BIO 101 Lab Manual 2017 - 2018 Final
BIO 101 Lab Manual 2017 - 2018 Final
Introduction Page 2
Practical 1: Introduction to Lab Safety Page 5
Practical 2: Testing different food samples for macromolecules Page 9
Practical 3: Effect of temperature and pH on enzymes Page 13
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Introduction
Laboratory practicals are meant to equip students with practical skills that are
essentialforundertaking scientific research. Through laboratory sessions students get a chance
to handle scientific equipment and get practical experience of the concepts taught in
lectures.Depending on the nature of the experiment students may either work individually or
in small groups. The objectives of laboratory practical sessions include; teaching scientific
methods and attitudes, to demonstrating concepts and principles taught in lectures and giving
the students a “feel” for the subject. Tutorial sessions are another opportunity for student
group work and close interaction between students and their instructors.In addition to
complementing material taught in lectures, tutorial sessions are meant to teach students other
essential skills such as writing and oral presentation skills. Unlike practicals which are
predesigned, tutorial sessions will be developed weekly based on the module team’s
assessment of students’ needs. Tutorial sessions will vary from discussions, quizzes, group
work to oral presentations. Attendance of both tutorial sessions and laboratory practicals will
be part of the module’sDP requirements and therefore attendance will be monitored for both
the practicals and tutorial sessions. It is the responsibility of students to sign attendance forms
for every one of these sessions.
Recommended Textbooks
Russell P.J., Hertz P.E., McMillian B. Biology: The Dynamic Science, 4th Edition.
Cengage, UK.
Sadava D., Hillis D.M., Heller H.C. and Berenbaum H. Life: The Science of Biology, 10th
Edition. Sinauer& Freeman, USA. Previous editions may also be used if you cannot afford a
new copy.
In addition to your textbook, you are required to bring the following to laboratory:
lab coat,
lab notebook,
drawing books,
calculators,
graph paper
Pencil and eraser
Stapler and staples
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ASSESSEMENT
The final mark awarded for BIO101 will be composed of final examination and continuous
assessment in a ratio of 40:60.
Your final examination (40% of your final mark) may include questions relating to anything
covered in lectures, practicals, tutorials and prescribed reading. Students wishing to score
an A or B+ grade on this course are expected to show significant evidence of independent
reading and conceptualisation.
Deadlines:
Late assignments submitted less than one week late will be penalised 5% for each day after
the submission deadline, work that is more than 3 days late will receive a ZERO mark.
Attendance:
You are expected to attend all lectures, laboratory and tutorial sessions. Students who fail
to attend a practical will receive zero for any assignment arising from that practical unless
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we receive a letter from the Faculty Administrator excusing them from that class (do not
bring medical certificates or any other excuses to us). Students guilty of unexplained
absences will not receive extra assistance to make up for their absence.
Copying (plagiarism):
Co-operation is encouraged but do not let it extend to copying. When we receive work that
has been copied, from students or published works, it will receive a ZERO mark, regardless
of who copied from whom and how much was copied. Make sure you keep your work
secure.
Members of Staff
Lecturer
Dr Kebaneilwe LEBANI
Office phone number: 4931533
Office: Biology Block 5, Office F36
E-mail address: lebanik@biust.ac.bw
Teaching Assistant
Will be allocated at the start of the semester
Name ______________________________ E-mail_________________________________
Laboratory Technicians
Lerato Goitsemang Lekgari
E-mail address: lekgarig@biust.ac.bw
Gakeonyatse Modirwagale
Email address: modirwagaleg@biust.ac.bw
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PRACTICAL 1: INTRODUCTION TO LAB SAFETY
Working in the biology laboratory can be interesting and exciting. But it can also be quite
dangerous if you are not alert and if proper safety precautions are not exercised at all times.
You are responsible for maintaining an enjoyable, instructional, and safe environment in the
biology laboratory. Unsafe practices endanger not only you but the people around you as
well. Read the following information about safety in the biology laboratory carefully. Review
applicable safety information before you begin each laboratory investigation. If you have any
questions about safety or laboratory procedures, be sure to ask your lecturer or instructor.
Biology lab safety rules are guidelines designed to help keep you safe when doing practicals
in the laboratory. Some equipment and chemicals in a biology laboratory can cause serious
harm. It is always wise to follow all lab safety rules. Don't forget, the most helpful safety rule
is to use plain old common sense. The following biology lab safety rules are a sample of the
most basic rules that should be followed when in biology lab.
Be Prepared:
Before you enter a biology lab, you should be prepared for and knowledgeable about any lab
exercises that are to be performed. That means you should read your lab manual to know
exactly what you will be doing. Review your biology notes and relevant sections in your
biology textbook before your lab begins. Make sure you understand all procedures and
purposes, as this will help you understand the lab activities you will perform. It will also help
you get your thoughts organized for when you have to write your lab report.
Be Neat:
When working in a biology lab, make sure you keep your area neat and organized. If you
happen to spill something, ask for assistance when cleaning it up. Also remember to clean
your work area and wash your hands when you are finished.
Be Careful:
An important biology lab safety rule is to be careful. You may be working with glass or sharp
objects, so you don't want to handle them carelessly.
Accidents do happen in a biology lab. Some chemicals have the potential to damage clothing.
With that in mind, you want to make sure that the clothing you wear is something you could
do without if it becomes damaged. As a precaution, always wear a lab coat when doing lab
practicals. You will also want to wear proper shoes that can protect your feet in case
something gets broken. Sandals or any type of open-toed shoes are not recommended.
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Be Cautious With Chemicals:
The best way to remain safe when dealing with chemicals is to assume that any chemical you
handle is dangerous. Be sure you understand what type of chemicals you are using and how
they should be properly handled. If any chemical comes in contact with your skin, wash
immediately with water and inform your lab instructor.
Always wear protective eyewear when handling chemicals. I know that safety goggles are not
stylish and can fit awkwardly on your face, but they should always be worn when you are
working with chemicals or any type of heating apparatus.
Be sure you know where to find all safety equipment in the biology lab. This includes such
items as the fire extinguisher, first aid kit, broken glass receptacles, and chemical waste
containers. Also be sure you know where all the emergency exits are located and which exit
route to take in case of an emergency.
There are several things in a biology lab that you must always avoid. Here are a few major
laboratory do nots.
Do Not:
Biology lab is an important aspect of any biology course. In order to have a good lab
experience, make sure that you follow these biology lab safety rules and any instructions
given to you by your lab instructor.
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Safety signs in the Biology Lab
Below is a table with common Biology safety signs and their meanings. Please read the table
attentively
The sign indicates the presence of a
biohazard. A biohazard is biological agent,
such as an infectious microorganism, or a
condition that constitutes a threat to humans,
especially in biological research or
experimentation
The sign indicates that the su substance is
explosive. Explosive substances cause a
sudden, almost instantaneous release of
pressure, gas, heat and light when subjected
to sudden shock, pressure, high temperature
or applied potential.
The sign indicates that there is open flame. You
may be working with flames from a Bunsen
burner, candle, or matches. Tie back loose hair
and clothing. Follow instructions from you
your
teacher about lighting and extinguishing
flames.
The sign indicates that open flame is
dangerous. Flammable materials may be
present. Make sure no flames, sparks, or
exposed heat sources are present.
The sign indicates a poisonous chemical. Do
not let any poisonous chemical come in contact
with your skin, and do not inhale its vapours.
Wash your hands when you are finished with
the activity.
This is a warning sign for high voltage. It
indicates that there is risk of an electric shock
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This sign is used to warn you that the surface
is hot.. It may be used in the laboratory to
indicate heated hot plates or other hot
surfaces. It is important to take appropriate
safety precautions when this sign is displayed
Environmental hazard sign. It indicates that a
substance can damage the surroundi
surrounding
natural environment e.g. air pollution, water
pollution, toxins, and radioactivity
radioactivity. The sign
is usually used to warn against environmental
pollution.
Your assigned teaching assistants will now go through writing a lab report with you. This
skill is very important as most of your laboratory sessions will require you to write a lab
report and have it assessed.
This is also a good time to go through designing an experiment. Discuss and take notes on the
following;
o Formulating a hypothesis
o How does a hypothesis differ from
fr an aim?
o What are experimental controls?
o Why do we need experimental controls?
o The effect of one factor and multiple
multi factors on experimental output
o Reporting and discussion of research output
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PRACTICAL 2: TESTING DIFFERENT FOOD SAMPLES FOR
MACROMOLECULES
Class: _______________
All living things are made up of macromolecules namely carbohydrates, lipids, proteins, and
nucleic acids. Carbohydrates range from simple forms such as Monosaccharides (e.g. glucose)
to more complex forms called polysaccharides (e.g. starch and cellulose). When you digest
starches from the food that you eat, they are broken down into monosaccharides, making energy
available to your body.
About 15% of your body is made up of lipids which are fats that are insoluble in water. Fats
commonly found in humans are triglycerides, phospholipids, and steroids. These lipids have
different functions within your body such as long-term energy storage, formation of cell
membranes, and regulation of hormonal changes. Another 17% of your body is made up of
proteinswhich perform a variety of functions throughout your body. They can be structural
proteins, enzymes (which catalyze reactions), or signalling proteins. These macromolecules are
obtained through the food that we eat.
In today’s lab practical we will test different food samples for carbohydrates, lipids and
protein following the scientific method.
SAFETY FIRST
1. Goggles Must Be Worn at All Times.
2. Iodine is poisonous and can stain clothing.
3. Biuret reagent contains sodium hydroxide. If you get the solution on your skin, wash
immediately with water.
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1. Add about 2mL of the sample to a test tube.
2. Add 2 mL of Benedict’s solution to the test tube.
3. Repeat this for each sample.
4. Place the tubes in a beaker of hot water for five minutes. Use test tube clamps to hold
hot test tubes. BE CAREFUL NOT TO BURN YOURSELF
5. Record your results for each sample.
What other substances might be interesting to test for reducing sugars? What would be your
expected results?
1. Use the white tile for this experiment, divide the tile into four. As shown
2. Add a small amount of each sample onto each section of the tile
3. Add 2 drops of iodine solution to each sample. Observe the change in colour.
4. Record your results for each sample.
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Testing for lipids
Ethanol extracts the lipids from the samples. When water is added a layer is formed at the top
as lipids are less dense than water giving a cloudy white appearance. You have;
The Sudan IV test can also be used to test for lipids? How does this test work? How is the
principle of the Sudan IV test similar and/or different from the one described in your
experiment?
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Results
Monosaccharides
(Glucose)
Polysaccharides
(Starch)
Lipids
Proteins
(16 marks)
Are the results as expected? Do you accept or reject your hypotheses? (2 marks)
Carbohydrates contain elements carbon, hydrogen, and oxygen. Proteins are made up of
carbon, hydrogen, oxygen, and nitrogen while lipids are mostly comprised of carbon and
hydrogen.
What type of chemical bond forms between the atoms of these macromolecules? Explain. (4
marks)
(Total: 30 marks)
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PRACTICAL 3: EFFECT OF TEMPERATURE
AND pH ON ENZYME ACTION
Enzymes are proteins which catalyse biological reactions. Some of them are “simple
proteins”, others are “conjugated proteins”. The latter can be broken down into the proteins
portions and another complex organic component (prosthetic group). In either case, the
enzyme catalysed reaction depends on a transient combination between the enzyme and the
substrate, whose reactivity is accelerated by the enzyme. This transient combination is called
the enzyme – substrate complex. It follows that the velocity of an enzyme-catalysed
reaction will depend on the number of combinations per unit time, between the enzyme and
the substrate. Factors that affect the reactivity of the active sites of the combination will also
influence the velocity of the reaction. If the amount of the substrate is in excess, there is a
linear relationship between the rate of reaction and the amount of enzyme. Further, the rate of
reaction rises to a plateau, if the amount of substrate is gradually increased in the presence of
a constant amount of enzyme. Like all chemical reactions, there is a dependence on
temperature. Similarly, enzymes catalysed reactions also depend on pH. The pH affects
enzyme catalysed reactions because enzymes are multivalent and dipolar.Their stability is
affected by pH leading to pH-dependent dissociation.
This experiment will be performed in two parts; in section A, the action of amylase on starch
and its dependence on temperature will be performed. In section B, the experiment will be
carried out at a different pH.
As the amylase break down the starch, it will cause the blue colour to disappear. Take note of
how long it takes in each case.
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What do you think would have been the result if another set of substrate and enzyme had
been kept at 100°C?
Label clean test tubes 1-6 and add 5ml of 1% starch solution to each tube
Add solutions to each tube as indicated below
TUBE CHEMICAL
1 1 ml 1M sodium bicarbonate solution
2 0.5 ml 1M sodium bicarbonate solution
3 1 ml water
4 0.5 ml water
5 1 ml 1M acetic acid
6 0.5 ml 1M acetic acid
When any of the samples fails to give a blue colour, this means that the starch in that tube has
been completely broken down into sugar by the amylase. Note the time for each tube when
this happens for each tube and stop sampling.
Write a detailed lab report based on the results obtained from the two experiments and in
your discussion include other factors that also affect enzyme action.
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PRACTICAL 4: HANDLING THE COMPOUND MICROSCOPE
A microscope is a precise instrument and should be treated accordingly. It may appear to be
very strongly made, but actually the slightest jerk could damage the working parts or the
optical system. Handle it with care and avoid subjecting it to sudden or severe impact
To prevent damage, do not hold the Do not carry the microscope with the
microscope by the stage [1] or observation power cord connected. Be sure to
tube [2]. disconnect the power cord when storing
the microscope after use.
1. Always use two hands to carry the instrument. Hold the arm of the microscope with
one hand and place the other hand under the base
2. When putting down the microscope on the bench top, put it down carefully so that the
delicate mechanism will not be damaged
3. Keep your fingers off the lens surface. Clean the eyepiece and objective lenses each
time that you use the microscope. Only use the proper lens cleaning paper, which
will be provided. Any other material might scratch the lenses.
4. Keep the stage of the microscope clean and dry. Wipe it immediately with a paper
towel if it becomes wet.
5. Always cover the object on the slide with a cover slip, in order to protect the objective
lens.
6. Never move the stage upwards while looking into the eyepiece. This is to avoid
breaking the slide. Be careful not to force the adjustment knobs beyond the end of
their travel
7. Do not use the course adjustment when the high power (HP) objective is in position.
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Parts of a microscope
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Identification of parts of a compound microscope
1. Ocular lens or eyepiece: the upper part of the arm holds the eyepiece. The
microscopes to be used are binocular. Each eyepiece has a magnification of 10X.
2. Diopter Adjustment Ring: The diopter adjustment is to compensate for the
difference in eyesightbetween your eyes.
3. Revolving Nosepiece: found on the upper part of the arm and holds the objective
lenses of differing magnifying power. Any objective can be rotated to the required
position above the stage. Note the position stops for each lens
4. Objective Lenses: there are usually four objective lenses on the microscope, 3.2X or
4X, 10X, 40X and 100X
5. Specimen Holder: platform on which slides are mounted for viewing; the control
knobs on the mechanical stage allow the operator to move the slide easily during
viewing. Always make sure you clip the slide in position properly.
6. Specimen holder axis feed knob: Move the specimen vertically and horizontally.
7. Aperture Iris diaphragm Ring: the diagram controls the amount of light that passes
to the specimen and can drastically affect the focus of the image.
8. Focusing knobs: Located on the slide of the microscope; outer (small) is the fine
focus and inner (large) is the coarse focus.
9. Pre-focusing knob:The pre-focusing knob controls the mechanism for preventing
collisionbetween the specimen and objective.
10. Light Intensity Adjustment Knob: the microscopes have built- in light sources. The
brightness control knob is located on the base and usually has a brightness range.
11. Main Switch: turns the light source on and off.
12. Par focal: Microscopes are par focal, meaning that the image remains in focus when
the objectives are changed, requiring only a slight change in fine adjustment for sharp
image.
13. Field of View: the area you can see at a given level of magnification.
14. Resolution: The resolving power of a lens or microscope is the smallest distance
separating two objects that allows the objects to be seen as two distinct objects rather
than a single entity.
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Using a compound microscope
You will now have an opportunity to look at a mounted letter E and a preparation of a
microorganism under the microscope. Practice. Practice. Practice.
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PRACTICAL 5: MICROSCOPY TWO
Firstly place a drop of liquid (this varies depending on the specimen) in the centre of a
clean slide and place the specimen on the drop.
The amount of liquid you use is critical. Too little liquid will not support and float the
cover slip. Too much liquid means that your specimen will not stay in your field of view
and you will get the microscope stage wet.
Cover Slip
Slowly lower
the coverslip
Slide
Holding the cover slip by its edges, lower it so that one edge contacts the drop of water
first, then lower the rest of the cover slip carefully so that all the air is expelled (as shown
in the above figure). It is often convenient to support the upper edge of the cover slip on
the point of your tweezers or a pin as it is lowered into place.
Do not squash down on the cover slip; it needs to float on the drop of liquid.
If you have got large air bubbles under the cover slip that obscure the specimen, make a
new preparation.
REMEMBER
USE ONLY THE FINE ADJUSTMENT KNOB FOR FOCUSING WHEN USING THE
HIGH POWER OBJECTIVES
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A. Human Epithelial Cells Specimen
Epithelial cells are the covering or lining cells in animals. The cells making up the lining of
the inside of your mouth are epithelial cells which are easily rubbed off. Using your own
epithelial cells, make a slide in the following manner.
Exercise
1. Draw and label 3-5 cells (6 marks)
2. Estimate the size of one cell (2 marks)
3. Calculate the drawing magnification (2 marks)
B. Onion Specimen
Exercise
4. Draw and label 3-5 cells (6 marks)
5. Estimate the size of one cell (2 marks)
6. Calculate the drawing magnification (2 marks)
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PRACTICAL 6: MOVEMENT OF SUBSTANCES ACROSS AND
THROUGH PLASMA MEMBRANES
INTRODUCTION
In general, substances move across plasma membrane by two principle kinds of transport
processes (Figure 2); Passive transport processes and active transport systems. Passive
transport processes do not require the expenditure of cellular energy, and active transport
processes require the expenditure of cellular energy.
Figure 2: transport of substances across the cell membrane by active and passive
transport
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Passive transport processes include diffusion through the bilipid layer, diffusion through
channels, facilitated diffusion (Figure 3.a), osmosis filtration and dialysis. In passive
transport processes substances move because of differences in concentration (or pressure)
from areas of higher concentration (or pressure) to areas of lower concentration (or pressure).
The movement continues until the concentration of substances (or pressures) reaches
equilibrium. These processes are the result of kinetic energy (energy of motion) of the
substances themselves and the cell does not expend energy to move the substances.
Figure 3: Illustrating the differences between facilitated transport and active transport
processes.
(Figure 4).
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HAEMOLYSIS AND CRENATION
Osmosis can be demonstrated by noting the effects of different water concentration on Red
Blood Cells. Red Blood Cells maintain their normal shape and volume when placed in an
isotonic solution (Solution 2).
2) An isotonic solution has the same salt concentration (0.9%
NaCl) as that found in red blood cells.
If however red blood cells are placed in a hypotonic solution (salt solution with less than
0.9%NaCl), a net movement of water into cells occurs, causing the red blood cells to swell
and eventually to burst (Solution 3). The red blood cells swell because the difference in
osmotic pressure inside the cell compared to outside the cell results in a net movement of
water into the cell. The rapture of blood cells in this manner
manner is termed haemolysis.
If instead red blood cells are placed in hypertonic solution (a salt solution with more than
0.9% NaCl), a net movement out of the red blood cells occur, causing the cells to shrink
(Solution 1). The red blood cells undergo the shrinkage
shrinkage known as crenation, because the
difference in osmotic pressure inside the cells compared to the outside of the cells results in
the net movement of water out of the cell.
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PROCEDURE
EXERCISE 1:
1. Mark three microscope cavity slides as follows: 0.9% NaCl, DW (Distilled Water),
3% NaCl.
2. Using a medicine dropper, place one drop of fresh (uncoagulated) blood on a
microscope slide that contains 0.5ml of 0.9% NaCl solution. Mix gently and
thoroughly with a clean toothpick.
3. Using a medicine dropper, place one drop of fresh blood on a microscope slide that
contains 0.5ml of distilled water. Mix gently and thoroughly with a clean toothpick.
4. Using a medicine dropper, place one drop of fresh blood on a microscope slide that
contains 0.5ml of 3% NaCl solution. Mix gently and thoroughly with a clean
toothpick.
5. Using a medicine dropper, place one drop of the red blood cells from the 0.5ml, 0.9%
NaCl a solution cavity slide, on another microscope slide, cover with a cover slip and
examine under the microscope.
6. Using a medicine dropper, place one drop of the red blood cells from the 0.5ml, 0.9%
DW cavity slide, on another microscope slide, cover with a cover slip and examine
under the microscope.
7. Using a medicine dropper, place one drop of the red blood cells from the 0.5ml, 3%
NaCl a solution cavity slide, on another microscope slide, cover with a cover slip and
examine under the microscope.
Observe the shape of the cells under each solution, what could have resulted in the observed
changes? Tabulate your results.
Generally, molecular weight of a substance gives some idea of the approximate diameter of
its molecules- the larger the molecule the greater the molecular weight. Steric configuration
and symmetry, or its lack, also influences the effective diameter of a molecule. Very large
molecules (e.g. proteins) have great difficulty entering a cell, while small molecules (e.g.
amino acids) may enter cells more freely.
The amount of time that it takes for hemolysis or crenation to occur is directly related to the
rate of osmosis across the cell membrane. Hemolysis time can be used as an index as to the
rate of diffusion of penetrating molecules into the cell. For example, red blood cells can be
suspended in hypertonic solutions of differing solutes. If the solute cannot penetrate through
the cell membrane and pull water with it, no hemolysis will occur. However, if the solute can
penetrate through the cell membrane, it does so because of its greater concentration outside of
the cell and increases the concentration of osmotically active molecules inside the cell. The
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water is pulled into the cell due to the newly established osmotic gradient and hemolysis
occurs. Therefore, the rate of haemolysis can also be used as an indicator of the diffusion rate
for particular penetrating.
Hemolysis of red blood cells is accompanied by changes in light absorbance and clarity of the
cell suspensions. As hemolysis occurs, the RBC membrane bursts open, releases its
hemoglobin, then settles to the bottom of the tube, causing the solution to clear.
PROCEDURE
1. Add 2 drops (or 100 µl) of red blood cells suspension to each test tube containing 2ml
of 0.3M solutions given below and note the start time.
Formamide 45
Propyl alcohol 60
Urea 60
Ethylene glycol 62
Thiourea 72
Glycerol 92
Glucose 180
Sucrose 342
Is there a correlation between the observed time of haemolysis and the known molecular
weight?
Write a full lab report on the results obtained from exercise 1 & 2.
If a 10% salt solution is separated from a 20% salt solution by a selectively permeable
membrane, in which direction will there be a net movement of water?
Plant fertilizer consists of numerous different solutes. A small dose of fertilizer can enhance
plant growth, but overfertilization can kill the plant. How might overfertilization have this
effect?
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PRACTICAL 7: RESPIRATION: ENERGY CONVERSION
During the process of aerobic respiration, relatively high-energy carbohydrates are broken
down in a step-wise fashion, ultimately producing the low-level products of carbon dioxide
and water and transferring released energy into ATP. But, what is the role of oxygen?
Seeds contain stored food material, usually in the form of some type of carbohydrate.
When a seed germinates, the carbohydrate is broken down by aerobic respiration,
liberating the energy (ATP) required for each embryo to grow into a seedling.
Two days ago, one set of dry black eye beans was soaked in water to start the germination
process. Another set was not soaked. In this experiment, you will compare carbon dioxide
production between germinating pea seeds, germinating pea seeds that have been boiled and
ungerminated (dry) black eye beans.
This experiment investigates the hypothesis that germinating seeds produce carbon dioxide
from aerobic respiration.
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Materials
Procedure
Place about 250 ml of tap water in a 600 ml Buchner flask, put the flask on a hot plate and
bring the water to a boil. Add some germinating black eye beans into the boiling water.
Continue to boil for 5 minutes. Tip out the boiling water and cool with cold water for about
10 minutes. Tip out the cooling water.
Make sure that the tubing from the centrifuge tube is places into a distilled water filled test
tube. Add enough water to the test tube to cover the end of the tubing that comes out of the
centrifuge tube. (This keeps gases from escaping from the centrifuge tube.)
Make a prediction about the carbon dioxide production in each of the three bottles
After 1 hour 15 minutes, pour the water in the test tube into the sink and replace it with an
equal volume of dilute phenol red solution.
Use distilled water to push any gases from the centrifuge tube into the test tube.
Phenol red solution, which should appear pinkish, will be used to test for the presence of
carbon dioxide within the respiration bottles. If carbon dioxide is bubbled through water,
carbonic acid forms:
When the phenol red solution is basic (pH >7), it is pink; when it is acidic (pH<7), the
solution is yellow.
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Record your observations
What happened during boiling that caused the results you found? (Think enzymes.)
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PRACTICAL 8: CELL CYCLE & CELL DIVISION
The meristamatic cells located in the root tips provide the most suitable material for the study
of mitosis. The chromosomes of monocotyledonous plants are large and more visible
therefore onion root tips are used to study mitosis.
Aim
The aim is to prepare a temporary mount of onion root tip to study mitosis.
Actively growing onions will be present in the lab for you to observe and harvest your own
roots.
The first step will be to ‘soften’ the roots so that they later can be spread on a microscope
slide.
1. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into a plastic micro-
tube. (One of the roots will be a spare one.)
2. Fill the tube about 2/3 full with 1N HCl from a dropper bottle.
*** Caution: Work with the HCl carefully, it is a strong acid. ***
3. Place the tube in a 60°C water bath, and allow the roots to incubate for 12 minutes.
4. After the 12 minute incubation period, remove the tube from the water bath.
Rinse the roots in H2O.
1. Using forceps, carefully transfer the root tips to a small petridish
2. Using a disposable pipette, carefully remove the HCl from the micro-tube and transfer it to
the “discard flask”.
3. Rinse the root tips 3 times with water from the dropper bottle, disposing of the rinses in the
discard flask.
Staining the chromosomes.
1. After removing the water from the third rinse, cover the root with Feulgen stain.
*** Caution: Feulgen stain will strongly stain skin and clothing. ***
2. Incubate the roots in the stain for 12 minutes. During this time the v tip of the root will
begin to turn red as the DNA stains the numerous small actively dividing cells at the tip.
Remove the stain and again rinse the roots.
Using a disposable pipette, carefully remove the Feulgen stain and discard it in the
discard flask.
Again, rinse the root tips 3 times with water.
Preparing the root tip squash.
1. Transfer a root to the centre of a clean microscope slide and add a drop of water.
2. Using a razor blade cut off most of the unstained part of the root, and discard it.
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3. Cover the root tip with a cover slip, and then carefully push down on the cover slide with
the wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide
sideways. The root tip should spread out to a diameter about 0.5 – 1 cm.
Observations of onion root tip squash.
Scan the microscope under the 10x objective. Look for the region that has large nuclei
relative to the size of the cell; among these cells you will find cells displaying stages of
mitosis. Switch to the 40X objective to make closer observations.
If you have managed to prepare a section of root tip that is actively growing then it should be
possible to observe all of the phases of cell division. Be aware that if your plant cells are not
actively growing then you are unlikely to see much cell division, so select your subject
carefully.
Submit a labelled drawing of your observations indicating what phase of mitosis the
cells are in.
Even in a growing root tip, the majority of the cells will likely be in interphase. In interphase
they will look like 'normal' plant cells, with a dark and well defined nucleus . Remember that
although interphase is the phase cells spend the most time in, they are not inactive. Cells will
be copying their DNA in preparation for cell division (referred to as Synthesis or S). Before
and after Synthesis, the cell will be growing (referred to as Gap 1 before Synthesis and Gap
2 after) which involves the production of proteins and organelles, including mitochondria.
Cells undergoing mitosis will look different depending on the stage they have reached. Cells
in prophase look similar to those in interphase but the nucleus will appear less well defined as
the nuclear membrane begins to break down. In metaphase, the chromosomes will appear
lined up along the middle of the cell, so individual chromosomes should be visible. In
anaphase chromosomes will be migrating to opposite ends of the cell, which you may be able
to see, or the cell may just look 'smudgy'. Finally, in telophase the cell will appear to have
two nuclei as the two sets of chromosomes are fully separated and the nuclear membrane
reforms. This can be tricky to spot as you need to make sure there is no cell wall separating
the two nuclei.
Cytokinesis is a separate stage of cell division from mitosis, where (in plants) a cell plate
forms across the middle of the cell, which eventually becomes a new cell wall. Cytokinesis in
plants is significantly different from cytokinesis in animal cells because of the presence of the
cell wall. When observing this under the microscope, plant cells may appear slightly 'pinched'
in the middle, but will also have a hazy or wavy line separating the two new daughter cells.
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PRACTICAL 9: POPULATION GENETICS
(CALCULATION OF ALLELE FREQUENCIES)
In the early twentieth century, Godfrey Hardy, a British mathematician; and Wilhelm
Weinberg, a German physicist, both working independently, developed a field of research
called population genetics. They studied the effect that heredity had on a population and
discovered that evolution occurs when the frequency of gene alleles changes. If evolution is
not occurring, then the ratio of heterozygous to homozygous individuals will not change. A
mathematical equation that can be used to determine the frequencies of alleles within a
population was developed. It was known as the Hardy-Weinberg Principle and is stated as:
p2 +2pq+q2=1
The frequencies of dominant and recessive alleles are represented by p and q, respectively.
For example, a diploid individual has two alleles at a particular locus. The two alleles ‘A’ and
‘a’, can result in only three possible genotypes: AA, Aa, and aa. The probability of receiving
‘A’ alleles from both parents is p x p or p2, and for the ‘a’ alleles, q x q or q2. Receiving the
Aa combination is described by 2pq, since it is possible for the ‘A’or ‘a’ allele to come from
either parent, thereby doubling the chance.
To apply the principle, at least one of the allele frequencies must be known. Let us use an
example of a genetically inherited trait: the ability to roll your tongue. The ability to roll your
tongue (R) is dominant over the inability to roll (r). Suppose in a class of 28 students, 23
students were rollers and 5 were not. Since tongue-rolling is a dominant trait, those with the
ability to roll may be either RR or Rr. However, those with the inability to roll can only have
one possible genotype, rr. Using this information, it is possible to calculate the frequency of
the recessive genotype (q2). Since 5 out of 28 could not roll their tongue, 5/28 = 0.18 or 18%
are non-rollers. Since q2 = 0.18, then q id the square root (√) of 0.18, or 0.42. This value is
the frequency of the recessive gene (regardless of genotype) within the class.
Since there are only two possible genes for this trait (R and r), it stands to reason that p+q =
100% or 1. Using some simple maths, the frequency of p can be calculated: 1 – q = p, or 1 –
0.42 = p; therefore p = 0.58. Using the Hardy Weinberg principle, the frequency of
heterozygotes can be calculated. Since 2pq = frequency of heterozygotes, then 2(0.58)(0.42)
= 0.49 or 49%.
If the relationship between p and q remains constant through randomly mating generations,
the population is said to be in Hardy-Weinberg equilibrium and no evolution occurs. In order
for a population to remain in Hardy-Weinberg equilibrium, seven conditions must be met:
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o Every member of the population produces the same number of offspring
o Mating is completely random
o The population is infinitely large
In reality, it is unlikely that any or all of these conditions will occur within a population. If
any of these conditions are not met, the proportions of heterozygotes to homozygotes can
change. Therefore, the Hardy Weinberg principle is most useful as a tool for measuring the
degree of genetic change or evolution occurring within a population.
Objective
Students will investigate genetically-transmitted traits (the ability or inability to taste certain
chemicals) and calculate the frequency of the recessive gene, dominant gene and the
genotypes within a population (the class).
Materials provided
Procedure
1. Make sure that you have rinsed your mouth before you come to your practical session.
2. Obtain a piece of PTC test paper. Place it on your tongue. Record whether it tastes
bitter or has no taste in your data table.
3. Repeat the procedure for the three remaining test papers. If possible rinse your mouth
between trials and record your results as you did before.
4. Tabulate the data for the entire class. Record the data in the table.
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Using the Hardy-Weinberg principle, calculate the frequency of the recessive genotype, the
frequency of the recessive gene, the frequency of the dominant gene, and the frequency of
heterozygotes in your class. Perform the calculations for all the chemicals tested.
There are several genetic traits that do not seem to be either beneficial or harmful. This
includes attached or detached earlobes. If you have time, you can analyse class data for these
traits. See if you can determine which is the dominant form, and which is the recessive form
of the trait. Apply the Hardy-Weinberg principle to determine the frequencies of these traits
within the class.
You are conducting a study of an isolated tribe in New Guinea and you find that there is
widespread resistance to a parasite of the genus cryptosporidium. You determine that the
gene for resistance is inherited in a recessive fashion. The incidence of resistance in a normal
population is 1/900. In New Guinea, it is 1/25. What are the carrier frequencies in the normal
population and in New Guinea, respectively? Assume that the populations are in Hardy-
Weinberg equilibrium.
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APPENDIX A: LABORATORY REPORT WRITING
TITLE
INTRODUCTION
- This section tells why the study was undertaken it establishes the framework for
the entire report
- Should have an openings statement and there should be flow of thoughts
(coherence) in your write up
- Define specialized terminology but your write up should not be a series of
definitions
- Briefly explain any specialized technique to be carried out or how a specialized
equipment works
- The background information must be related to the investigation
- Support all statements of fact and opinion with evidence i.e. cite literature; but not
websites, primary sources only
- Never set out to prove, verify or demonstrate the truth of something. Rather set
out to test, document or describe.
- Your introduction must lead to an aim/ objective/ research question /hypothesis/
prediction
- Your introduction should be brief and well thought out.
METHODS
- Must give sufficient detail on how the investigation was conducted. The detail
should allow someone else or even yourself to repeat the investigation.
- Should be passive and in past tense
- Should be written in continuous /essay format not point form
- Dot not list the materials used separately, these should rather be reflected in the
write up as you outline what was done
- Do not give unnecessary details and do not departure from the given instructions
- Include
. description of study site
. how the investigation was conducted
. variables measured
. variables to be computed and how this would be done, e.g. use of formulae
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RESULTS
DISCUSSION
- This is where you discuss the pattern(s) highlighted in the results section. Avoid
veering away from objective/research question/hypothesis/prediction.
Remember this is a discussion, so it is not acceptable to simply restate the results.
- What does the literature say? For example, studies with similar results and those
with different results.
- Do the results support your hypothesis? Are the results as you had predicted?
- What is the relevance, application and implications of your results to the everyday
life.
- Should end with a CONCLUSION. The conclusion should be in line with the
objective/research question/hypothesis/prediction, results and discussion.
- No new information in conclusion section.
- State your findings, have your aims been fulfilled and have your results lewd you
to accept or reject your hypothesis?
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CITATIONS IN THE REPORT
In the narrative text, join the names with the word ‘and.’
E.g. As Leiter and Maslach (1998) demonstrated……
When work has three authors, cite all authors the first time the reference occurs.
E.g. Kahneman, Knetsch, and Thaler (1991) found……
In the subsequent citations per paragraph, include only the surname of the first author
followed by “et. al.” (Latin for “and others”) and the year of publication.
E.g. Kahneman et al. (1991) found….
When work has four or more authors, cite the last name of the first author et al.
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E.g. Nyamukondiwa, Mazebedi, Williams &Mokobela (2012) is cited as
Nyamukondiwaet al. (2012)
REFERENCES
Foukaridis, G.N., Osuch, E., Mathibe, L. & Tsipa, P., 1995. The ethnopharmacology and toxicology of
Urginea sanguinea in the Pretoria area. Journal of Ethnopharmacology, 49, pp.77-79.
Jaleel , C.A., 2009. Non-Enzymatic Antioxidant Changes in Withania somnifera with Varying Drought
Stress Level. American-Eurasian Journal of Scientific Research, 4(2), pp.64-67.
Durrell , K. & Ricklefs, R.E., 1990. Ecology. 3rd ed. New York: Freeman and Company.
Fransworth, N.R. & Soejarto, D.D., 1991. Global importance of medicinal plants. In O. Akerele, V.
Heywood & H. Synge, eds. Conservation of medicinal plants. Cambridge: University Press. pp.25-51
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GENERAL
- If your investigation did not involve manipulation of any variable, then it was not
an experiment. Refer to it as a study or an investigation.
- Write to illuminate not impress: use the simplest words and phrasing consistent
with the goal
- Always distinguish fact from possibility. Don’t state your opinion as though it
were fact.
- Stick to the point
- Say exactly what you mean, and don’t waffle. Good scientific writing is precise.
Sloppy writing often implies sloppy thinking.
- Number the pages of the report.
- Type in font 12, Times New Roman
- Write scientific names properly (Genus name starts with upper case and species
name with a lower case letter. Italicize or underline the scientific name).
- Use past tense.
- Write in impersonal form.
- Write numbers zero to nine in words and those above nine in figures.
- e.g, etc. & and other such abbreviations should be spelt out in full.
- sms language is not allowed.
- Have a title page for the report.
- Do a spelling and grammar check
- EDIT, EDIT, EDIT
-
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APPENDIX B: BIOLOGICAL DRAWING GUIDELINES
The study of biology requires that a considerable amount is time spent observing
structures and discovering their interrelationships and functions. Biological drawings
enhance observation skills and enable one to communicate what has been observed.
Careful observation of the object often yields good drawings. Poor drawings are often a
result of poor observation.
1 Draw what you see, not what you think should be there.
3. Use clean lines for diagrams (do not sketch). All drawings are to be done on
plain whitepaper, with one drawing per face. Leave a 2 cm margin on the left of the page.
4. Drawing should be large enough to show all parts without crowding. You
have a whole sheet of paper; do not be afraid to use it. You must draw each cell the same
size as what you see.
6. You do not need to draw every cell in the field of view which you have in
focus, but you must draw representative groups or filaments of cells. If there are different
types of cells, be sure to include at least one of each type. Draw them so that you can
recognizethem later.
8. Keep your drawings to the left of the page. Put all the labels to the right of
the diagram. All labels should be printed. Use a ruler for label lines and make sure you do
not cross your label lines.
9. Do not shade your drawing. If you wish to indicate a darker area, use dots.
Indicate the thickness of a plant cell wall by using two lines.
10. Include a printed and underlined title immediately below the drawing in the
centre of the page. The Total Magnification follows the title in parentheses [e.g. BONE CELL
(100X)]. Include name of specimen, part drawn, orientation e.g. longitudinal section, Cross
section or whole mount
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11. Ensure that each drawing has a drawing Magnification. Drawing
Magnification is calculated with following steps:
Number of times that cell/specimen fits across the diameter of field of view
You MUST show the formulas and your calculations at the bottom of your drawing.
3.2X 5.25
4X 4.0
5X 3.5
10X 1.6
40X 0.4
100X 0.16
Hints:
Move the slide around to get a good overview of the specimen, don not just
concentrate on one part. Also view the slide under different magnifications and light
amounts.
You should label any structure which can be identified or is specified in the
laboratory manual.
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3. If you and another student are viewing the same organism, looking at the same field
of view, make sure that your drawings are not alike. In other words, choose different
organisms in the field of view. It will definitely not be proper for you to let someone
"look at" your drawings. You will lose points if someone else's drawings resemble
yours too closely. Also, your drawings should not appear identical to those found in
the textbook or Internet sources!
4.
Figure 1: (a) Young anther (ts); (b) Enlarged view of one microsporangium.
The drawings above illustrate a tissue map in (a) and a cellular map in (b).
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