But never directly copy or quote sentences from your sources. The sentences should be
paraphrased with your own words and sources must be given as references at the end of the lab
report. All scientific names (genus and species) must be italicized or underlined.
The purpose of experiment should be given at the end of Introduction section. In few sentences
explain why the experiment was performed-what was the scientific problem and objectives of
experiment. A good introduction will answer these questions. What knowledge already exists
about this subject? What was the specific purpose of the experiment?
Materials and Methods
Firstly, in this sec1tion the materials used in th eexperiment were desribed. Then, the
experimental procedures were explained with sufficient detail for someone else o obtain the same
results. The steps of procedure in the lab manual should not be copied exactly. Rather, the
procedure should be rewritten to provide essential information. In addition, how the data was
collected and manipulated should be clearly described in this section. Always the past passive
voice is used in materials, methods and results sections.
Results
The scientific data obtained in the experiments is presented in this section. Results contain both
quantitative measurements and qualitative observations. The number should never stand alone.
They must be accompanied by appropriate units (M, mM, mm, cm, g, mg, µg, sec, min, h, ml,
l, etc.). This section also includes calculations, tables, graphs, and figures. Whenever possible
present your data in a table or a graph. But never include data twice. If the results are presented
in a table, then do not show the same data graphically, and visa versa.
Discussion
This section includes analysis and interpretation of the data, relating them to existing theory and
background information. In this part the answer of the question “What is the significance or
meaning of the results? Is tried to find. For a good discussion part, the following questions
should be answered. What do the results indicate clearly? What have you found? What questions
might we raise? Find logical explanations for problems in the data. Explain what you know with
certainly based on your results and draw conclusions.
Suggestions and recommendations for the improvement of techniques may be described here.
On the basis of your own data draw your conclusions at the end of this section in few sentences.
References
All sources of information should be cited in this part. In the text part reference is given as
(Smith et al, 2001). If the source has three or more authors, then the abbreviation “et al” can be
used as co-workers.
• Reference from a book:
Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular cloning: a laboratory
manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Romano, A. H., Saier, M. H., Jr (1992). Evaluation of the bacterial

phosphophenolpyruvate: sugar phosphotransferase system. I. Physiological and
organismic considerations. In The Evolution of Metabolic Function, pp. 171-204.
Edited by R.P. Mortlock, Boca Raton, FL: CRC Press.

• Reference from a journal:

Arciola, C.R., Collamati, S., Donati, E., Montanaro, l. (2001). A rapid PCR method for
the detection of slime-producing strains of Staphylococcus epidermis and S. eureus in
periprosthesis infections. Diagn Mol Pathol 10, 130-137

• Reference from Internet:

Franchesca, P. “Algae: the forgotten treasure of tidepools”
(www.sonoma.edu/biology/algea/algea.html), May, 1997.

Anonymous. “Algal bioassays” (www.dep.state.fl.us/biol/AA.html) no date given.

To prevent writing errors, read and think about what you write. Learn to reread and edit your
work.

SAFETY PROCEDURES
1. Notify your instructor immediately if you are pregnant, color blind, allergic to any insects
or chemicals, taking immunosuppressive drugs, or have any other medical condition
(such as diabetes, immunologic defect) that may require special precautionary measures
in the laboratory.
2. Upon entering the laboratory, place all books, coats, purses, backpacks, etc. İn
designated areas, not on bench tops.
3. Locate and, when appropriate, learn to use exits, fire extinguisher, fire blanket,
chemical shower, eyewash, first aid kit, broken glass container, and cleanup materials
for spills.
4. In case of fire, evacuate the room and assemble outside the building.
5. Do not eat, drink, smoke, or apply cosmetics in the laboratory.
6. Confine long hair, loose clothing, and dangling jewelry.
7. Wear shoes at all times in the laboratory.
8. Cover any cuts or scrapes with a sterile, water-proof bandage before attending lab.
9. Wear eye protection when working with chemicals.
10. Never pipet by mouth. Use mechanical pipetting devices.
11. Wash skin immediately and thoroughly is contaminated by chemicals or microorganisms.
12. Do not perform unauthorized experiments.
13. Do not use equipment without instruction.
14. Report all spills and accidents to your instructor immediately.
15. Never leave heat sources unattended.
16. When using hot plates, note that there is no visible sign that they are hot (such as red
glow). Always assume that hot plates are hot.
17. Use an appropriate apparatus when handling hot glassware.
18. Keep chemicals away from direct heat or sunlight.
19. Keep containers of alcohol, acetone, and other flammable liquids away from flames.
20. Do not allow any liquid to come into contact with electrical cords. Do not attempt
to disconnect electrical equipment that crackles, snaps, or smokes.
21. Upon completion of laboratory exercises, place all materials in the disposal areas
designated by your instructor.
22. Do not pick up broken glassware with your hands. Use a broom and dustpan and discard
the glass in designated glass waste containers; never discard with paper waste.
23. Wear disposable gloves when working with blood, other bodily fluids, or mucous
membranes. Change gloves after possible contamination and wash hands immediately after
gloves are removed.
24. Place gloves, swabs, toothpicks, etc. that may have come into contact with body fluids
in a disposable autoclave bag.
25. Leave the laboratory clean and organized for the next student.
26. Biohazard symbol indicates procedures that may pose health concerns.
27. The caution symbol points out instruments, substances, and procedures that require
special attention for safety

Figure 1. Biohazard symbol Caution symbol

Laboratory 2
TECHNIQUES IN MICROSCOPY
1. BACKGROUND
Microscope has been developed to help explore the world of living things too small to be seen
with the naked eye. Since an unaided eye cannot detect anything smaller than 0.1 mm (10-4
meters) in diameter, cells, tissues and many small organisms are beyond our visual capability
The history of microscope begins with Zacharias Jansen’s light
microscope that was the first light microscope in 1590s. The
first big microscopy advances came in 1665, when Robert
Hooke published the Micrographia, a collection of copper-
plate illustrations of objects he had observed with his own
compound microscope. He coined the term ‘cell’ when looking
at a piece of cork under 30x magnification.
Figure 1. Hooke’s
Microscope
In the late 1660s, Antony van Leeuwenhoek began to grind his own lenses and make simple
microscopes. The optical properties of lenses have been known for the last 300 years B.C., but
this knowledge were not used to the fullest until the seventeenth century when Antonio Van
Leeuwenhoek (1632-1732), a Dutch, and his colleagues discovered a simple workable
microscope. He observed animal and plant tissue, sperm cells and blood cells, minerals, fossils,
and much more.
The dawn of the 20th century witnessed the development of an alternative to the light
microscope known as the electron microscope. These devices used electrons rather than light
to generate an image of the target. Electron microscope uses electron beams (generally from
gold) that cover the objects and increase the magnifying ability of the light microscopes. The
German physicist, Ernst Ruska developed the first prototype of electron microscope in 1931, a
transmission electron microscope (TEM). The development of this technology was
representative of a huge advance in the field, given that these microscopes were capable of
delivering a much higher resolution.
TYPES OF MICROSCOPES
Light Microscopes
There are two main types of light microscopes; compound and stereo microscopes. Compound
microscopes are so called because they are designed with a compound lens system. The
objective lens provides the primary magnification which is compounded (multiplied) by the
ocular lens (eyepiece). Many types of microscopes fall under the category of light microscopes,
which are used to enhance contrast of image. Examples of light microscopes include brightfield
microscopes, darkfield microscopes, phase-contrast microscopes, differential interference
contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and
two-photon microscopes.
A. Brightfield microscope: The most commonly used type of microscope, is a compound
microscope with two or more lenses that produce a dark image on a bright background.
B. Dark field microscope: It increases contrast without staining by producing a bright image
on darker background, especially useful for viewing live specimens.
C. Phase contrast microscope: Density differences in the specimen cause light rays to come
out of “phase”. The microscope enhances these phase differences so that some regions of the
specimen appear brighter or darker than others. The technique is widely used to observe living
cells and organelles.
D. Differential interference contrast microscope: Optical methods are used to enhance
density differences within the specimen so that certain regions appear brighter than others. This
technique is used to view living cells, chromosomes and organelle masses.
E. Fluorescence microscope: A fluorescence microscope uses fluorescent chromophores
called fluorochromes, which are capable of absorbing energy from a light source and then
emitting this energy as visible light. Fluorochromes include naturally fluorescent substances
(such as chlorophylls) as well as fluorescent stains that are added to the specimen to create
contrast.
F. Confocal microscope: Whereas other forms of light microscopy create an image that is
maximally focused at a single distance from the observer (the depth, or z-plane), a confocal
microscope uses a laser to scan multiple z-planes successively. This produces numerous two-
dimensional, high-resolution images at various depths, which can be constructed into a three-
dimensional image by a computer. As with fluorescence microscopes, fluorescent stains are
generally used to increase contrast and resolution. Confocal microscopes are thus very useful
for examining thick specimens such as biofilms, which can be examined alive and unfixed
G. Two-photon microscope: It uses a scanning technique, fluorochromes, and long-
wavelength light (such as infrared) to visualize specimens

Stereoscopic Dissecting Microscopes

A stereo microscope is an optical microscope that provides a
three-dimensional view of a specimen. It is also known by
other names such as dissecting microscope and stereo zoom
microscope. Dissecting microscope parts include separate
objective lenses and eyepieces. As a result, you have two
separate optical paths for each eye. The slightly different
angling views to the left and right eyes produce a three-
dimensional visual. Because it gives the three-dimensional
view it is also called as the dissecting microscope.
Figure 2. Stereoscopic microscope
Electron Microscopes
The maximum theoretical resolution of images created by light microscopes is ultimately
limited by the wavelengths of visible light. Most light microscopes can only magnify 1000x,
and a few can magnify up to 1500x, but this does not begin to approach the magnifying power
of an electron microscope (EM), which uses short-wavelength electron beams rather than light
to increase magnification and resolution. There are two basic types of EM: the transmission
electron microscope (TEM) and the scanning electron microscope (SEM).
A. The transmission electron microscope (TEM): The TEM is somewhat analogous to the
brightfield light microscope in terms of the way it functions. However, it uses an electron beam
from above the specimen that is focused using a magnetic lens (rather than a glass lens) and
projected through the specimen onto a detector. Electrons pass through the specimen, and then
the detector captures the image
B. The scanning electron microscope (SEM): SEMs form images of surfaces of specimens,
usually from electrons that are knocked off of specimens by a beam of electrons. This can create
highly detailed images with a three-dimensional appearance that are displayed on a monitor.

PARTS OF MICROSCOPE
Ocular Lens (Eyepiece Lens): The ocular lens is
the lens you look through. If your microscope has
one ocular, it is a monocular microscope. If it has
two, it is binocular. In binocular microscopes, one
ocular is adjustable to compensate for the
differences between your eyes. Ocular lenses can
be made with different magnifications usually 10x
or 15x power.
The Body Tube: The body tube is the hollow
housing through which light travels to the ocular.
It connects the eyepiece to the objective lenses.
Figure 3. A Binocular Light microscope
Objective Lenses: The objective lenses are a set of three or four lenses mounted on a rotating
turret at the bottom of the body tube. Rotate the turret and note the click as each objective comes
into position. The objective gathers light from the specimen and projects it into the body tube.
Magnification ability is stamped on each lens.
Scanning (small) lens 4x High-power (large) lens 40x

Low-power (medium) lens 10x Oil immersion (largest) lens 100x

Stage: The horizontal surface on which the slide is placed is called the stage. It may be
equipped with simple clips for holding the slide in place or with a mechanical stage, a geared
device for precisely moving the slide. Two knobs, either on top of or under the stage, move
the mechanical stage.
Condenser Lens: The substage condenser lens system, located immediately under the stage,
focuses light on the specimen. An older microscope may have a concave mirror instead.
Diaphragm Control: The diaphragm is an adjustable light barrier built into the condenser.
This diaphragm has different sized holes and is used to vary the intensity and size of the cone
of light that is projected upward into the slide. It may be either an annular or an iris type. With
an annular control, a plate under the stage is rotated, placing open circles of different diameters
in the condenser light path to regulate the amount of light that passes to the specimen. With the
iris control, a lever projecting from one side of the condenser opens and closes the diaphragm.
Light Source: The light source has an off/on switch and may have adjustable lamp intensities
and color filters. Use medium to low voltages whenever possible to prolong lamp life.
Base and Body Arm: The base and body arm the cast metal parts of the microscope.
Coarse Focus Adjustment: Depending on the type of microscope, the coarse adjustment
device either raises or lowers the body tube or the stage to focus the optics on the specimen.
This rack and gear mechanism facilitate a relatively large movement with only a partial
revolution of the adjustment knob.
The Focus Adjustment: The fine adjustment changes the specimen-to-objective distance very
slightly with each turn of the knob and is used for all focusing of the 40x objective. It has no
noticeable effect on the focus of the scanning objective (4x).
IMAGE QUALITY OF THE LIGHT MICROSCOPES
Magnification is a measure of how much larger a microscope (or set of lenses within a
microscope) causes an object to appear. For instance, the light microscopes typically used in
high schools and colleges magnify up to about 400 times actual size. So, something that was 1
mm wide in real life would be 400 mm wide in the microscope image.
The resolution of a microscope or lens is the smallest distance by which two points can be
separated and still be distinguished as separate objects. This capacity is termed as the resolving
power of the lenses or the resolving power of the microscope. The higher the resolution of the
microscope, the higher is the ability to distinguish details of the object. Microscope quality
depends upon the capacity to resolve, not magnify, objects. Magnification without resolving
power, however, is not worthless in the field of microscopy.
Contrast, is defined as the difference in light intensity between the image and the adjacent
background relative to the overall background intensity. To show detailed parts of the
specimens, dyes or pigments can be used to differentiate them from the background.
The Microscope and Your Eyes
Students often wonder if they should remove their glasses when using a microscope. If you are
nearsighted or farsighted, there is no need to wear your glasses. The focus adjustments will
compensate. If you have astigmatism, however, you should wear your glasses because
microscope lenses do not correct for this problem. If your microscope is monocular, you will
probably tend to use it with one eye closed. Eyestrain will develop if this is continued for long.
Learn to keep both eyes open as you look through the microscope and ignore what you see with
other eye. This will be hard at first. Remove all light-colored papers from your field of view or
try covering your eye with your hand.

MICROSCOPE SLIDE TECHNIQUES

Negative Staining

Bacteria are quite colorless and transparent. A better way to observe bacteria for the first time
is to prepare a slide by a process called negative, or back-ground, staining. This method consists
of mixing the microorganisms in a small amount of nigrosine or india ink and spreading the
mixture over the surface of a slide. These stains do not penetrate the microorganisms. Instead
they stain the background, leaving the organisms transparent and visible in a darkened field.
This can be useful for determining cell morphology and size. No heat is applied to the slide,
there is no shrinkage of the cells, and, consequently, more accurate cell-size determinations
result than with some other methods.

2. LAB EXERCISES

2.1.Making Slides and Using a Microscope

2.1.1. Materials
Microscope Slide, coverslip, compound microscope, newspaper

2.1.2. Protocol

1. Figure 4 shows how to prepare a specimen as a wet mound on a microscope slide. Take a
newspaper or an old printed page and cut out a lowercase letter e or a or the number 3, 4 or
5.
2. Clean a microscope slide with a tissue, add a drop of water to the center, and place the letter
in the drop.
3. Add a coverslip and place the slide in its normal orientation on the microscope stage with
the scanning objective in place.

(a) (b) (c)

Figure 4. Procedure for making a wet-mount slide. (a) Place a drop of water on a clean slide.
Steps Used in Viewing a Slide
1. Check that the ocular and all objective lenses as well as the slide are clean
2. Turn the illuminator on and open the diaphragm. Center the specimen over the stage
opening.
3. Look through the ocular. Starting with the scanning objective as close to the slide as possible
with looking through the oculars, back off with the coarse adjustment knob until the
specimen is in sharp focus.
4. Readjust the light intensity and center the specimen in the field of view by moving the slide.
Close down the iris diaphragm and, if possible, adjust the substage condenser until the edges
of diaphragm are in focus.
5. Switch from the scanning objective to the low-power (10x) objective. The lens stop should
click when the objective is in place. Sharpen the focus, adjust the centering of the specimen,
and readjust the condenser height and diaphragm opening.
6. Switch to the high-power (40x) objective. Adjust the focus with the fine focus adjustment
only. If you use the coarse adjustment, you may hit the slide and damage the high-power
objective.
7. If you have a binocular microscope, adjust the ocular lenses for the differences between your
eyes. Determine which ocular is adjustable. Close the eye over that lens and bring the
specimen into sharp focus for the open eye. Open the other eye, and close the first. If the
specimen still is not in sharp focus, turn the adjustable ocular until the specimen is in focus.
You need not repeat this procedure when you look at other specimens, but should do it each
time you get the microscope from the cabinet because other students may also be using your
microscope and adjusting it for their eyes.

2.1.3. Study Questions

2.2. Making Slides for Negative Staining

2.2.1. Materials
Microscope Slide, coverslip, compound microscope, Nigrosine solution or india ink,
Slant cultures of bacteria, Inoculating straight wire and loop

2.2.2. Protocol
1. Organisms are dispersed into a small drop of nigrosine or india ink. Drop should not
exceed 1/8” diameter and should be near one end of the slide.
2. Spreader slide is moved toward drop of suspension until it contacts the drop causing the
liquid to be spread along its spreading edge.
3. Once the spreader slide contacts the drop on the bottom slide, the suspension will spread
out along the spreading edge as shown in figure 5.
4. Spreader slide is pushed to the left, dragging the suspension over the bottom slide. After
the slide has air-dried, it may be examined under oil immersion.

Figure 5. Negative staining procedure

3. REFERENCES
David Bardell "The Biologists' Forum: The invention of the microscope," BIOS 75(2), 78-
84, (1 May 2004). https://doi.org/10.1893/0005-3155(2004)75<78:TIOTM>2.0.CO;2
Abramowitz, M., Keller, H. E., Spring, K. R., “Basic Concepts in Optical
Microscopy”(https://micro.magnet.fsu.edu/primer/anatomy/anatomy.html), Nov, 2015
Laboratory 3
MEASURING TECHNIQUES
1. BACKGROUND
We learn about the world by making observations and comparing them with other observations.
The major difference between the types of comparisons we make in everyday life and the
discipline known as science is that science requires a more rigorous, focused and systematic
approach. By using a common system of units of measurement and by making precise and
accurate measurements, scientists ensure that procedures and results can be reported and
repeated everywhere in the world.
In this laboratory, we aim to use standard laboratory measuring devices to learn making
accurate measurements of length, volume and mass.
Whether making measurements or performing experiments, scientists use many common
laboratory equipments.

Figure 1. Laboratory equipments

2. LAB EXERCISES

2.1.Lab Exercise 1
2.2.1. Protocol

1. Identify the commonly used equipments in the laboratory.

2. Write down the names of 4 equipments you found on your bench.
Equipment 1 …………………………
Equipment 2 …………………………
Equipment 3 …………………………
Equipment 4 …………………………

Most scientific data are expressed in the units of metric system (Table 1). This system of units,
known as the International System of Units, is commonly referred to as the SI system. The SI
is made up of 7 base units that define the 22 derived units with special names and symbols.
Table 1. SI units
Name SI unit
Length Meter (m)
Mass Kilogram (kg)
Time Second (s)
Electric current Ampere (A)
Temperature Kelvin (K)
Amount of substance Mole (mol)
Luminosity Candela (cd)

The prefixes used with SI units indicate either a fraction or a multiple of a unit, depending in
part on the size of what is being measured and the degrees of accuracy of the measurement.
SI unit prefixes always express a power of ten by which the basic unit has been multiplied
(Table 2).
Table 2. SI prefixes
Factor Name Symbol Factor Name Symbol
1024 yotta Y 10-1 deci d
1021 zetta Z 10-2 centi c
1018 exa E 10-3 milli m
1015 peta P 10-6 micro µ
1012 tera T 10-9 nano n
109 giga G 10-12 pico p
106 mega M 10-15 femto f
103 kilo k 10-18 atto a
102 hecto h 10-21 zepto z
101 deka da 10-24 yocto y

When measuring the number, mass, length, or volume of objects, scientists write numbers in
scientific notation. A number is expressed in scientific notation when it is written as a product
of a decimal number between 1 and 9 and number 10 raised to the proper number.
100→ 1 102 3468.5→ 3.4685 103 0.0069→ 6.9 10-3
 2.2.2. Study Question
What do you observe in the laboratory? List materials.

2.3. Lab Exercise 2

2.3.1. Protocol
1. Convert the following measurements to scientific notation and indicated unit.

Table 3. Scientific notation, SI unit conversion table

Scientific Notation SI Unit conversion

0.00013 g mg
0.00000625 L ml
2323000 m km
10 μg kg
400 μl nl
1345 μg mg
1.645 km m
When making measurements, it is important to be as accurate and precise as possible.
Accuracy is a measure of how close an experimental measurement is to the true,
accepted value. Precision refers to how close repeated measurements (using the same
device) are to each other.

Ruler shown in left is marked in centimeters.

The mark could be estimated as 1.67. This
measurement has significant figures, one certain
(6) and one estimated (uncertain) (7). All certain
digits (including the estimated one) are called
significant figures.

Mass is measured using a balance or scale. The gram is the standard unit of mass. The
most common subdivision of gram used in laboratory are milligram (mg) and
microgram (μg). To protect the balance or for convenience for weighting, a paper or a
plastic container may be used during weighting. The weight of the container must be
subtracted to get the ‘net’ weight of the substance.

2.3.2. Study Questions

What is precise measurement?
What is accurate measurement?

2.4. Lab Exercise 3

2.4.1. Materials

Beaker, Scale/Balance, Spoon, Sugar

 2.4.2. Protocol

1. Obtain a 100-mL beaker and determine the mass of this beaker.

2. Afterwards add a spoon of sugar to the beaker. Do not do this over the balance!
3. Determine the new combined mass of both the beaker and the sugar.
4. Repeat this with 2-5 spoons of sugar.
5. Draw a graph using this data.
6. Determine the mass of the sugar using this data.

Table 4. Gross weight, net weight measurement table

Gross weight Net weight
1 spoon
2 spoons
3 spoons
4 spoons
5 spoons

2.4.3. Study Questions

What is the difference between spoons?

What is net weight and gross weight?

2.5. Lab Exercise 4

Life as you know depends on water and the types and concentrations of particles
dissolved in it. To determine the concentration of dissolved substances, you must be able
to measure volume accurately. For measuring large volumes of fluid, graduated cylinders
are used. There are two types of cylinders used, glass cylinders and cylinders made from
organic polymers. Water adheres to the glass rather than polymers and therefore water at
the top of a column will ‘climb’ to the sides creating a bowl like meniscus. To obtain a
precise measurement, volume must be read at the bottom of the ‘bowl’.
2.5.1. Materials
Graduated cylinder, Water

2.5.2. Protocol

1. Partially fill a 100-ml graduated cylinder with water and set it on table.
2. Measure the volume and height of volume from 3 positions indicated in the picture.
3. Record the results. Which one is the most accurate?
 Position Volume Height

Pipettes are glass vessels that are constructed and calibrated so as to deliver a precisely
known volume of liquid. Pipettes are usually used to measure liquid volumes of 10 ml or
less, although some types can measure 100 ml or more at a given temperature. The markings
on the pipet means that it was calibrated To Deliver (TD) a preset amount of liquid at 25°C.

2.5.3. Study Questions

What is the difference between 3 positions that you observed?
What is the correct position? Why?

2.6. Lab Exercise 5

2.6.1. Materials

Beaker, Erlenmeyer flask, Pipettes, Balance

2.6.2. Protocol

1. Obtain about 40 mL of distilled water in a 150-mL beaker.

2. Allow the water to sit on the desk while you weigh and record the weight of an empty,
dry 25-mL Erlenmeyer flask (tare) to the nearest 0.1 mg.
3. Using your pipette, pipette exactly 10.00 mL of water into this flask and weigh the flask
with the water. Obtain the weight of the water.
4. Using the equation below obtain the volume of water delivered and therefore the volume
of your pipette.

5. Normally, density is given in units of grams per milliliter (g/mL) for liquids, grams per
cubic centimeter (g/cm3) for solids, and grams per liter (g/L) for gases. Repeat this
procedure in triplicate-that is, deliver and weigh exactly 10.00 mL of water three
separate times.
Research laboratories need to measure small volumes accurately and repeatedly. To do this,
micropipettes are used. Micropipettes are devices to deliver small volumes. They have ranges
from 0.1μl to 1000μl and even fractions between. A plastic tip is used to hold the liquid and the
tip can be ejected easily.

2.6.3. Study Questions

Is there any difference between measurements? Why?

If your measurement different than expected, what could be the reasons?

2.7. Lab Exercise 6

2.7.1. Materials

Micropipettes, Parafilm, Balance

2.7.2. Protocol

1. Take a predetermined amount of water with your micropipette for ranges 1μl-10μl;
20μl-200μl and 200μl- 1000μl.
2. Put the water on a parafilm leaflet. Weight the sample.
3. Calculate the error of the micropipette. How precise are the micropipettes?

2.7.3. Study Questions

What are the error rates?

Which one is accurate and precise?

How precise are the micropipettes?

3. REFERENCES
Al Akhawayn University CHE1401 Laboratory Manual, 2015 p1-11
Helms, D.R, and S.B. Miller, Principles of Biology: A Laboratory Manual for Biology 110
Apex, NC: Contemporary Publishing, 1978
Dickey, J. Laboratory Investigations for Biology, Menlo Park, CA. Addison Wesley Longman,
1995.
Laboratory 4

ASEPTIC TECNIQUE And BACTERIAL GROWTH MEASUREMENT

1. Transfer of Bacteria: Aseptic Technique

A microbiological medium (media, plural) is the food that we use for culturing bacteria, molds,
and other microorganisms.

It can exist in three consistencies: liquid, solid, and semi-solid.

Liquid media are used for the propagation of large numbers of organisms, fermentation studies,
and various other tests.

Solid media are made by adding a solidifying agent, such as agar, gelatin, or silica gel, to a
liquid medium. A good solidifying agent is one that is not utilized by microorganisms, does not
inhibit bacterial growth, and does not liquefy at room temperature.

Aseptic is defined as an environment or procedure that is free of contamination by pathogens.

Because microorganisms are so small, large populations are generally needed in order to
monitor microbial activity. Aseptic techniques help ensure that only one type of microorganism
is present in a container (pure culture). These methods also ensure that the microorganisms do
not escape from the container, contaminating the laboratory, and possibly causing disease.

All culture media must be sterilized before use. Sterilization is usually carried on using an
autoclave. Containers of culture media, such as test tubes or petri plates should not be opened
until you are ready to work with them.

Aseptic transfer and inoculation are usually performed with a sterile, heat-resistant,
noncorroding Nichrome wire attached to an insulated handle.

• - When the end of the wire is bent into a loop, it is called an inoculating loop.
• - When straight, it is an inoculating needle.

According to different purposes, cultures can also be transferred by sterile cotton swabs,
pipettes, glass rods, or syringes.

The use of aseptic technique minimizes the risk of contamination of cultures and also reduces
the risk of micro-organisms from the laboratory cultures escaping to the environment.
Table 1: Potential points of contamination and the techniques employed to minimize the risk.

2. Measurement of Growth of Bacteria

Measurements of microbial growth is a way to measure growth of microbial populations. There

are various ways to measures microbial growth for the determination of growth rates and
generation times. Microbial growth can be measured by several methods such as cell count, cell
mass and cell activity.

1) Cell count this method involves the measurement of growth either by microscopy or by
using an electronic particle counter or indirectly by a colony count.

2) Cell mass in this growth can be measured directly by weighing or by a measurement of

nitrogen concentration in cells or indirectly by the determination of turbidity using
spectrophotometer.
3) Cell activity in this growth can be measured indirectly by analysis of the degree of
biochemical activity to the size of population.

2.1. Turbidity Estimation of Bacterial Numbers

Turbidity measurement depends on the fact that as the number of cells in a solution increase,
the solution becomes increasingly turbid (cloudy). The solution looks turbid because light
passing through it is scattered by the microorganisms present and the turbidity is proportional
to the number of microorganisms in the solution. The turbidity of a culture can be measured
using a spectrophotometer. Spectrophotometers use prisms or diffraction gratings supplying
a narrow band of wavelengths to the sample. It measures the amount of transmitted light, the
light that makes it from the light source through the sample to the detector.

**More cells = more turbidity; more turbidity = less light passing through the suspension

**T% is percent transmission - fewer cells present (less turbidity) will allow more light to pass
through, the T% is higher when the cell number is lower.

**Absorbance is inversely related with T %. More light is absorbed when more cells are present
- absorbance goes up as turbidity (or cell number) goes up.

Optical density (OD) is a measure of the light scattered by the bacterial suspension which
manifests itself as absorbance for a given wavelength. Higher OD means lower transmittance
value, higher number of cells and vice versa. Using a spectrophotometer to measure the optical
density at 600 nm (OD600) of a bacterial culture to monitor bacterial growth has always been
a central technique in microbiology. Three of the more common applications where bacterial
OD600 is used are the following:
 • Determination and standardization of the optimal time to induce a culture during
bacterial protein expression protocols.
• Determination and standardization of the inoculum concentration for minimum
inhibitory concentration (MIC) experiments.
• Determination of the optimal time at which to harvest and prepare competent cells.

Turbidity measurements can usually be made without destroying or significantly disturbing the
sample. For these reasons, turbidity measurements are widely used to follow the growth rate of
microbial cultures. The same sample can be checked repeatedly, and the measurements plotted
on a semilogarithmic plot versus time. From these, it is easy to calculate the generation time
and other growth parameters of the growing culture.

3. LAB EXERCISES
3.1. Lab Exercise 1
3.1.1. Materials
LB agar plates, lab marker
3.1.2. Protocol
• Divide your petri dishes into 4 sections by using marker, and label the quadrants like
A, B, C, D or 1, 2, 3, 4 (Attention!! Label the bottom not the lid of plate).
• Put your finger (without gloves) on to the first quadrant for few seconds.
• Clean your finger with 70% ethanol, and then put your finger onto to the second
quadrant.
• Touch the lab bench or your mobile phone and do the same thing as previously
explained in the third and fourth sections.
• Incubate the plates overnight at 37oC.
• Attention! Petri dishes should be placed upside down to prevent condensation
dropping on to cultures.

3.2. Lab Exercise 2

3.2.1. Materials
Tubes containing LB broth, E.coli culture , Spectrophotometer cuvettes (tubes),
Spectrophotometer
3.2.2. Protocol

• In this section, you will measure the OD values of E. coli culture.

• Transfer 1ml LB into the spectrophotometer cuvette (This is your blank).
• Transfer 1 ml of culture into the cuvette of the spectrophotometer.
• Measure the absorbance of culture at 600 nm wavelength.
Laboratory 5
ORGANIC MOLECULES
1. BACKGROUND
All known life is made out of a small group of chemical compounds called organic molecules.
Common organic molecules include proteins, carbohydrate, lipids, and nucleic acids. Each of
these classes of molecules has specific properties that can be identified by simple chemical
tests.
In this laboratory you will learn to identify three of the four major types of organic molecules:
carbohydrates, fats and proteins.
A. Carbohydrates
Carbohydrates is named so because many have formula Cn(H2O)n. The basic structural unit of
carbohydrates is the monosaccharide (or single sugar). Monosaccharides are classified by the
number of carbons they contain for example, trioses have three carbons, pentoses have five
carbons, and hexoses have six carbons. They may contain as few as three or as many as 10
carbons.

(a) (b) (c) (d) (e)

Figure 1. (a) Glyceraldehyde, a representative triose with an aldehyde reactive group. (b)
Ribose, a representative pentose with an aldehyde reactive group. (c) Fructose, a representative
hexose with a ketone reactive group. In solution, ribose and fructose occur predominantly in
ring forms (d,e) When heated, sugars in the ring form interconvert to chain form. Usually sugars
need to be in the chain form to interact with colorimetric substances.

Monosaccharides are also characterized by the presence of a terminal aldehyde group (Figure
1 a,b) or an internal ketone group (Figure 1c). Both of these groups contain a double-bonded
oxygen atom that reacts with Benedict’s reagent to form a colored precipitate.
When two monosaccharides are joined together, they form a disaccharide. If the reactive
aldehyde or ketone groups are involved in the bond between the monosaccharide units (as in
sucrose, Figure 2a), the disaccharide will not react with Benedict’s reagent. If only one group
is involved in the bond (as in maltose, Figure 2b), the other is free to react with the reagent.
Sugars with free aldehyde or ketone groups, whether monosaccharide or disaccharides, are
called reducing sugars (these sugars are oxidized (lose electrons to) by the Cu2+ in Benedict’s
reagent, which then becomes reduced (gains electrons); hence the name reducing sugar).
In this laboratory, you will use Benedict’s reagent to test for the presence of reducing
sugars.

Free
reactive
group

(a) (b)

Figure 2. (a) In sucrose, both reactive groups are involved in the bond between
monosaccharides, preventing a reaction with Benedict’s reagent. (b) In maltose, only one
reactive group is involved in the bond between monosaccharides, leaving the other group free
to react with Benedict’s reagent. Here the sugars are shown in the ring form. The reactive of
maltose is highlighted.

Monosaccharides may join together to form long chains (polysaccharides) that may be either
straight or branched. Starch is an example of a polysaccharide formed entirely of glucose units.
Starch does not show a reaction with Benedict’s reagent because the number of free aldehyde
groups (found only at the end of each chain) is small in proportion to the rest of the molecule.
Therefore, you will test for the presence of starch with Lugol’s reagent (iodine/potassium
iodide, I2KI).
B. Testing for Lipids
Organic molecules that are characterized by low solubility in water, that is, are relatively
hydrophobic. Lipids are soluble in nonpolar solvents such as chloroform (CHCI3). Although
lipids include fats, steroids and phospholipids, this exercise will focus primarily on fats.
Triglycerides are most common form of fat that consist of three fatty acids attached to a glycerol
molecule (Figure 3). Triglycerides are found predominantly in adipose tissue and store more
energy per gram than any other types of compounds.
At room temperature, some lipids are solid (generally those found in animals) and are referred
to as fats while others are liquid (generally those found in plants) and are referred as oils.
Vetetable oil, a liquid fat, is a mixture of triglycerides.
Since both solid and liquid fats are nonpolar, you will test for their presence by using Sudan
IV, a nonpolar dye that dissolves in nonpolar substances such as fats and oils but not in polar
substances such as water.

Figure 3. A triglyceride is composed of three

fatty acids and a glycerol molecule. Each bond is
formed when a molecule of water is removed by
condensation.
C. Testing for Proteins and Amino Acids
Proteins are made up of one or more polypeptides, which are linear polymer of smaller
molecules called amino acids (Figure 4). Amino acids derive their name from the amino group
and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids
are joined together by peptide bonds between the amino group of one amino acid and the
carboxyl group of a second amino acid.

(a)

(b)

Figure 4. (a) Structure of amino acid (Glycine). Note the presence of amino (-NH2) group and
a carboxyl group (-COOH) group. (b) Two amino acids are joined by a peptide bond when a
molecule of water is “split out” from their amino and carboxyl groups. A polypeptide is made
up of many amino acids joined in this way.
D. Analyzing Unknowns Qualitatively
Use knowledge gained in previous exercises to identify the organic molecules present in
unknown solutions.

2. LAB EXERCISES

2.1. Lab Exercise 1: Benedict’s Test for Reducing Sugars

When Benedict’s reagent is heated with a reactive sugar, such as glucose or maltose,
the color of the reagent changes from blue to green to yellow to reddish-orange,
depending on amount of reactive sugar present. Orange and red indicate the highest
proportion of these sugars (Benedict’s test will show a positive reaction for starch only
if the starch has been broken down into maltose or glucose units by excessive heating).

2.1.1. Materials

Benedict’s reagent, test tubes, solutions (starch, sucrose, fructose, glucose), milk, onion
juice, water, water bath.

2.1.2. Protocol

1. Label your test tubes and add 2 ml of the solutions listed in Table 1, matching each
number to the number on the tube.
2. Add one dropperful (approximately 2 ml) of Benedict’s reagent to each tube.
3. Mix the reagent and the sample by agitating the solution in each tube from side to side.
Record the original color of each tube’s contents in Table 1, under “Benedict’s Test”.
4. Heat the test tubes in a boiling water bath for 3 minutes. Record any color changes in
Table 1 under “Benedict’s Test”

2.1.3. Study Questions

Which is a reducing sugar, sucrose or glucose?

Which contains more reducing sugars, potato juice or onion juice?

Is there a difference between the storage of sugars in onions and potatoes?

2.2. Lab Exercise 2: Lugol’s Test for Starch

Lugol’s reagent changes from a brownish or yellowish color to blue-black when starch
is present, but there is no color change in the presence of monosaccharides or
disaccharides.

2.2.1. Materials

Lugol’s reagent, test tubes, solutions (starch, sucrose, glucose), potato, water.

2.2.2. Protocol

1. Prepare another test tubes as indicated in step 1 above and add 1 ml of the solutions
listed in Table 1, matching each number to the number on the tube.
2. Record the original color of each tube’s contents in Table 1.
3. Add several drops of Lugol’s reagent (I2KI) to each tube, mix, and immediately record
in Table 1 any color changes that take place. Do not heat the test tubes in the Lugol’s
test.

2.2.3. Study Questions

In the Iodine test, which of the solutions is a positive control? Which is a negative control?

Which colors more intensely, onion juice or potato juice?

Table 1. Data table for Benedict’s and Lugol’s Test

Benedict’s Test Lugol’s Test

Tube Original color Final color Original color Final color

Before Boiling After Boiling Before Adding I2KI After Adding I2KI

1. Glucose

2. Starch

3. Sucrose

4. Water

5. Milk

6. Fructose

7. Potato

2.3.Lab Exercise 3: Sudan IV Solubility Test

2.3.1. Materials
Sudan IV reagent, test tubes, sunflower oil, water.
2.3.2. Protocol

1. The familiar “grease spot” is the basis of very simple test for fats. On a piece of unglazed
paper, such as brown wrapping paper, place one drop of oil and one drop of water.
Allow the drops to dry.
2. Describe the difference between the oil spot and the water spot after a period of drying.
3. Label five tubes in sequence 1 through 5. Add 1 dropperful (1ml) of each substance you
have listed in Table 2 to the appropriate tube.
4. Add 3 drops of Sudan IV to each tube.
5. Mix and then add 2 ml of water to each tube. If fats or oils are present, these will appear
as floating red droplets or as a floating red layer colored by Sudan IV.
6. In Table 2, record the reactions that occur in each of the test tubes.
Table 2. Data table for the Sudan IV Solubility Test

Substance 1. Water 2. Water + oil 3. Oil

Sudan IV Solubility Reaction

 2.3.3. Study Questions

What do you conclude about the solubility of lipids in polar solvents such as water?

What do you think about the solubility of lipids in non-polar solvents such as chloroform?

2.4. Lab Exercise 4: Testing for Amino Acids with Ninhydrin

Ninhydrin reagent turns purple or violet in the presence of the free amino groups in
amino acids. In the presence of proline, however, it turns yellow. Proline reacts
differently because its amino group is not free but is, instead, part of the ring structure
of the molecule (Figure 5a,b).

Figure 5. (a) The amino acid Leucine, with free amino group. (b) Proline, with amino
group incorporated into a ring.

2.4.1. Materials

Ninhydrin reagent, test tubes, filter paper, Pasteur pipette, solutions (alanine, arginine,
proline), water.

2.4.2. Protocol

1. Obtain a piece of filter paper and divide it into four quadrants with a pencil. Letter the
quadrants A, B, C and D.
2. Place one drop of each solution (labeled A, B, C and D) onto the filter paper in the
quadrant with the corresponding letter. Allow the spots to dry.
3. Apply one drop of ninhydrin to each spot. Caution: Ninhydrin is poisonous; avoid
contact with your skin. Allow the paper to dry at room temperature for 20 to 30 minutes.
(The reaction will occur more quickly if the paper is lighly passed over a warm hotplate)
4. One of the solutions contains, proline, two of the solutions contain amino acids other
than proline, and one is distilled water. In table 3, indicate the content of each solution.
 Table 3. Data table for Ninhydrin Test
Solution Final color with Ninhydrin Type of Molecule in Solution

A. Blank

B. Arginine

C. Alanine

D. Proline

2.4.3. Study Questions

Which contains more protein (C-N bonds), egg albumen or honey?

Do free amino acids have peptide bonds? Explain why or why not

2.5. Lab Exercise 5: Analyzing Unknowns Qualitatively

2.5.1. Materials

Unknown solutions, Benedict’s reagent, Lugol’s Reagent, Ninhydrin reagent, Sudan IV

reagent, test tubes, water bath, filter paper, Pasteur pipette.

2.5.2. Protocol

Table 4. Data table for Analyzing Unknowns Qualitatively

Unknown Positive (+) or Negative (-) Results

Tested
Benedict’s Lugol’s Biuret Test Ninhydrin Sudan IV
Test Test Test Test

3. REFERNCES

Slavin, J., & Carlson, J. (2014), Carbohydrates. Advances in Nutrition, 5(6), 760–761. doi:
10.3945/an.114.006163

Holland, M. J., Dobi, K., (2019), Laboratory Notes for Bio 1003 Organic molecules - Laboratory tests,
retrieved from “http://faculty.baruch.cuny.edu/jwahlert/bio1003/organic_tests.html”
Laboratory 6

OSMOSIS AND DIFFUSION

1. BACKGROUND

Cell Membrane
The plasma membrane of the cell separates the inside of the cell from the outside, extracellular
matrix, and protects the cell from the environment. The plasma membrane is selectively
permeable phospholipid bilayer. It is, among other molecules, mainly made up of lipids,
specifically, phospholipids. Phospholipids are lipid molecules that have two portions,
hydrophilic head groups and hydrophobic fatty acid tail groups. The head groups contain
phosphate and are polar, thus allowing them to dissolve in water, which is also a polar group.
Fatty acid tail groups are hydrophobic, meaning that they are nonpolar and cannot dissolve in
water. Phospholipids have both polar, hydrophilic, and nonpolar groups, hydrophobic, making
them an amphiphilic molecule. In polar environments, like water, phospholipids form a
spherical liposome structure and keep the hydrophilic groups exposed to water while protecting
the hydrophobic portions from the water.

Figure 1. Liposome and bilayer formation of phospholipids. Phospholipids have both

hydrophobic tails and hydrophilic heads. In a polar environment, hydrophobic tails come
together, exposing the hydrophilic heads to polar environment.

The selective permeability of the plasma membrane comes from its amphiphilic nature. The
molecules that can dissolve in water, hydrophilic, polar molecules, cannot pass through the
nonpolar, hydrophobic, center of the bilayer. Charged atoms (ions), or polar molecules (ex.
glucose) are repelled by the nonpolar center. However, they can pass through the plasma
membrane with the help of membrane proteins that are specialized for the transport of these
molecules. On the other hand, hydrophobic molecules such as lipids can pass through the
membrane, as can small non-polar molecules (such as oxygen gas or carbon dioxide).
Maintaining the steady-state of a cell is achieved only through regulated movement of materials
through the cytoplasm, across organelle membranes and across the plasma membrane. This
regulated movement facilitates communication within the cell and between cytoplasm and the
external environment. The cytoplasm and extracellular environment of the cell are aqueous
solutions. They are composed of water, which is the solvent, or dissolving agent, and numerous
organic and inorganic molecules, which are solutes, or dissolved substances. Organic
membranes and the plasma membrane are selectively permeable, allowing water to freely pass
through but regulating the movement of solutes.
Molecular Transport
The cell actively moves dissolved substances across membranes, expanding adenosine
triphosphate (ATP) to accomplish the movement, which is called Active Transport. Passive
Transport is the passage of molecules without the expenditure of ATP from the cell if only the
membrane is permeable to those substances. In diffusion, the driving force of the movement is
the concentration difference. A molecule will diffuse from high concentration to low
concentration. If undisturbed, this molecule will continue to move to low concentration until it
reaches equilibrium, in which both concentrations are the same. However, membranes are not
permeable to all molecules. Atoms, ions and small, nonpolar molecules can pass freely through
the membrane. However, larger and polar molecules cannot freely diffuse through the
membrane. In this case, proteins on the membrane help the movement of these molecules across
the membrane without the expenditure of ATP. This type of transport is called Facilitated
Diffusion.
Osmosis
Osmosis is a type of diffusion in which the water molecules move through a membrane from
high concentration to low concentration. The difference in either side of the membrane occurs
if there is an unequal distribution of at least one dissolved substance that cannot pass through
the membrane and will generate osmotic pressure, cause the water to move to the side where
the dissolved substance is in higher concentration. This substance that generates osmotic
pressure is called “Osmotically Active Substance (OAS)”.
Tonicity
There are three terms that are used when referring to two solutions separated by a selectively
permeable membrane; hypertonic, hypotonic and isotonic. Hypertonic solutions will have
higher concentrations of OAS compared to the solution on the other side of the membrane, it is
also referred to as “having higher osmolarity” (Hyper-: excessively, above normal). Hypotonic
solutions will be the opposite of hypertonic solutions. Hypotonic solutions will have lower
concentration of OAS, and lower osmolarity, compared to the solution on the other side of the
membrane. (Hypo-: below normal). When the concentrations of OAS of two solutions are in
equilibrium, thus having equal osmolarity, the solutions are isotonic and there will be no
movement of the water across the membrane. (Iso-: equal).
Figure 2. The movement of water in hypotonic, hypertonic and isotonic solutions. (a) The
concentration of OAS in the cell is higher compared to the environment, causing the water to
move into the cell and cell to swell. (b) The cell is in a hypertonic solution, the environment
has higher OAS concentration. This causes the water to move out of the cell and shrinkage of
the cell. (c) The cell is in isotonic solution, the osmolarities of both the cell and the environment
are the same. There is no significant water movement in or out of the cell.

2. LAB EXERCISES

2.1. Lab Exercise 1: Diffusion

In this section, you will investigate the permeability of dialysis tube, which is a membrane made
of regenerated cellulose fibers formed into a flat tube. If two solutions containing dissolved
substances of different molecular weights are separated by this membrane, some substances
may readily pass through the pores of the membrane, but others may be excluded. You will be
given a dialysis tube containing starch and glucose and investigate the permeability of dialysis
tube for both of these molecules with Benedict’s and Lugol’s reagents.
2.1.1. Materials
Dialysis tube, Beaker, Eppendorf tubes, Starch, Glucose, Benedict’s reagent, Lugol’s
reagent
2.1.2. Protocol

1. Place the dialysis tube inside the beaker filled with dH2O
2. Wait 10-15 minutes
3. Take out the dialysis tube
4. Label 2 Eppendorf tubes for Lugol’s test and Benedict’s Test
5. Add 500 µL of the water sample from the beaker into both of the labeled tubes.
6. Do the Lugol’s and Benedict’s test for both of the samples
7. Record color changes
2.1.3. Study Questions
Do you expect color change in both samples?
What is the reason of color change or not in your samples?
What would you expect to observe in glucose solution within beaker and membrane with
respect to color change with Benedict’s reagent, if we checked the solution in every 30
minutes?

2.2. Lab Exercise 2: Osmosis

In this section, you will investigate the movement of water through the dialysis tube. As
mentioned above, the water will move to the side with the higher concentration of OAS.
You will be given two dialysis tubes, one has starch, other has dH2O, and compare the
differences in their weights before and after they wait in different environments

2.2.1. Materials
Dialysis tubes, Beakers, Starch, Balance

2.2.2. Protocol

1. Weight both of the dialysis tubes and record their weights

2. Place the dialysis tubes into the beakers. Dialysis tube containing starch goes into the
beaker filled with H2O and vice versa
3. Wait 10-15 minutes
4. Take out both tubes and weight

2.2.3. Study Questions

What is the final weight of dialysis tubes? Is that increased-decreased or same compare to
initial weight? Why?

2.3. Lab Exercise 3: Tonicity

You will investigate the movement of water through the dialysis tube in different
environments. You will be given 4 beakers, one filled with dH2O and the other 3 are
unknown in terms of their tonicity, and 4 identical dialysis tubes containing a certain amount
of starch. Place them into the 4 beakers and determine the tonicities of three unknown
beakers.
2.3.1. Materials
Dialysis tubes, Beakers, Balance
2.3.2. Protocol:

1. Label and weight the dialysis tubes

2. Place the dialysis tubes into 4 beakers
3. Wait 10-15 minutes
4. Take the tubes out and weight
5. Determine the tonicities of unknown solutions depending on the weight changes

2.3.3. Study Questions

What should be final weight of dialysis tubes in hypertonic, hypotonic and isotonic
solutions?

3. REFERENCES
Dickey, J. Laboratory Investigations for Biology, Menlo Park, CA. Addison Wesley
Longman, 1995.
Al Akhawayn University CHE1401 Laboratory Manual, 2015 p1-11
Helms, D.R, and S.B. Miller, Principles of Biology: A Laboratory Manual for Biology 110
Apex, NC: Contemporary Publishing, 1978
Laboratory 7

MITOSIS

1. BACKGROUND

An important characteristic of living cells is their ability to divide, producing two daughter
cells, which are genetically identical. Prior to division, cells undergo a growth process in which
molecules, such as fats, proteins, and nucleic acids, are synthesized from food molecules using
energy derived from respiration. Molecular synthesis alone, however, is not sufficient to ensure
proper growth. Molecules must assemble or be assembled into eukaryotic cellular components,
such as plasma membranes, ribosomes, mitochondria, and chromosomes. Before dividing, a
cell contains hundreds of mitochondria, thousands of ribosomes, and literally trillions of small
molecules, such as amino acids and sugars. Cells in balanced, continuous growth double their
components and then divide these components in half, producing two equal daughter cells.
Most eukaryotic cells have only one nucleus, and its division involves a special mechanism.
The important contents of the nucleus are the chromosomes, the carriers of hereditary
information. (Different organisms have different number of chromosomes in their nuclei: for
example, the donkey has 66, humans have 46, and fruit flies, 8.)
During the growth period, these chromosomes make copies of themselves. Each duplicated
chromosome consists of two strands of genetic information called sister chromatids (Figure 1).
Each sister chromatid consists of a single long DNA molecule that is coiled, folded, and
wrapped around structures called nucleosomes. These are composed of proteins called histones.
Each microscopic chromosome in an onion cell contains a highly folded DNA molecule that is
about a meter long. Mitosis is the process by which the nucleus equally divides its contents,
including the copies of chromosomes, to form two daughter nuclei. As a cell enters mitosis, its
nuclear envelope breaks down, a spindle opposite poles of the cell attach to each of the two
chromatids. The fibers attach to each chromatid at centromere, a locally constricted region of a
chromosome where the chromatids are held together, and a kinetochore plate is found.

Figure 1. How DNA is

organized in a chromosome. A
chromosome consists of two
sister chromatids joined at
centromeres during metaphase.
As a cell progresses through mitosis, the centromeres split, and the spindle fibers pull the
separated sister chromatids to opposite poles of the cell. The cell then divides along the
centerline, producing two daughter cells that each have a copy of all the chromosomes that were
in the mother cell prior to growth and division (Figure 2).

Figure 2. Chromosome positions during stages of mitosis in a hypothetical animal cell.

Following mitotic nuclear division, also called karyokinesis, the cytoplasm divides by
cytokinesis. Fundamentally different cytokinesis mechanisms are found in animal and plant
cells. In animal cells, the cytoplasm divides by constricting inward in a process called
furrowing. In the furrow region, the protein actin, the same one involved in muscle contraction,
encircles the cell. This contractile ring gradually pinches the cell in half, forming the daughter
cells.
In plant cells, there is no constriction process. Instead, membrane vesicles containing cell wall
components and derived from the Golgi apparatus migrate to the center of the cell and form a
plate (phragmoplast) across the center of the mother cell. These vesicles fuse with each other
and the plasma membrane to form the end membranes of two new daughter cells. Hence, the
phrase cytokinesis by cell plate formation is used.
The alternating periods of growth (interphase) and division (phrases of mitosis and cytokinesis)
are called the cell cycle. Interphase can be divided into three subphases known as G1, S and
G2. During the S phase the DNA of the chromosome duplicates.
 2. LAB EXERCISES

2.1. Lab Exercise 1: Mitotic Cells in Onion Tip

2.1.1. Materials
Compound microscope, prepared slides with onion root cells
2.1.2. Protocol

1. Observe a prepared slide of a whitefish blastula under scanning power with your
compound microscope. Note that several sections of the blastula are on the slide.
2. Center a cell containing chromosomes in the field of view and observe it first under
medium power and then with the high-power objective. Sketch the cell, indicating such
features as the spindle, chromosomes, centromeres, and asters.
3. Locate another cell in which the chromosomes are visible. Changes are that the
chromosomes are not aligned in the same patterns as in the previous cell.
4. Look at various cells on the blastula slide and identify the mitotic stages. You should
find examples of interphase, prophase, metaphase, anaphase and telophase. Use Figure
3 to help you identify the stages. Sketch each stage and label the structures.

Figure 3. Stages of mitosis in an onion root tip as seen in two types of preparations.
2.1.3. Study Questions
Why is regulation of cell division necessary?
What are the stages of cell cycle?
Why the parent cell and both daughter cells must have the same number of chromosomes?
What is the difference between cytokinesis in animals and plants?
 3. REFERENCES
Alberts, B., Bray, D. Lewis, J., Raff, M., Roberts, K., and Watson, J. D. Molecular Biology of
the Cell 3rd ed. New York: Garland, 1994.
Komberg, R., and Klug, A. The nucleosome, Scientific American, 244 (2): 52-64, 1981
Mcintosh, R. R., and McDonald, K.L. The mitotic spindle, Scientific American, 261 (4): 48-56,
1989.
Mazia, D. The Cell Cycle. Scientific American, 230(1): 54-68, 1974.
Laboratory 8

MEIOSIS

1. BACKGROUND

Meiosis is a sort of cell division that decreases the number of chromosomes in the parent cell
by half and generates four gamete cells. This division is required to generate egg and sperm
cells for sexual reproduction. During reproduction, as the sperm and egg combine to form a
single cell, the number of chromosomes is regenerated in the offspring.
Meiosis starts with a parent cell which is diploid because it has two copies of each chromosome.
The parent cell makes two separate cycles of nuclear division following one round of DNA
replication. At the end, four daughter cells that are haploid, meaning they contain half the
number of chromosomes of the diploid parent cell are obtained.
Meiosis and mitosis as two cell division processes have both similarities and differences.
Firstly, mitosis which is a cell division process results in two identical daughter cells in contrast
to meiosis producing four daughter cells with haploid chromosome number. Meiosis starts after
one round of DNA replication in cells of the male or female sex organs. The process is divided
into two phases as meiosis I and meiosis II that both have multiple phases. Meiosis I is a kind
of cell division restricted to germ cells, while meiosis II resembles to mitosis. Moreover,
contrary to mitotic division, meiosis results in genetic variation. Apart from those differences,
mitosis and meiosis have similarities like the production of new cells and requirement of a
single cell to divide.

Figure 1. Schematic representation of mitosis vs meiosis (Pearson education, 2009).

Figure 2. Schematic representation of meiosis II (Thomson Learning, 2001).
A. Meiosis I
The first meiotic division starts with prophase I. During prophase I, the complex of DNA and
protein named as chromatin condenses to generate chromosomes. The pairs of replicated
chromosomes are called as sister chromatids which remain joined at a central point called the
centromere. A large structure named as the meiotic spindles are generated from long proteins
called microtubules on each side, or pole, of the cell. Between prophase I and metaphase I,
homolog chromosome pairs are found in tetrad structure. This tetrad formation enables any pair
of chromatin arms to overlap and to fuse in a process crossing-over or recombination.
Recombination is a process which breaks, recombines and rejoins sections of DNA to obtain
new combinations of genes. In metaphase I, homolog chromosome pairs align on either side
of the equatorial plate. Then, in anaphase I, the spindle fibers contract and pull the homologous
pairs, each with two chromatids, away from each other and toward each pole of the cell. During
telophase I, the chromosomes are enclosed in nuclei. The cell now undergoes a process called
cytokinesis that divides the cytoplasm of the original cell into two daughter cells. Each daughter
cell is now haploid and has only one set of chromosomes, or half the total number of
chromosomes of the original cell.
B. Meiosis II
The second part of the meiosis resembles to mitotic division by which each of the haploid cells
from meiosis I are directed to meiosis II. During prophase II, the chromosomes become
condensed, and a new set of spindle fibers forms. The chromosomes begin to move toward the
Figure 3. Schematic representation of meiosis II (Pearson Education, 2009)

equator of the cell. Continuing with metaphase II, the centromeres of the paired chromatids
align along the equatorial plate in both cells. Then in anaphase II, the chromosomes are
separated by pulling of spindle fibers at the centromeres. The spindle fibers pull the separated
chromosomes toward each pole of the cell. Finally, during telophase II, the chromosomes are
enclosed in nuclear membranes. Cytokinesis follows the division of the cytoplasm to two cells.
At the conclusion of meiosis, there are four haploid daughter cells that go on to develop into
either sperm or egg cells.

2. LAB EXERCISES
2.1. Lab Exercise: Meiosis in Plant Cells - Meiotic cells in Lily Anther

Figure 4. Schematic representation of meiosis stages (Pearson Education, 2009).

Lily is a flowering plant which have both male and female organs. Anthers are the male
reproductive structures of flowering plants that produce pollen by meiosis.
 2.1.1. Materials
Compound microscope, prerapred slides for meiosis-I, meiosis-II, mature pollen, pollen
tetrad

2.1.2. Protocol

1. Observe a prepared slide of a lily anther under scanning power with your compound
microscope. Note that several sections of the lily anther are on the slide.
2. Look at first meiotic division, second meiotic division, pollen tetrads, mature pollen
slides.
3. Identify differences between meiosis I and meiosis II.

Figure 5. Stages of meiosis in a lily anther

2.1.3. Study Questions

What are differences between mitosis and meiosis?
How does the alternation of meiosis and fertilization in the life cycles of sexually
reproducing organisms maintain the normal chromosome count for each species?
How are the chromosomes in a cell at metaphase of mitosis similar to and different from
the chromosomes in a cell at metaphase of meiosis II?
What is the original source of variation among the different alleles of a gene?
3. REFERENCES

Meiosis (2014), Nature Education

Retrieved from https://www.nature.com/scitable/definition/meiosis-88/ on Nov 12,
2019
Ohkura H. (2015). Meiosis: an overview of key differences from mitosis. Cold Spring
Harbor Perspectives in Biology, Vol. 7, No 5.
Hall, P. (2009). Pearson Education. United States of America.
Thomson, M. (2001), Thomson Learning, London.
Laboratory 9
DNA ISOLATION
1. BACKGROUND
For further molecular biology and genetics analysis in the studies, DNA extraction is the first
step. After isolation of DNA, it can be used for many downstream applications such as; PCR
(polymerase chain reaction, RFLP (restriction fragment length polymorphism), Southern
Blotting, study for DNA-protein interactions, gene expression, genome analysis, genome
sequencing, etc. Basically, DNA extraction is divided into three; homogenization, degradation,
and recovery of DNA.
HOMOGENIZATION
In the homogenization step, the main goal is to disrupt tissue structure, the cell wall and
membrane structure of the cells leading to releasing of all contents to the medium. The obtained
suspension is called homogenate including decomposed cells, membrane pieces, organelles,
and other cell components. There are many homogenization methods dividing into two groups;
physical and chemical fragmentation methods.
Physical Methods
Physical methods include the mechanical method, mortar, and pestle, liquid homogenization,
ultrasonication, osmotic shock, and freeze-thaw.
A. Mechanical Disruption: Mechanical methods rely on the usage of rotating blades to grind
and dispense large amounts of complex tissue, such as liver or muscle. The Waring blender and
the Polytron are commonly used for this purpose. Unlike the Waring blender, which is similar
to a standard household blender, the Polytron draws the tissue into a long shaft containing
rotating blades. The shafts vary in size to accommodate a wide range of volumes and can be
used with samples as small as 1 mL.
B. Mortar and Pestle: Manual grinding is the most common method used to disrupt plant cells.
The tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of
the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this
method is the fastest and most efficient way to access plant proteins and DNA. The tissue also
can be ground using mortar and pestle in the lysis buffer without frozen in liquid nitrogen.
C. Liquid Homogenization: Liquid-based homogenization is the most widely used cell
disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or
tissue suspension through a narrow space, thereby shearing the cell membranes. Three different
types of homogenizers are in common use; Dounce homogenizer, Potter-Elvehjem
Homogenizer and French Press.
D. Ultra Sonication: Ultrasonic homogenizers, also called as disintegrators or sonificators, are
based on the electric effect while generating the high energy or ultrasonic wave, interacting
with the sample. Energy, resolved after explosion/implosion of gas microbubbles, effectively
destroys solid particles such as cells. Ultrasonic devices are mainly used to homogenize small
pieces of soft tissues (brain, blood, liver, etc.). Tough and dense tissues are not recommended
to homogenize using this equipment.
E. Osmotic Shock: Transfer of the cells from a solution with the high osmotic potential to a
hypotonic solution causes the flowing of water inside the cell from outside resulting in bursting
of cell membrane. This method is suitable for the cells without cell wall. It is suitable for blood
cells.
F. Freeze-Thaw: The freeze-thaw method is commonly used to lyse bacterial and mammalian
cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and
then thawing the material at room temperature or 37 ᵒC. This method of lysis causes cells to
swell and ultimately break as ice crystals form during the freezing process and then contract
during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite
lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins
located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in
some protocols.
Chemical Methods
Chemical methods include chemicals used in DNA isolation for both degradation steps and
recovery steps. In the DNA extraction procedure, buffers are used to avoid unwanted chaos in
the cell lysate. Their usefulness stems mostly from their ability to resist changes in pH. Besides
this, due to including other chemical agents, buffers serve degradation and lysis steps. There
are different buffers for DNA extraction (ex; STE, CTAB buffers).
Depending on the tissue type, aim, cost, time and resource, the contents and concentrations are
changed and modified. Below the purpose of using chemicals is indicated.
Organic solvents are used for the degradation of cell stabilization and structures including cell
membrane, lipids, protein structures to make DNA more exposed. For this purpose, usage of
organic solvents such as ethyl-acetate, tolüene degrades membrane lipids. Using detergents
such as SDS (sodium dodecyl sulfate), CTAB (Cetyl trimethylammonium bromide) removes
proteins and lipoproteins of the membrane.
NaCl is used to remove polysaccharides. Usage of salts during precipitation of DNA increases
the solubility of polysaccharides in ethanol thus prevents co-precipitation with DNA. PVPP is
used to eliminate polyphenols during DNA purification.
ß-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction
because it is a strong reducing agent that can remove tannins and other polyphenols often
present in the crude plant extract. It may also help to denature proteins by breaking disulfide
bonds between cysteine residues.
Phenol and chloroform are used for DNA extraction for years and years. The phenol,
chloroform (and also isoamyl alcohol) are added in a specific ratio of 25:24:1. Phenol dissolves
organic impurities, like proteins, etc. Chloroform provides density to phenol so that it settles
below water during phase separation. Isoamylalcohol is used to prevent phosgene from the
reaction of chloroform with air. The Phenol:Chloroform:Isoamylalcohol (PCI) solution is
added to the cell extract after the removal of debris. After proper mixing, centrifugation is done
to separate the phases. Two phases are formed; the upper aqueous phase that contains DNA and
the lower phenol phase that contains organic impurities. Thus two phases are separated by a
very clearly defined boundary of coagulated proteins. The aqueous phase is precipitated,
allowing the DNA to be pelleted after rounds of purifications.
Enzymes are used for degradation of proteins, lipids, and structures. For example, Proteinase
K is used for protein degradation, lysozyme is used for hydrolysis of glycosidic bonds of
bacterial cell walls. RNAse is an enzyme that degrades the RNA and is used to eliminate RNA
contamination in the DNA sample.
EDTA is added to extraction buffers. It binds to +2 charged ions and breaks the stabilization of
membranes. It binds to Mg+2 and inhibits DNAse enzyme which degrades DNA.
The main role of isopropanol is to precipitate the DNA by engaging with water molecules and
not allowing DNA to be dissolved in water.
Cellular and histone proteins bound to DNA can be removed either by adding protease enzymes
or by having precipitated the proteins with sodium acetate or ammonium acetate or extracting
them with a phenol-chloroform mixture prior to the DNA-precipitation. If desired, the DNA
can be resolubilized in a slightly alkaline buffer or in ultra-pure water. Generally, the acetate-
compound strength is high to keep up with the alkaline environment for keeping the DNA
solubilized.
DNA ISOLATION METHODS
Many different methods and technologies are available for the isolation of genomic DNA. In
general, all methods involve disruption and lysis of the starting material followed by the
removal of proteins and other contaminants and finally recovery of the DNA. Removal of
proteins is typically achieved by digestion with Proteinase K, followed by salting-out, organic
extraction, or binding of DNA to a solid-phase support (either with anion-exchange or silica
technology). DNA is usually recovered by precipitation using ethanol or isopropanol. The
choice of a method depends on many factors; the required quantity and molecular weight of the
DNA, the purity required for downstream applications and the time and expense. Several of the
most commonly used methods are detailed below. However, many different methods and
variations of these methods exist. Home-made methods often work well for researchers who
have developed and regularly use them. However, these methods usually lack standardization
and therefore yields and quality of them are not always reproducible. Reproducibility is also
affected when the method is used by different researchers, or with different sample types.
Preparation of Crude Lysates
An easy technique for isolation of genomic DNA is to incubate cell lysates at high temperatures
(e.g., 90℃ for 20 minutes), or to perform a Proteinase K digestion, and then use the lysates
directly in downstream applications. Considered “quick-and-dirty” techniques, these methods
are only appropriate for a limited range of applications. The treated lysate usually contains
enzyme-inhibiting contaminants, such as salts, and DNA is often not at the optimal pH.
Furthermore, incomplete inactivation of Proteinase K can result in false-negative results and
high failure rates. It is not recommended to store DNA prepared using this method, as the high
levels of contamination often result in DNA degradation.
Salting-out Methods
Starting with a crude lysate, “salting-out” is another conventional technique where proteins and
other contaminants are precipitated from the cell lysate using high concentrations of salt such
as potassium acetate or ammonium acetate. The precipitates are removed by centrifugation, and
the DNA is recovered by alcohol precipitation. Removal of proteins and other contaminants
using this method may be inefficient, and RNAse treatment, dialysis and/or repeated alcohol
precipitation are often necessary before the DNA can be used in downstream applications. DNA
yield and purity are highly variable using this method.
Organic Extraction Methods
Organic extraction is a conventional technique that uses organic solvents to extract
contaminations from cell lysates. The cells are lysed using a detergent and then mixed with
phenol, chloroform and isoamylalcoholç The correct salt concentration and pH must be used
during extraction to ensure that contaminants are separated into the organic phase and that DNA
remains in the aqueous phase. DNA is usually recovered from the aqueous phase by alcohol
precipitation. This is a time-consuming and cumbersome technique. Furthermore, the procedure
uses toxic compounds and may not give reproducible yields. DNA isolated using this method
may contain residual phenol and/or chloroform, which can inhibit enzyme applications such as
PCR. The process also generates toxic waste that must be disposed of with care and in
accordance with hazardous waste guidelines. In addition, this technique is almost impossible to
automate, making it unsuitable for high-throughput applications.
Cesium Chloride Density Gradients
Genomic DNA can be purified by centrifugation through a cesium chloride (CsCl) density
gradient. Cells are lysed using a detergent and the lysate is alcohol precipitated. Resuspended
DNA is mixed with CsCl and ethidium bromide (EtBr) and centrifuged for several hours. The
DNA band is collected from the centrifuge tube, extracted with isopropanol to remove EtBr and
then precipitated with ethanol to recover DNA. This method allows the isolation of high-quality
DNA but is time-consuming, labor-intensive and expensive (an ultracentrifuge is required),
making it inappropriate for routine use. This method uses toxic chemicals and is also impossible
to automate.
2. LAB EXERCISES

2.1. Lab Exercise: Genomic DNA Isolation

2.1.1. Materials
Tissue sample, extraction buffer, mortar and pestle, ß-Mercaptoethanol, heater, centrifuge,
vortex, Chloroform:Isoamylalcohol, isopropanol, ethanol, TE buffer.
2.1.2. Protocol

1. Take about 200 mg tissue, add 600 uL extraction buffer (CTAB or STE) and
homogenize it with suitable homogenization step (blender, mortar and pestle, liquid
nitrogen or any other homogenization method)
2. After homogenization, add 100 uL ß-Mercaptoethanol to sample and vortex
3. Incubate samples at 65℃ for 45-60 minutes.
4. Centrifuge the samples after incubation at 14000 rpm for 10 minutes
5. Transfer the supernatant (liquid phase) to a new tube
6. Add 600 uL Chloroform:Isoamylalcohol (CI) and vortex well. Be careful CI is
harmfull!
7. After vortex, centrifuge the samples at 14000 rpm for 10 minutes.
8. Transfer the supernatant to a new tube.
9. Add 250 uL cold isopropanol to each tube and leave it for 15 minutes at 4℃.
10. Centrifuge at 14000 rpm for 10 minutes. Remove the isopropanol from the tubes
(supernatant).
11. Wash samples by adding 500 uL ethanol to each tube.
12. Centrifuge at 14000 rpm for 3-5 minutes (spin).
13. Remove the ethanol. Open the lids of tubes and leave them at room temperature until
they dry. Or leave at drying oven at 65℃ for 15-25 minutes until they dry.
14. Resuspend the pellet in 15-25 uL TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA,
pH 8.0).
15. Incubate tubes at 65℃ for 45-60 minutes and store in -20℃.

2.1.3. Study Questions

What DNA is used for in scientific applications?
What is the purpose of homogenization?
What are the types and differences between them?
What are the functions of each chemicals for homogenization in chemical methods?
What are the step of DNA isolation?
How many methods are there for DNA isolation?
Compare all methods and which one is the most advantageous? Why?

3. REFERENCES
Douglas Hayworth, Ph.D. Thermo Scientific
Vijai Singh and Vinay Kumar (2012). An optimized method of DNA isolation from highly
mucilage-rich okra (Abelmoschus Esculentus L.) or PCR analysisç Advances in Applied
Science Research, 3 (3):1809-1813ç
Dr. Vikash Kumar. Dubey Proteomics & Genomics
Laboratory 10
AGAROSE GEL ELECTROPHORESIS
1. BACKGROUND
Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins.
Most every molecular biology research laboratory routinely uses agarose gel electrophoresis
for the preparation and analysis of DNA (PCR products, restriction products, isolated
DNA/RNA materials).
In this experiment we will be using agarose gel electrophoresis to determine the presence of
isolated DNA sample and to make quantitative and qualitative check of the DNA.
Electrophoresis is a method of separating substances based on the rate of movement while under
the influence of an electric field. Agarose gel electrophoresis is a separation technique which is
based on charge and size of the molecules.
Agarose is a polysaccharide purified from seaweed. Agarose which is linear polymer agarose
is a polysaccharide, whose monomeric unit is a disaccharide of D-galactose and 3,6- anhydro-
L-galactopyranose (Figure 1). An agarose gel is created by suspending dry agarose in a buffer
solution, boiling until the solution becomes clear, and then pouring it into a casting tray and
allowing it to cool. The result is a flexible gelatin-like slab. When it starts to cool, it undergoes
cross-linking (H-bonding) and results in formation agarose gel matrix. Extent of cross-linking
depends on percentage of agarose (higher percentage results in higher cross linking thus more
sieving effect due to small pore size).

a b

Figure 1. (a) Monomeric unit of the agarose. (b)Formation of gel matrix

DNA is negatively charged because of the negative charge on the sugar-phosphate backbone of
DNA polymers (Figure 2). This causes migration of DNA molecules towards the positive
electrode when placed in an electrical field. The rate of movement towards the positive end of
the electrical field is affected by the composition of the material the DNA is placed in. The
migration rate of the DNA is mainly affected by the factors such as size of the DNA, agarose
concentration used and conformation of the DNA. The pores of the agarose gel restrict the
movement of the DNA and creates an environment in which each individual DNA
molecule/fragment’s rate of movement varies based on its length. Smaller DNA molecules
move through the agarose faster than larger molecules. DNA itself is not visible within an
agarose gel. The DNA will be visualized using a dye that binds to DNA.
Figure 2. DNA structure

During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a
positive and negative electrode (Figure 3). The DNA to be analyzed is forced through the pores
of the gel by the electrical current. Under an electrical field, DNA will move to the positive
electrode (red) and away from the negative electrode (black). Several factors influence how fast
the DNA moves, including; the strength of the electrical field, the concentration of agarose in
the gel and most importantly, the size of the DNA molecules.

a b

Figure 3. (a) Agarose gel electrophoresis system. (b) Details of gel system.

2. LAB EXERCISES

2.1.Lab Exercise
 2.1.1. Materials
• Agarose
• 1x TAE Buffer
• EtBr (DNA stain)
• 6x Sample Loading Buffer
• DNA ladder standards
• Electrophoresis chamber and Power supply
• Gel casting tray and combs
• Staining tray
• Gloves
• Pipette and tips
• DNA samples
1X TAE Buffer Recipe:
• 4.84 g Tris Base
• 1.14 ml Glacial Acetic Acid
• 2 ml 0.5M EDTA (pH 8.0)
• bring the total volume up to 1L with water
Add Tris base to ~900 ml H2O. Add acetic acid and EDTA to solution and mix. Pour mixture
into 1 L graduated cylinder and add H2O to a total volume of 1 L.
Note – for convenience a concentrated stock of TAE buffer (either 10X or 50X) is often made
ahead of time and diluted with water to 1X concentration prior to use.
6X Sample Loading Buffer Recipe:
• 1 ml sterile H2O
• 1 ml Glycerol
• enough bromophenol blue to make the buffer deep blue (~ 0.05 mg)
for long term storage, keep sample loading buffer frozen.

2.1.2. Protocol

2.1.2.1.Preparing the agarose gel (2%, total volume 300 ml)

(Amount and volumes change depending on size of agarose gel system in the laboratory.)
1. Measure 6 g Agarose powder and add it to a 500 ml flask
2. Add 300 ml 1x TAE Buffer to the flask and mix (the total gel volume can change
depending on the size of the casting tray).
3. Melt the agarose in a microwave (or hot water bath) until the solution becomes clear.
(if using a microwave, heat the solution for several short intervals - do not let the
solution boil for long periods as it may boil out of the flask).
4. Meanwhile, seal the ends of the casting tray with two layers of tape.
5. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly.
6. Add 2.5 μl EtBr into the gel solution when it cools to 55°C (Do not touch EtBr with
bare hands).
7. Pour the melted agarose solution into the casting tray and place the combs in the gel
casting tray.
8. Let cool until it is solid (it should appear milky white) (10-15 mins depending on
concentration of the gel).
9. Carefully pull out the combs and remove the tape.
10. Place the gel in the electrophoresis chamber.
11. Add enough 1x TAE Buffer so that there is about 2-3 mm of buffer over the gel.

Note – gels can be made several days prior to use and sealed in plastic wrap (without
combs). If the gel becomes excessively dry, allow it to rehydrate in the buffer within
the gel box for a few minutes prior to loading samples.

2.1.2.2.Loading the gel

Figure 4. Examples of gel loading.

1. Add 1 µl of Sample Loading Buffer to each DNA sample (15-20 μl) (isolated DNA or
PCR products) If you have low volume of the sample you can add distilled water.
2. Load samples, positive control and ladder into wells of the gel.
3. Record the order each sample will be loaded on the gel, including who prepared the
sample, controls and ladder.
4. Carefully pipette 20 µl of each sample/Sample Loading Buffer mixture into separate
wells in the gel.
2.1.2.3.Running the gel
1. Place the lid on the gel box, connecting the electrodes.
2. Connect the electrode wires to the power supply, making sure the positive and negative
are correctly connected (Remember – “Run negative to positive because DNA is
negatively charged)
3. Turn on the power supply to about 100 volts. Maximum allowed voltage will vary
depending on the size of the electrophoresis chamber
4. Make sure the current is running through the buffer by looking for bubbles forming on
each electrode.
5. Make sure that the current is running in the correct direction by observing the movement
of the blue loading dye – this will take a couple of minutes (it will run in the same
direction as the DNA).
6. Let the power run until the blue dye approaches the end of the gel.
7. Turn off the power.
8. Disconnect the wires from the power supply.
9. Remove the lid of the electrophoresis chamber.
10. Using gloves, carefully remove the tray and gel.
11. Use gel imagination system to get image under UV light.
12. Compare your results with the ones below (Figure 5).

a b

Figure 5. (a) Genomic DNA. (b) Degraded genomic DNA.

2.1.3. Study Questions

What is a marker or ladder? Why is this considered a standard in this lab?

Why is a ladder or standard necessary part of this lab? Why does each lab team need to run
their own ladder or standard?

How does the size of the DNA fragment affect its movement or migration through the
agarose gel during electrophoresis?

Name three components found in the sample loading buffer. What is the purpose of each
of these components?

Predict what would happen if you forgot to add the sample loading buffer?

Why do you add 1X TAE buffer to the gel box?

What would happen if you added water instead of the 1X TAE buffer and ran the gel with
the water?

3. REFERENCES
Lee PY, Costumbrado J, Hsu CY, Kim YH (2012). Agarose electrophoresis fort he
separation of DNA fragments. J Vis Exp. (62):3923
Wave JS, Fourney RM. (1990). Agarose gel electrophoresis of linear genomic DNA in the
presence of ethidium bromide: band shifting and implications for forensic identity testing.
TAG, 1(4):193-6.
Voytas D. (2001). Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. Chapter 2:
Unit2.5A. doi: 10.1002/0471142727.mb0205as51.
Laboratory 11
PHOTOSYNTHESIS
1. BACKGROUND
Literately, photosynthesis as a term defines building up processes by light. The process that
synthesizes organic compounds from inorganic components in the presence of light is called
photosynthesis. Green plants, algae and some kinds of bacteria get the energy directly from the
sun and use to synthesize proteins, fats, nucleic acids and carbohydrates. In simple,
photosynthesis inputs are carbon dioxide and water; the outputs are carbohydrate and oxygen:

Thereby, photosynthesis converts radiant energy into the chemical energy. Photosynthesis has
a crucial role in the regulation of carbon dioxide and oxygen cycle. All plants and animals take
the oxygen from atmosphere during respiration resulting in conversion of organic compounds
(carbohydrate) finally to carbon dioxide and water. Liberated energy is stored in ATP and used
for energy required reactions. Photosynthesis takes those outputs as input and produces
carbohydrate and oxygen (Figure 1).

Figure 1. Carbon dioxide cycle.

In plants all plastids are derived from proplastids (small organelles) which are found in
immature cells of plant meristem (Figure 2).

Figure 2. Plastid types. Figure 3. Chloroplast

 Photosynthesis is conducted in chloroplasts (Figure 3). Photosynthesis consists of two set
of reactions: light and Calvin cycle reactions (dark reactions). In photosynthetic electron
transfer reactions (light reactions) the light energy (photon) excites chlorophyll. Chlorophyll
obtains electrons from water and oxygen is released as a side product. Electrons are transferred
through protein complexes of the photosystem II and I which are embedded in the thylakoid
membrane. Electrons are transferred to NADP+ and forms NADPH. Meanwhile, protons are
released to thylakoid space. ATP synthase which is found in the thylakoid membrane use H+
gradient to produce ATP in the stroma.
In Calvin cycle reactions (dark reactions) the outputs of the light reactions are used. ATP as
energy source and NADPH as a reducing power are used to generate carbohydrates from carbon
dioxide and water. It starts in the stroma and continues in the cytosol. Calvin reactions occur in
the stroma. Calvin reactions consist of three reaction groups: carbon fixation, reduction and
regeneration (Figure 4).
The main output of Calvin cycle: Glyceraldehyde 3-phosphate (G3P) is used to synthesize
fatty acid, glucose or glycerol. In the stroma, G3P is used to synthesize starch. In cytosol, it is
used to synthesize sucrose (Figure 5).

Figure 5. Assimilation of carbon and

Figure 4. Calvin cycle. synthesis of sucrose and starch.
C4 plants
Under dry and hot conditions, plants close their stomata to decrease loss of water. However,
this decreases CO2 level in the leaf. In this case, Rubisco adds oxygen instead of CO2 to the
ribulose biphosphate and liberates CO2 which is the first step of the photorespiration resulting
in no energy source. Thus, the plants lose one-third of CO2 which they fixed. Certain plants
such as corn and sugar cane have adaptation to decrease photorespiration. They have bundle
sheat cells which are surrounded by mesophyll cells. Mesophyll cells protect the bundle sheat
cells from air. All rubisco of the plant are found in bundle sheat cells in where all carbon fixation
reactions occur only. Mesophyll cells use energy provided from chloroplast to transfer CO2
from mesophyll cell’s cytosol to bundle sheat cell. In the cyctosol, CO2 combines with 3C
molecule and forms 4C carbon molecule. 4C molecule diffuses to bundle sheat cell and it is
broken down into CO2 and 3C molecule. 3C molecule turns back to the mesophyll cells. Those
kinds of plants that capture initial CO2 in 4C molecule are called 4C plants. All others are 3C
plants that capture CO2 in (3-Phosphoglyceric acid) form of 3C in Calvin cycle (Figure 4 and
Figure 6).
Figure 6. Differences of the cells arrangement between C3 and C4 plants.
Pigments
In addition to water, carbon dioxide, and light energy, photosynthesis requires pigments.

As we know, chlorophyll is the primary light-absorbing pigment in autotrophs. Chlorophyll is

found inside chloroplasts. Actually, plant leaves contain different type of pigments including
chlorophylls, carotenes,and xanthophylls (Figure 7). Plant pigments give color to leaves,
flowers, and fruits and are also important in controlling photosynthesis, growth, and
development. During the summer when leaves contain large amounts of chlorophyll (thereby
they are green), the presence of the other pigments is not obvious to the eye. During the fall,
after most of the chlorophyll has been degraded, these other pigments can be more readily
observed, and the leaves of many plants take on the variety of colors that are typical of fall
foliage.
Chlorophylls
A. Chlorophyll A: The molecule which makes photosynthesis possible, by passing its
energized electrons on to molecules which will manufacture sugars. All plants, algae, and
cyanobacteria which photosynthesize contain chlorophyll a. Chlorophyll b occurs only in
"green algae" and in the plants.
Figure 7. Plant pigments
B. Xantophylls: Structural components of the light harvesting antenna in chloroplasts. They
are accessory pigments for harvesting light at wavelengths that chorophyll cannot, and transfer
the light energy to chlorophyll. They also absorb excess light energy and dissipate it in order to
avoid damage in what is termed the Xanthophyll Cycle (Color: Yellow).
Chromatography
Chromatography is used to separate mixtures of substances into their components. All forms
of chromatography work on the similar principle.
They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase
(a liquid or a gas). The mobile phase flows through the stationary phase and carries the
components of the mixture with it. Different components travel at different rates. Thin layer
chromatography is what exactly it says - using a thin, uniform layer of silica gel or alumina
coated onto a piece of glass, metal or rigid plastic. In this lab section you will separate plant
pigments using Thin Layer Chromatography (TLC).

Figure 8. Diagrams for TLC system

Different components are required to conduct the procedure along with the phases involved.
 1. Thin Layer Chromatography Plates: Ready-made plates are used which are chemically
inert and stable. The stationary phase is applied on its surface in the form of a thin layer.
The stationary phase on the plate has a fine particle size and also has a uniform
thickness.

2. Thin Layer Chromatography Chamber: Chamber is used to develop plates. It is

responsible to keep a steady environment inside which will help in developing spots.
Also, it prevents the solvent evaporation and keeps the entire process dust-free.

3. Thin Layer Chromatography Mobile phase: Mobile phase is the one that moves and
consists of a solvent mixture or a solvent. This phase should be particulate-free. The
higher the quality of purity the development of spots is better.

4. Thin Layer Chromatography Filter Paper: It has to be placed inside the chamber. It is
moistened in the mobile phase.

Methodology/Principle Explanation
A pencil line is drawn near the bottom (1-1.5 cm) of the silica plate and a small drop of a
solution of the mixture (for ex. plant extract) is placed on it. Any labelling on the plate to show
the original position of the drop must also be in pencil. If any of this was done in ink, dyes from
the ink would also move as the chromatogram developed (Figure 8).
When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered
beaker. It is important that the solvent level is below the line with the spot on it.
The reason for covering the beaker is to make sure that the atmosphere in the beaker is
saturated with solvent vapour. To help this, the beaker is often lined with some filter paper
soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the solvent from
evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different components of the dye mixture travel
at different rates and the mixture is separated into different colored spots.
The solvent is allowed to rise until it almost reaches the top of the plate. That will give the
maximum separation of the dye components for this particular combination of solvent and
stationary phase.

Rf Value
In order to help identify the compounds present Rf value is calculated. The distance traveled
by the solvent, and the distance traveled by individual spots are measured.
Rf = distance travelled by component /distance travelled by solvent (Figure 9).
 When you repeat the experiment under exactly the same conditions, then the Rf value for each
component would always be the same. However, if anything changes (the temperature, the
exact composition of the solvent etc.), then Rf value changes.

Figure 9. Rf value calculation

For example: A sample run 2.1 cm through the plate. Solvent run 2.8 cm through the plate.
Then Rf value of the sample is calculated as 0.75.
To sum up; the mix of pigments in a leaf may be separated into bands of color by the
technique of TL chromatography. In this technique, the mixture containing the pigments to be
separated is first applied as a spot or a line to the paper about 1.5 cm from the bottom edge of
the paper. The paper is then placed in a container with the tip of the paper touching the suitable
solvent system. Solvent is absorbed by the layer and moves up the layer. When the solvent
crosses the area containing plant pigment extract, the pigments dissolve in and move with the
solvent. The pigments are carried along at different rates because they are not equally soluble.
Thus, the less soluble pigments will move slower up the paper than the more soluble pigments.
This is known as developing a chromatogram.
Silica-gel thin-layer chromatography (TLC) is an analytical method in which the stationary
phase is a thin coating of the very polar silica gel (oxides of silicon) on a glass or plastic plate,
and the mobile phase is a solvent (or mixture of solvents) that is less polar than silica gel.
Typical solvents are hydrocarbons like hexane (C6H14, very nonpolar), acetone (CH3COCH3,
moderately polar), and ethanol (C2H5OH, very polar).

5. LAB EXERCISES

5.1. Thin Layer Chromatography

5.1.1. Materials
• Spinach (5-10 leaves) 95% • Graduated cylinder (25 ml)
• EtoH (35-40 ml) • Ruler
• Mortar and pestle • Pencil
• TLC plate • Stretch fim
 5.1.2. Protocol
1. Take 5-10 small spinach pieces. Use mortar and pestle to ground spinach leaves in 10-
15 ml 95% EtOH (If it evaporates add a little more EtOH).
2. Take the thin layer chromatography plate. Use ruler and pencil to draw a baseline and
mark center of the baseline.
3. Take a small drop of plant extract and put it on the center using micropipet, Pasteur
pipet or toothpick like in third figure.
4. View of small drop on the center. Leave it dry (5-10 min).
5. Take a 25 ml graduated cyclinder. Put 10 ml 95% EtOH in it. Place the TLC plate in the
cylinder. Use a plug or a stretch film to cover the top of the cylinder. Wait 15 min.
6. After 15 min, take the TLC plate out of the cylinder and leave it to dry for 15 min
(Figure 10). Decide/write which spot corresponds to which pigment. Then, calculate the
Rf values of your components (Figure 11).

1 2

3 4

5 6

Figure 10. Visual representation of the protocol steps.

Figure 11. Examples of TLC plates run in different solvents and table of Rf value in a solvent.
Remember polarity and solubility of the component in the solvent are important factors.

5.1.3. Study Questions

1. How do you define photosynthesis?

2. What are the phases of photosynthesis? What are inputs and outputs of those phases?
What are the phases of the Calvin cycle (name)?
3. What is the important of the Calvin cycle?
4. What is the feature of C4 plants?
5. What are the plant pigments?
6. Name plant plastids.
7. Why are the leaves of the green plants colorful during fall?
8. What is the general principle of TLC?
9. Is silica plate polar or non-polar? What is importance of polarity?

6. REFERENCES
Elias Kaiser, Viviana Correa Galvis, Ute Armbruster; Efficient photosynthesis in dynamic
light environments: a chloroplast's perspective. Biochem J 15 October 2019; 476 (19):
2725–2741. doi: https://doi.org/10.1042/BCJ20190134
Alvarez S., Naldrett M.J. (2016) Plant Structure and Specificity – Challenges and Sample
Preparation Considerations for Proteomics. In: Mirzaei H., Carrasco M. (eds) Modern
Proteomics – Sample Preparation, Analysis and Practical Applications. Advances in
Experimental Medicine and Biology, vol 919. Springer, Cham. https://doi.org/10.1007/978-
3-319-41448-5_4
Sajewicz M, Kowalska T, Sherma J. Sample Preparation for Thin Layer Chromatography.
Adv Chromatogr. 2017;53:301-329. PMID: 29461697.
Laboratory 12
FERMENTATION AND ANAEROBIC RESPIRATION
1. BACKGROUND
Fermentation
If oxygen is absent, many cells are still able to use glycolysis to produce ATP. Two ways this
can be done are through fermentation and anaerobic respiration. Fermentation is the process by
which the electrons and hydrogen ions from the NADH produced by glycolysis are donated to
another organic molecule.
The Point of Fermentation
The reason this is done is to produce NAD+ which is needed to keep glycolysis going. Unless
the cell has some sort of electron transport system, the NADH is not usable. At the same time
NAD+ is needed for glycolysis and it is much less expensive in terms of energy for the cell to
simply take the NADH that would normally go to the mitochondrion and use it to regenerate
the NAD+. This is shown in the figure for ethanol fermentation in yeast (Figure 1).

Figure 1. Ethanol fermentation in yeast.

Notice that the NADH produced by glycolysis donates its hydrogen ions and electrons that in
aerobic respiration would have ended up powering electron transport phosphorylation (Figure
2).

Figure 2. Cellular respiration.

Other Fermentation Pathways

There are a number of fermentation pathways that different cells use. Yeast cells produce ethyl
alcohol by fermentation. Certain cells of our body, namely muscle cells, use lactic acid
fermentation, while depending on the organism some of the other products of fermentation
include acetic acid, formic acid, acetone and isopropyl alcohol.

Fermentation and Running

In our bodies certain muscle cells, called fast twitch muscles, have less capability for storing
and using oxygen than other muscles. When you run and these muscles run short of oxygen,
the fast twitch muscles begin using lactic acid fermentation. This allows the muscle to continue
to function by producing ATP by glycolysis.
The muscles get enough ATP for quick spurts or shall we say sprints, but quickly become
fatigued as their stores of glycogen are used up. Eventually you cramp. This is in part because
the muscles lack sufficient ATP to continue contracting. Also, lactic acid builds up and must
be metabolized by the liver. Runners who sprint actually have more muscle cells specialized
for lactic acid fermentation than do long distance runners.
What are Yeasts?
A yeast is a unicellular fungus which reproduces asexually by budding or division, especially
the genus Saccharomyces which is important in food fermentations. Yeasts and yeast-like fungi
are widely distributed in nature. They are present in orchards and vineyards, in the air, the soil
and the intestinal tract of animals. Like bacteria and moulds, they can have beneficial and non-
beneficial effects in foods. Most yeasts are larger than most bacteria. The most well-known
examples of yeast fermentation are in the production of alcoholic drinks and the leavening of
bread. For their participation in these two processes, yeasts are of major importance in the food
industry.
Some yeasts are chromogenic and produce a variety of pigments, including green, yellow and
black. Others are capable of synthesizing essential B group vitamins.
Although there is a large diversity of yeasts and yeast-like fungi, (about 500 species), only a
few are commonly associated with the production of fermented foods. Varieties of the
Saccharomyces cervisiae genus are the most common yeasts in fermented foods and beverages
based on fruit and vegetables. All strains of this genus ferment glucose and many ferment other
plant derived carbohydrates such as sucrose, maltose and raffinose. In the tropics,
Saccharomyces pombeis the dominant yeast in the production of traditional fermented
beverages, especially those derived from maize and millet.
Conditions Necessary for Fermentation
Most yeasts require an abundance of oxygen for growth, therefore by controlling the supply of
oxygen, their growth can be checked. In addition to oxygen, they require a basic substrate such

as sugar. Some yeasts can ferment sugars to alcohol and carbon dioxide in the absence of air
but require oxygen for growth. They produce ethyl alcohol and carbon dioxide from simple
sugars such as glucose and fructose.
Yeasts are active in a very broad temperature range - from 0 to 50 ᵒC, with an optimum
temperature range of 20 to 30 ᵒC.
The optimum pH for most micro-organisms is near the neutral point (pH 7.0). Moulds and
yeasts are usually acid tolerant and are therefore associated with the spoilage of acidic foods.
Yeasts can grow in a pH range of 4 to 4.5 and moulds can grow from pH 2 to 8.5, but favour
an acid pH.
In terms of water requirements, yeasts are intermediate between bacteria and moulds. Bacteria
have the highest demands for water, while moulds have the least need. Normal yeasts require a
minimum water activity of 0.85 or a relative humidity of 88%.
Yeasts are fairly tolerant of high concentrations of sugar and grow well in solutions containing
40% sugar. At concentrations higher than this, only a certain group of yeasts – the osmophilic
type – can survive. There are only a few yeasts that can tolerate sugar concentrations of 65-
70% and these grow very slowly in these conditions.
Anaerobic respiration occurs in some prokaryotic organisms of environments without oxygen.
They use ETC but final electron acceptors are not oxygen.
e.g. Sulfate reducing marine bacteria use sulfate ion at the end of respiratory chain as the
electron acceptor to produce H2S as the by- product.
Fermentation is a way of harvesting chemical energy w/o using either oxygen or any ETC.
A. Ethyl Alcohol Fermentation: The first step of ethyl alcohol fermentation releases CO2 from
the pyruvate which is then converted to acetaldehyde. The second step involves reducing
acetaldehyde to ethanol by NADH. NAD+ is regenerated for continuity of glycolysis (Figure
3).
B. Lactic Acid Fermentation: Pyruvate is reduced directly by NADH to form lactate as an
end-product, with no release of CO2 (Figure 4).

Figure 3. Ethyl alcohol fermentation. Figure 4. Lactic acid fermentation.

2. LAB EXERCISES

2.1. Lab Exercise: Yeast Fermentation

2.1.1. Materials

Glass conical flasks, dried yeast, glucose, sucrose, water, string, ruler and balloons
2.1.2. Protocol

1. Label glass conical flasks A, B, C, D and E so that you don’t mix them up. Draw the
table below in your notebook so you can record your results.
 Table 1. A table to record the results

2. In stages, you will be adding the quantities of water, sugar and yeast from Table 2 into
each of the 5 glass conical flasks.
Stage 1. Put 200ml of water into each conical flask.
Stage 2. Add the sugar. Follow the amounts in table 2, so for example, conical flask A
will have no sugar, conical flask B will have half a teaspoon of sugar and so on. Shake
each conical flask so that the sugar dissolves.
Stage 3. Add yeast to conical flask A, B, C and D but importantly not to conical flask
E.
Table 2. The amount of water, sugar and yeast in each conical flask

3. Stretch a balloon over each bottle opening, making sure the seal is tight.
4. Now take your first measurement of each balloon diameter. The easiest way is to wrap
a piece of string around the balloon, cutting it with the scissors at the point where the
 ends meet and then measuring the length of the string with a ruler. You can then record
this zero-time point measurement in your notepad.
5. Incubate all samples at 37oC.
6. You will need to take the measurement of all 5 balloons every 15 minutes to record how
much the balloons inflate. You should record each measurement in your notepad. Over
the course of a few hours, you will see how the amount of sugar affects how quickly
and how much the balloons inflate.

2.1.3. Study Questions

What is cellular respiration?

What are the two stages of cellular respiration?
What is fermentation?
What is ATP? Why is ATP important?
How do the products differ between respiration and fermentation?
In which situation is air present -during respiration or fermentation?
How many stages are there in fermentation? Which stage that is present in cellular
respiration is not present in fermentation?
In which process, respiration or fermentation, are carbohydrates not completely broken
down? How does this affect the amount of ATP released?
Why is fermentation important for yeast?

7. REFERENCES

Jeremy M. Berg, John L. Tymoczko and Lubert Stryer, Biochemistry, 6th edition, W.H.
Freeman and Company, 2007, pages 205-237.

Fugentius Lugemwa, Decomposition of Hydrogen Peroxide, Chemical Educator, April

2013, pages 85-87.

Daniel Q. Duffy, Stephanie A. Shaw, William D. Bare, Kenneth A. Goldsby, More

Chemistry in a Soda Bottle, A Conservation of Mass Activity, Journal of Chemical
Education, August 1995, pages 734-736.

Jessica L Epstein, Matthew Vieira, Binod Aryal, Nicolas Vera and Melissa Solis,
Developing Biofuel in the Teaching Laboratory: Ethanol from Various Sources, Journal of
Chemical Education, April 2010, pages 708–710.
Laboratory 13

ENZYMES
1. BACKGROUND
Most of the chemical reactions that take place within a cell include protein catalysts called
enzymes. Enzymes, like other catalysts, speed up the rates of chemical reactions by lowering
the activation energy of that reaction.
They bind reactants (substrates) and hold them in a certain orientation. This case maximizes
the chances that a certain chemical reaction will occur. In the end, the enzymes convert the
substrates into products (Figure 1).

Figure 1. Enzyme converts substrates into the products.

The ability of enzymes to function depends on the three-dimensional shape of the protein. All
proteins have a shape which is due to various types of chemical interactions that occur among
amino acid side chains, and between amino acid side-chains and the surrounding environment.
There interactions are ionic interactions among charged side chains, hydrogen bonds,
polar/nonpolar interactions, disulfide bonds. Some of the amino acids of the enzyme are
arranged to form a pocket- like structure called an active site (Figure 1).
The process of enzyme catalysis:
1. The substrate(s) binds to the active site to form an enzyme-substrate complex.
2. The reaction occurs converting the substrate(s) into product(s), forming an enzyme-product
complex.
3. The products are released from the active site, leaving the enzyme in its original, unaltered
form.
Enzymes show very high degree of specificity for their substrates. They bind specific substrates
and catalyze specific reactions.
The ability of an enzyme to convert substrate into product is referred to as enzyme activity, and
is often used as a synonym for reaction rate (since as enzyme activity increases, more substrate
is converted into product per unit time). Enzyme activity is not constant. There are several
factors that affect enzyme activity. Some of them are: enzyme concentration, substrate
concentration, pH, temperature.
Factors Affecting Enzyme Activity
A. Enzyme concentration on enzymatic activity: Each enzyme molecule requires x amount
of time to produce one unit of product. Two enzyme molecules would produce two units in that
time, three enzyme molecules would produce three units of product, and so on. Therefore, the
more enzyme is available, the more substrate can be converted into product quickly. Therefore,
in general, (assume all other factors are constant) as enzyme concentration increases, there is a
proportional increase in reaction rate (Figure 2).

Figure 2. The relationship between enzyme concentration and enzyme activity.

B. Substrate concentration on enzymatic activity: To convert substrate into product, the
substrate must first bind with the enzyme. This is usually achieved simply by random collisions
between enzyme and substrate as these particles diffuse around in solution. The frequency of
these collisions can be influenced by several factors. One of them is simply the amount of each
substance that is present in solution. For example, the more concentrated the substrate is in a
solution, the more frequently substrate molecules will randomly collide with the enzyme, and
thus the more often substrate will be converted into product. So, in general (assume that all
other factors, such as enzyme concentration, are constant), one would expect that the rate of an
enzyme increases as substrate concentration increases (Figure 3).

Figure 3. The relationship between substrate concentration and reaction rate (Note that as
substrate concentration increases, rate increases, but the change in rate becomes progressively
less until a maximum rate is reached when the enzyme becomes saturated with substrate.).
C. Temperature on enzymatic activity: Temperature is the average kinetic energy of a
system. Kinetic energy is the energy in motion. This means that at higher temperatures particles
tend to be moving more quickly than at lower temperatures. In solids, molecules remain in
roughly the same position in space but vibrate more. In liquids and gases, where particles are
free to move from one location to another, these particles tend to do so at greater speeds. Since
particles are moving more quickly, they also tend to collide with one another more frequently
and with greater energy. Therefore, the rates of chemical reactions tend to increase as
temperature increases.
Many enzymes show an unusual relationship between reaction rate and temperature. Although
over much of the range of temperatures biological organisms experience there is an increase in
enzyme activity with increased temperature there is often a decrease in reaction rates at very
high temperatures (e.g., above 70 °C). There could be several reasons for this. For example, the
increase in temperature may weaken and destabilize the bonds that link enzymes with necessary
cofactors. However, the most important factor to consider is that the shape of the enzyme can
be influenced by temperature (denaturization) (Figure 4).

Figure 4. The relationship between temperature and the rate of enzyme catalyzed reactions.
D. pH on enzymatic activity: pH is an index of hydrogen ion (H+ concentration). The H+
concentration of a water-based solution can vary due to the presence of the solutes. pH of a
solution (environment) affects the ionic interactions of the protein. Since the catalytic ability of
an enzyme is so tightly linked to the specific shape and chemical properties of its active site,
alteration of normal ionic bonding patterns within the protein tends to reduce catalytic function.
Therefore, there is an optimal pH where the right degree of H+ binding and dissociation of
various acidic and basic amino acids exists such that the active site of the protein has the shape
for maximum catalytic activity (Figure 5).
Figure 5. Variation in the rate of enzyme-catalyzed reactions with pH.

Measurement of Enzyme Concentration

There are complex methods for directly measuring some proteins (e.g. Radioimmunoassay),
but there is a convenient method that can be used to measure enzyme concentration (using
spectrophotometry).
If all else is constant (substrate and coenzyme concentrations, temperature, and pH), the rate of
an enzyme catalyzed reaction is proportional to the concentration of enzyme in the solution.
The rate at which substrate is converted into product is proportional to enzyme concentration.
So, if you ran an experiment where you allowed solutions of different enzyme concentrations
to react with substrate for a fixed amount of time, the amount of increase in the concentration
of product present in the solution at the end of the experiment should be proportional to the
concentration of the enzyme. So, we could use changes in the concentration of product formed
over time as an indirect means of measuring enzyme concentration.
In many cases we can measure changes in product concentration in a solution with
spectrophotometric methods (as we will do in this week). According to Beer’s Law the
concentration of a solute in solution (in this case, the product of the reaction) is proportional to
the absorbance of light by the solution. Therefore, the rate at which the absorbance changes for
an enzyme-substrate solution is proportional to the rate that product is being formed. Therefore,
since rate of product formation is proportional to enzyme concentration, the rate that absorbance
changes during the reaction is proportional to enzyme concentration.
• Expressing enzyme concentration in conventional terms (e.g., number of particles per
unit volume), the concentration of enzymes is often expressed in terms of enzymatic
activity (how much substrate can be converted into product in a given amount of time).
• The standard unit used to quantify the amount of enzyme (based on enzyme activity) is
called the international unit of enzyme activity (U), and is equal to the amount of
enzyme needed to convert 1 μmole of substrate into product in 1 min. Enzyme
concentration, then, would be the measure of enzyme activity divided by volume (e.g.,
U/L).
 To understand this week’s experiment, make sure you understand clearly spectrophotometric
assay’s principle (Watch this video on youtube
https://www.youtube.com/watch?v=pxC6F7bK8CU ) (Figure 6 and Figure 7).

Figure 6. A view of spectrophotometer machine

Spectrophotometry is used to measure how much a substance absorbs light by measuring the
intensity of light as a beam of light passes through sample solution (Figure 7). You can detect
how much of light is absorbed or transmitted by a known substance. So, you can directly
correlate that more absorbance means more amount of the substance. To do this, you should
know the optimum absorbance wavelength of the substance. For example, p-nitrophenol
absorbs the light optimally at ~400-405 nanometer (nm). So, if you have an experiment related
to measure how much amount of p-nitrophenol is available in the solution, the
spectrophotometer is set up at 405 nm for measurements.

Figure 7. Basic principle of the spectrophotometer.

Beer-Lambert Law
It states that there is a linear relationship between the absorbance and the concentration of a
sample
A=εlc
• A is the measure of absorbance
• ε is the molar extinction coefficient or molar absorptivity (or absorption coefficient)
 • l is the path length
• c is the concentration
The molar extinction coefficient is given as a constant and varies for each molecule. ε has the
units: L·mol-1·cm-1. The path length is measured in centimeters. Because a standard
spectrometer uses a cuvette that is 1 cm in width, l is always assumed to equal 1 cm. Since
absorption, ε, and path length are known, we can calculate the concentration c of the sample.
Example: Chemical X has a maximum absorbance of 275 nm. ε275=8400M−1cm−1 and the
path length is 1 cm. Using a spectrophotometer, you find the that A275=0.70. What is the
concentration of Chemical X?
Answer:

A=εlc
-1 -1
0.70 = (8400 M cm )(1 cm)(c)
-5
C= 8.33x10 mol/L

2. LAB EXERCISES

2.1. Lab Exercise: GST Assay

Glutathione S Transferase (GST) is an enzyme involved in detoxification of a wide range of
compounds and is involved in reducing free radical damage in red blood cells. The enzyme is
easily purified by affinity chromatography and has been as a fusion partner for many
recombinant proteins. Identification of GST is done by westernblotting or more easily by
enzymatic assay.
Enzyme Reaction: Glutathione-SH + CDNB → Glutathione-S-CDNB
The reaction is measured by observing the conjugation of 1-chloro, 2,4-dinitrobenzene (CDNB)
with reduced glutathione (GST). This is done by watching an increase in absorbance at 340nm.
One unit of enzyme will conjugate 10.0nmol of CDNB with reduced glutathione per minute at
25 ᵒC.

2.1.1. Materials
• 100 mM CDNB dissolved in ethanol and stored in microfuge tubes
• 100 mM reduced glutathione
• Assay buffer –PBS adjusted to pH=6.5
GSH is prepared in ethanol and can be stored at -20 ᵒC for one month. CDNB can be
frozen/thawed for no more than five times. Allow all powders to come to room temperature
prior to measuring to reduce condensation of solids.
For each assay you will perform prepare one ml of assay buffer.
Assay Buffer Recipe:
• 980 µl PBS pH 6.5
• 10 µl of 100 mM CDNB
• 10 µl of 100 mM glutathione
Mix-the solution may be cloudy at first but should clear up after mixing.

2.1.2. Protocol

1. For each sample and a blank, place 900 µl of enzyme cocktail into 1.5 ml plastic cuvettes.
2. Incubate at 30 ᵒC in spectrophotometer for 5 min.
3. To the blank cuvette add 100 µl PBS and zero spec.
4. Add 100 µl of sample cuvettes and mix.
5. Measure absorbance at 340 nm for five min.

2.1.3. Study Questions?

How does the formation of the enzyme-substrate complex explain the reduction in the
activation energy of chemical reactions?
What effect does an increase in the enzyme concentration have on the rate of an enzyme-
catalyzed reaction?
What is the GST enzyme reaction principle? What are the functions of CDNB and
glutathione in the experiment?

3. REFERENCES
Ainsworth S. (1977) Enzymes as Biological Catalysts. In: Steady-State Enzyme Kinetics.
Palgrave, London. https://doi.org/10.1007/978-1-349-01959-5_1
Kurochkina N. (2019) Enzymes. In: Protein Structure and Modeling. Springer, Singapore.
https://doi.org/10.1007/978-981-13-6601-7_3
Richards, F. M. and Wyckoff, H. W. (1971). In The Enzymes, Vol. IV, 3rd edn (ed. P. D.
Boyer), Academic Press, New York and London, p. 647
Robinson P. K. (2015). Enzymes: principles and biotechnological applications. Essays in
biochemistry, 59, 1–41. https://doi.org/10.1042/bse0590001