Centre for Pre-U Studies

MF 011
GENERAL BIOLOGY II

Laboratory Manual (Revised)

Lecturer : Mr. James L
 MF011 – General Biology II

Content Page

Content Page

Guidelines : Report writing, submission and lab conduct. 2

Practical 1 : Investigation of the Carbohydrates Metabolised by Yeast. 6

Practical 2 : Observation of the Structure of the Mammalian Kidney using 8

Kidney Model and Tissue Slides.

Analysis of kidney filtration process using simple filtration system

Practical 3 : Observation on Blood Smear 13

Practical 4 : Reflexes 14
Practical 5 : Testing the Hardy-Weinberg Equilibrium 15
Practical 6 : DNA Extraction from Human Buccal Cells 22

Practical 7 : The Effect of Penicillin on Bacterial Growth 24

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 MF011 – General Biology II

Guidelines for writing laboratory reports

Laboratory reports should be written according to the format below (failure to do so will result
in marks being deducted):

 Formatting
Font Type: Times Roman
Font size: 12
Spacing: 1.5, justified
Pages : 5 (minimum) - 10 (maximum) [pages must be numbered]

 Title page
You are required to use the lab report submission page available on the LMS and are
to include these details: lab no., title of experiment, students’ names and ID, date of
experiment as well as your group number.

 Introduction
This section serves to acquaint the reader with the subject and justify the objective(s)
of the experiment. There should be two parts to the introduction: first, a clear
description of the principle(s) underlying the experiment; and second, a statement of
how you will conduct the experiment to study the underlying principle(s). You must
include the source of references/adaptations for the points made (in-text references).
Approximately ONE page is recommended.

 Objective(s)
The objective(s) addressed in the experiment must be clearly stated and numbered.

 Chemical / apparatus
List only the important materials used. Commonly used apparatus or glassware (e.g.
clamp, forcep, beaker, measuring cylinder) need not be mentioned. Do measure
magnitude/volume of apparatus used, when applicable.

 Procedures
The description of experimental methods must contain sufficient information to allow
another person to duplicate the experiment. All methods should be written in past
tense, passive voice and should be written in your own words (paraphrase). Some lab
sessions may have several parts and as such, you may use subheadings for these
parts (i.e. Practical 2 which has 2 parts – A. Kidney model examination and B.
Nephron kidney slide observation).

 Results
Results may be combined with discussion in order to provide a more coherent flow
(can also be separated from discussion). This section must contain sufficient
information (and calculations if necessary) to fully describe the outcome of the
experiment. Where necessary, the use of tables is encouraged. Tables must contain
enough information within them and with their respective titles or legends to be
understandable without referring to the text. Drawings and labelling must be done
using pencil only. A maximum of 2 drawings per A4 size paper is advised. Photos
maybe included as thumbnail to drawings.

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 MF011 – General Biology II

 Discussion
This section contains an explanation of the meaning of the results. The principles,
relationships, and general truths shown by the results should be presented without
reiterating the results. Use text to emphasize important points in your results, to
connect results with one another, and to restate the trend of the idea (the objective
already mentioned in the ‘Introduction’) while connecting them to textbook, reference
books or other reference materials such as journals or online articles as
references/supporting materials. You must include the source of
references/adaptations for the points made, including diagrams taken (in-text
references). Exceptions or lack of correlation and important precautionary measure(s)
should be pointed out and unsettled points (if any) explained. The theoretical or
practical implications of the experiment should be discussed. Approximately TWO to
THREE (maximum no) pages are recommended.

 Conclusion
The conclusion should be stated briefly (usually in a SINGLE PARAGRAPH). It would
usually be answering the objectives of the experiment.

 Questions
All questions must be answered in a separate section, regardless of whether the
calculation or answer has been mentioned. Keep answers short and precise (these
are not mini-essay questions).

 References
Students must indicate the references made. References are important to support
the points made and giving credit to the original author. One may follow the
alphabetical format (sorted according to last names) or numerical format (sorted
according to the order of citation in the main text). Each report should contain at least
FIVE references.

Assessment Criteria

Criteria Wtg.
Title Page 2
Introduction 10
Objectives 2
Chemical / apparatus 6
Procedures 10
Results 10
Discussion 25
Conclusion 5
Answers to Questions 15
Referencing 10
Lab Report Presentation 5
TOTAL 100

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 MF011 – General Biology II

Submission of laboratory reports

 Students are required to conduct experiments in groups of 4 to 5 persons. Only 1 report

is submitted per group for every experiment.

 Students are required to submit laboratory reports within 1 (ONE) weeks after each day
of laboratory session. Failure to do so will result in an automatic 0.

 Plagiarism is strictly forbidden. If found, students will be automatically awarded a 0.

 Each laboratory report is to be submitted together with a copy of the “Laboratory

Report Submission Form”. Only if you require an acknowledgement receipt of your
laboratory report, please prepare a duplicate copy of the form. This form can be
downloaded from the LMS.

Laboratory practical rules

 Questions are HIGHLY encouraged in lab sessions.

 All students are to ensure proper attire (long pants/long skirts only, covered shoes,
clipped/tied hair (if long), laboratory coat) (SHORTS, SKIRTs and SLIPPERS ARE
FORBIDED) at all times during the lab session. Failure to do so will result in the removal
of penalised student from the laboratory and the student will be awarded 0 mark.

 No caps/hats, sunglasses, food, drinks, mobile phones and headphones allowed in the
laboratory.

 Be serious in the lab. Laughing is allowed but no horse-play is allowed.

 All students are not allowed to wear lab coats outside the lab.

 Attendance is COMPULSORY. It is the responsibility of the student to ensure that

his/her chosen laboratory session schedule does not clash with other course(s)
undertaken during the semester.

 All students are required to read and understand the procedures of the corresponding
experiment(s) before coming for their laboratory sessions. Students are expected to be
proactive during the lab sessions.

 All students must be punctual for their laboratory sessions. Those absent for any
laboratory session must produce medical certificates (from Laurent Blue
Clinics/Government Hospitals) or written letters stating their reasons for being absent.
If a valid reason is not shown, the student will be given an automatic 0 mark for that
session. Acceptance of reasons will be subject to the sole discretion of the lecturer.

 All students are to enter and leave the laboratory only with the permission of the lecturer
or laboratory technician.

 All students are to ensure to equip themselves with felt tip permanent marker pens for
labelling, and camera(s) (optional) to photograph work and data.

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 MF011 – General Biology II

 The lab benches should not have personal belongings other than essential stationary
during the lab sessions (in the lab bench area).

 Students are to be seated according to group number and they are responsible to clean
up their respective apparatus and lab bench after each lab session. Failure to do so
will result in deduction of marks.

 Safety measures are to be observed (i.e. gloves, masks etc.) at all times especially
when handling dangerous biohazard materials.

 Do not wear gloves to touch the door of the laboratory, take off your gloves before
leaving.

 Students are to report if accident occurs at any point during the session to either the
lecturer or lab staff for further assistance.

 All students are to wash hands thoroughly before and after practical sessions (in the
lab).

 Group leaders are responsible to keep respective group members in check of these
rules.

 Students are to ask questions, if query arises regarding the lab practical itself, or
assessment criteria, before, during and/or after the class. It is highly encouraged that
students are to discuss amongst group members & classmates before referring to the
lecturer.

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 MF011 – General Biology II

Practical 1
An Investigation of the Carbohydrates Metabolised by Yeast

1.0 Background information

Yeast, Saccharomyces cerevisiae, can metabolise carbohydrates under two different

conditions. When oxygen is present, aerobic respiration occurs yielding a large amount of
energy for the organism and producing carbon dioxide and water as waste products. However,
in anaerobic conditions, when oxygen is in short supply the yeast will break down the
carbohydrate into ethanol and carbon dioxide with a much reduced energy output (alcoholic
fermentation).
Specific enzymes catalyse both of these forms of respiration in addition to most metabolic
processes where the efficiency of the yeast is in metabolising different carbohydrates can be
monitored by observing the time taken for Methylene blue to be discoloured.
In this experiment, you will be investigating the relative efficiency with which different
carbohydrates can be metabolised by yeast.

2.1 Materials

Boiling tubes
Carbohydrates (0.5M glucose, 0.5M fructose, 0.5M lactose, 0.5M sucrose)
Graduated pipettes
Distilled deionised water
10% (w/v) Baker’s yeast (boiled and unboiled yeast suspensions)
Methylene blue (0.005% v/v) (Precaution: Methylene blue is harmful. Avoid contact with
eyes and skin. It will stain skin or clothes)
Water bath (40oC)

2.2 Methodology

1. Prepare and label 6 duplicate of boiling tubes and fill in the tubes in accordance to Table
1.0 below.
2. Add the carbohydrates solution first, followed by three drops of methylene blue and lastly,
add the yeast suspension into each of the tubes. Note the time (starting point) upon adding
the yeast culture. You may vortex or shake each of the tubes to mix the content.

TABLE 1.0 Composition of Different Carbohydrates in Boiling Tubes.

Tube number
Component (volumes in mL)
1 2 3 4 5 6
0.5M glucose 2.0 - - - - -
0.5M fructose - 2.0 - - - -
0.5M lactose - - 2.0 - - -
0.5M sucrose - - - 2.0 - -
0.5M starch - - - - 2.0 -
Double-deionised water - - - - - 2.0
20% (w/v) unboiled yeast suspension 1.0 1.0 1.0 1.0 1.0 1.0
20% (w/v) boiled yeast suspension 1.0 1.0 1.0 1.0 1.0 1.0

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3. Immediately, place all the boiling tubes in the 40ºC water bath. Do not disturb the tubes
again by shaking, but note the time taken for the blue colour to disappear from each tube,
leaving behind the creamy colour of the yeast. A thin film of blue at the surface of the tube
can be ignored and should not be moved. (You may observe the tubes in every 1 to 2
minutes intervals depending on how fast the colour change takes place).
4. Record the duration taken in Table 1.1.
5. The experiment may be repeated by simply shaking all the tubes again until the blue colour
returns (if time permits). Take an average of the times obtained, if experiment is repeated.

TABLE 1.1 Yeast Carbohydrate Metabolism.

Tube number Time for methylene blue to go colourless
Trial 1 Trial 2 (if time permits)
1
2

3.0 Questions

1. What causes the methylene blue solution to go colourless?

2. Why was distilled water used?
3. If the hydrogen atoms for the reduction of methylene blue come from glucose, and if
boiled yeast was used instead, should the tube containing methylene blue and glucose
with boiled yeast become decolourized at all? Why?
4. Why, do you think, the colour retuned on shaking the tubes?
5. Cyanide is a poisonous substance, leading to harmful effects toward humans as well
as many other organisms. If the unboiled yeast suspension in this experiment were to
be treated with cyanide compound, describe the possible effect(s) of this compound
toward the metabolic pathway of these cells.

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 MF011 – General Biology II

Practical 2
Observation of mammalian kidney model and tissue slides
Analysing kidney filtration using simple filtration system

1.0 Background information

The excretory system of mammals centres on a pair of kidneys. In humans, each

kidney tubules are about 10 cm long and is supplied with blood by a renal artery and drained
by a renal vein. Blood that flows through the kidneys is voluminous.

The kidneys account for less than 1 % (w/w) of the human body mass but receive
roughly 25% (v/v) of the blood exiting the heart. Various important structures that exist in the
kidney perform its main function in homeostasis for osmoregulation. Nephron, which is a
functional unit of kidney plays a major role in the processing and production of renal fluid. Each
nephron consists of a Bowman’s capsule containing glomerulus, a bundle of capillaries, and a
long differentiated tubule which leads to a collecting duct. Malpighian corpuscle made up of
both the Bowman’s capsule and glomerulus together.

In this experiment, you will be investigating the general structure of a mammalian

kidney while analysing a simple filtration system. Blood is filtered across the walls of the
glomerulus into the cavity (capsular space) of Bowman’s capsule, leaving behind large
molecules such as blood cells or plasma proteins. As the renal filtrate flows along the tubule of
the nephron, important and useful molecules are selectively reabsorbed back into the
bloodstream, whilst harmful substances are also being secreted into the tubule from the blood
vessel surrounding the tubules.

2.1 Materials

Microscope
Mammalian kidney model
Kidney nephron slides
Whatman filter paper (8 nm pore size)
Conical flask
Filter funnel
Biuret solution
Benedict’s solution
Iodine in potassium iodide (KI) solution
Test tubes

2.2 Methods

a) Observation of major structures in a mammalian kidney

1. Observe the kidney model presented to you and identify the various important
structures in the kidney that is vital for osmoregulation.
2. Draw these main structures and label them accordingly. Indicate the position of
nephrons and tubules in your figure based on your understanding.

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b) Examination of tissue slides under a microscope

1. Observe and examine a prepared horizontal or transverse section of mammalian kidney
slide.
2. First, hold it up to the light and examine it under a lens and note the demarcation
between cortex and medulla. Figure 2.0 shows on a distorted scale on how the
nephrons and collecting ducts are disposed relative to the cortex and medulla.

FIGURE 2.0 Mammalian kidney sectioned to show the position of the nephrons and
their blood supply. For clarity, the nephron and blood vessels are shown
separately; in reality they are intimately associated. A Bowman’s capsule
and its associated glomerulus together constitute a Malpighian body.
Note how the nephron and its blood supply are orientated relative to the
kidney as a whole, and also which parts of the nephron are in the cortex
and which ones in the medulla.

3. Examine the cortex under low power. Notice that it contains Bowman’s capsules,
numerous tubules in section, and capillaries.
4. Examine the detailed structures of cortex under high power. Select one that most
resembles Figure 2.1 in its general appearance. The capsule contains a glomerulus
and you may see afferent and efferent arterioles which take blood to and from it. Notice
the gap between the bunch of glomerular capillaries and the outer epithelium of the
capsule: this is the capsular space into which blood is filtered to form renal fluid. The
renal fluid then passes along the tubules and eventually to the collecting ducts.

FIGURE 2.1 A Malpighian body as it may appear in a microscopic section of the

kidney. This drawing is idealised; rarely would a section pass through
the afferent and efferent vessels and the proximal tubule.

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5. The tubular structures visible in a section of the cortex are a mixture of proximal and
distal convoluted tubules and collecting ducts as shown in Figure 2.2. Below are the
details for the three structures:
a) Proximal tubule:
Outer diameter about 60μm, large lining cells, cell membranes between adjacent
cells not visible, relatively few nuclei visible in cross section of tubule, small
irregular lumen, brush border, three times as long as distal tubule, thus more
abundant in section.

b) Distal tubule:
Outer diameter about 20 - 50μm, smaller lining cells, cell membranes between
adjacent cells not visible, more nuclei visible in cross section of tubule, large
regular lumen, no brush border visible, shorter than proximal tubule, thus less
abundant in section.

c) Collecting duct:
Outer diameter about 25 - 60μm, wall similar to distal convoluted tubule except
that cell membranes between adjacent cells are clearly visible.

FIGURE 2.2 Tubules, collecting duct and capillaries as they appear in a microscopic
section of the cortex of the kidney. Plasma membranes are not usually
visible between adjacent cells in the walls of the tubules, hence their
absence in this drawing. Red blood cells are sometimes present in the
blood capillaries.

6. Observe and examine the medulla under high power. It contains the loop of Henle which
connects the proximal with the distal convoluted tubule. Notice the descending and
ascending limbs of the loop of Henle and the long straight capillaries, vasa recta, which
run parallel to them as shown in Figure 2.3. Below are the details for the descending
and ascending limbs of the loop of Henle as well as the collecting ducts: (Note: The wall
of the thin limb is confusingly similar to that of the capillaries (vasa recta). However, the
capillaries can usually be distinguished by the fact that they contain red blood cells).
a) Descending limb (except for the lowest third):
Outer diameter about 14 - 20μm, lining of thin pavement epithelium, nuclei
flattened and bulging into lumen (known as thin limb).

b) Ascending limb (plus lowest third of descending limb)

Outer diameter about 30 - 35μm, lining of cuboidal epithelium, cell membranes
between adjacent cells not visible, rounded nuclei (known as thick limb).
c) Collecting duct:

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Outer diameter about 50 - 200μm, lining cells cuboidal to columnar, cell

membranes between adjacent cells are clearly visible with rounded nuclei.

FIGURE 2.3 The loop of Henle, collecting duct and capillaries as they appear in a
microscopic section of the medulla of the kidney. The thin limb is the top
two-thirds of the descending limb of the loop of Henle; the thick limb is
the rest of the descending limb plus the whole of the ascending limb.
Plasma membranes are not usually visible between adjacent cells in the
walls of the loop of Henle, hence their absence in this drawing.

7. Prepare high power drawings for each section, cortex and medulla. (Detailed drawing
as seen under high power microscope showing accurate cell/tissue characteristics).
Draw only a few cells.
8. Each drawing must have a complete title which gives the following information:
a. Name of organ
b. Type of section
c. Magnification

c) Analysing kidney filtration (Note: You would have to troubleshoot accordingly. Explore and
‘enjoy’ errors/ambiguities in your results)

1. Add 1 mL of test solution as table 2.1 below. Label them accordingly:-

TABLE 2.0 Kidney filtration experiment test tube composition

Content (in Test tubes
mL) 1 2 3 4
1% Protein 1.0 - -
1 % Glucose - 1.0 -
Starch - - 1.0
ddH2O - - - 1.0

2. Add 1 mL of Biuret solution to the test tube labelled protein. Record your observations.
3. Add 1 mL of Benedict’s solution into the test tube labelled glucose. Place the test tube
in the water bath (100oC) and leave for 5 minutes, shaking. Record your observations.

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 MF011 – General Biology II

4. Add 1 drop of iodine in potassium iodide solution into test tube labelled starch. Record
your observations.
5. Repeat steps 2 – 4 for test tube 4 (separately).
6. Discard the tested solutions and rinse the test tubes clean.
7. Re-prepare the solutions as table 2.1, but this time, prepare for a volume of 4.0 mL
instead.
8. Prepare a filter paper on a Buchner funnel and a conical flask. Pour content of each
test tube to the filter paper and collect the filtered solution in the conical flask.
9. Repeat steps 2 - 4 onto the filtrate.
10. Record your observations accordingly in a table 2.1.

TABLE 2.1 Results tabulation of the kidney filtration simulation.

Ensure that observation is taken before and after filtration.
Tube Colour intensity Colour intensity
number (before filtration – test tube) (after filtration - filtrate)
(- : no presence, + : least intense, (- : no presence + : least intense,
++ : intense, +++ : most intense ) ++ : intense, +++ : most intense )
1
(Biuret)
2
(Benedict’s)
3
(Iodine)
4
(All 3)

3.0 Questions

1. Among the tested substances, which of the compounds passed through the filter
paper?
2. Was there a difference in the intensity of the colour that was observed from your initial
tests? If so, why? If not, why? Suggest a method to quantify your method.
3. Explain why some of the compounds did not pass through the filter paper.
4. What is the function of test tube 4?
5. How does the filtration in this activity resemble the activity of the kidney?
6. Explain the difference in the roles played by the different tubules present in the nephron
structure of a kidney.
7. Why do mammals need to get rid of their excretory products?

Suggested reference
Histology Text books

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Practical 3
An Observation on Blood Smear and Structure of Blood Vessels

A. An Observation on Blood Smear

Learning Outcome
By the end of this practical you should be able to:
 Use a microscope with skill and precision
 Show all the structures that can be seen in the defined part of a specimen
 Make clear, accurate, labelled, scale drawings of specimens

Material
 Microscope
 Eyepiece graticule and stage micrometer
 Prepared slides of human blood stained with Leishman’s or Wright’s stain

Human Blood Smear

Blood is the major transport medium of mammals. In mammals blood consists of red blood
cells, white blood cells and platelets suspended in a fluid medium, plasma.

No staining is required to see the red blood cells, but staining is necessary to distinguish the
platelets and various types of white blood cells. Leishman’s stain or Wright’s stain are usually
used. Each of these contains eosin and methylene blue.

Methodology
3. Observe the slide at 40X. Draw and label accordingly.

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Practical 4
Reflexes

Learning Outcome
By the end of this practical you should be able to:
 Describe and explain reflex arc that occurs during a knee-jerk reflex, swallowing reflex,
iris reflex and blinking reflex
 Evaluate the procedure, discussing why it is difficult to quantify this investigation
 Describe and explain the reflex arc that occurs during reaction timing

Introduction
A reflex is a rapid, unconscious, stereotyped response to a stimulus. Most reflexes involve the
brain, but some use only the spinal cord. Either way, reflexes can give us important information
about the functioning of the nervous system, and abnormalities in our reflex responses can help
doctors to diagnose certain disorders of the nervous system.

Procedure
Knee Jerk
1) The subject should sit on a table with his or her legs hanging loosely over the edge.
With a small tendon hammer, the experimenter should tap the tendon just below the
knee cap. There is no need to tap hard. If the tap is applied correctly, the extensor
muscle in the front of the thigh contracts and the leg gives a little kick.
2) Practice eliciting the knee jerk until you get it right every time. Try varying the intensity
and location of the stimulus. Note that a response is elicited only by applying a sudden
tap in just the right place.
3) Draw a diagram of the nervous pathways responsible for the knee jerk. Bear in mind
that when an extensor muscle contracts its antagonist (the flexor) should relax, and
that when the leg gives a kick more than one extensor muscle may be involved.

Ankle jerk
1) The subject should kneel on a chair with his or her feet hanging loosely over the edge.
The experimenter should now tap the tendon at the back of the foot, just above the
heel. Describe the response.
2) Repeat tapping the response tendon at approximately two taps per second. Does the
response decline, get larger or stay the same? Explain.

Swallowing reflex
1) Swallow the saliva in your mouth cavity, and immediately afterwards try to swallow
again – and again. You will probably find it difficult to swallow the second time, and
even more difficult the third time. Why do you think this is?
2) Now drink a glass of water and note that you have no difficulty swallowing several times
in rapid succession. Propose an explanation for the difference.

Pupil reflex
1) Point a torch or bench light at your partner’s eye in a darkened room with the torch /
bench light switched off. Watch his / her pupil.
2) Switch on the torch / bench light. Observe the pupil constricting. How is this achieved?

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Practical 5
Testing the Hardy-Weinberg Equilibrium

1.0 Background Information

In 1908, G.H. Hardy and W. Weinberg separately introduced a mathematical model

that shows the relationship between the frequency of an allele and the frequency of a genotype
in a hypothetical population that has reached genetic equilibrium. Collectively this is commonly
used population genetics equilibrium is known as the Hardy-Weinberg equilibrium. It states that
“under certain conditions, after one generation of random mating, the frequency of dominant
alleles and the frequency of recessive alleles at a single gene locus in a population remain
constant from generation to generation under certain conditions”. These conditions are:-
 A large population (so that any change in allelic frequency is negligible, or to minimize
genetic drift)
 No natural selection
 Random mating (all individuals are sexually active and equally fertile)
 No mutation (in case of mutation, the rate of forward mutation is the same as the rate
of backward mutation)
 No migration/gene flow (no immigration or emigration)
Based on the equilibrium and conditions, Hardy and Weinberg developed an equation
based on the theory that the total frequency of alleles of a gene is 100% (generally
represented as 1.0). Hence, the Hardy-Weinberg equation

Equation (7.1):-
p + q = 1.0
where, p = frequency of dominant allele; q = frequency of recessive allele

However, in diploids, alleles occur in pairs. Thus a 2 nd derivation (commonly known as the
Hardy Weinberg population genotype frequency)

Equation (7.2):-
p2 + 2pq + q2 = 1.0
where, p2 = frequency of homozygous dominant allele genotype;
2pq = frequency of heterozygous genotype;
q2 = frequency of homozygous recessive genotype.

Hence, the Hardy-Weinberg equilibrium allows us to predict the population’s genotype

frequency from its allelic frequencies.

The application of the Hardy-Weinberg principle in relation to an X-linked gene (i.e. the
one controlling eye colour in Drosophila melanogaster and colour blindness in humans) is
slightly different. The allelic frequencies are estimated from the frequencies of genotypes
in males; and the frequencies of the genotypes in females are obtained by applying the
Hardy-Weinberg principle to these estimated allelic frequencies (assumption: allelic
frequencies are the same in the 2 sexes). For example, in human colour blindness (X-linked
recessive mutation), if the frequency of normal vision (C) is p = 0.88 and the frequency of
colour blindness (c) is q = 0.12, then under the Hardy-Weinberg assumptions, refer to table
7.1:-

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TABLE 7.1 Allelic frequencies in colour blindness

Sex Genotype Frequency Phenotype
Males C p = 0.88 Normal vision
(single allele
because either XCY C q = 0.12 Colour blind
c
or X Y -
hemizygous)
CC p2 = 0.77 Normal vision
Females Cc 2pq = 0.21 Normal vision
Cc q2 = 0.02 Colour blind

In this exercise we will demonstrate the contention of the Hardy-Weinberg Theorem

that genetic recombination as a result of sexual reproduction will not, by itself, cause any
change in allele frequencies or genotype frequencies in a population from one generation to
the next. Obviously, it is difficult to demonstrate this principal using real organisms: depending
on the organism, it could take days, weeks, months or even years to show that allele and
genotype frequencies remained constant for several generations - or even for a single
generation. Furthermore, the Hardy-Weinberg equilibrium depends on the absence of any
forces operating which might change allele frequencies in a population. These forces—
mutation, migration, selection, and chance effects—are almost never absent from natural
populations and it would be very difficult even in the laboratory to erect a set of conditions that
might reliably exclude them.

The Hardy-Weinberg Law can be mathematically demonstrated in the following table.

If p equals the frequency of allele A and q is the frequency of allele a, union of gametes would
occur as follows:

p q

p p2 pq

q pq q2

One of the predictions of the Hardy-Weinberg Law refers to the genotypic

frequencies after one generation of random mating. In the above table, the genotypic
frequencies for AA is p2, the genotypic frequency for Aa is 2pq and the genotypic frequency
for aa will be q2. These are the values that are predicted by the law. The second prediction
is that the frequencies of the two alleles will remain the same from generation to generation.
To prove it, finish the Hardy-Weinberg derivation worksheet.

Assuming Hardy-Weinberg equilibrium is in effect we can calculate the allelic

frequencies from the genotypic frequencies. To calculate the allelic frequency from the
genotypic frequency, first figure out from the genotypes where all the A alleles are found, (AA
and ½ of the alleles of Aa individuals will be A). So (where f stands for frequency), p = f(AA) +
½ f (Aa). Since p2 represents the frequency of AA and 2pq represents the frequency of Aa then
when we substitute those frequencies into the equation we get, p = p2 + ½(2pq). Using
analogous calculations we can calculate the frequency of the a (q) allele. In absence of any
factors that change the allelic frequencies, the genotypic and allelic frequencies will remain the

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same from generation to generation.

The Hardy-Weinberg equilibrium also assumes random mating in the population, and
once again this is a situation that is rarely found in nature and difficult to achieve in the lab.
Therefore, for the purposes of our demonstration, we are going to work not with real organisms
but with a model—in this case dried beans.

Case study
Background

Around the Taman Negara area, we found that there are 20% black and 80% grey
squirrels. The gene freqeuncy in the population is written as

pp : 2pq : qq

Or

p2 + 2pq + q2 = 1

where p = the frequency of the dominant allele (black) , and q = the frequency of the
recessive allele (gray). It follows that p + q = 100% of all the genes in the gene pool.

Materials

Two different colour beans

3 empty beakers
1 plastic bag

Procedure

1. Count 100 beans of each color.

2. The red beans represent the allele for black fur, and the green beans represent the allele
for gray fur. The plastic bag represents the environment in Taman Negara where the
squirrels randomly mate.

3. Label one beaker “Black Fur, FF” for the homozygous dominant genotype. Label a second
beaker “Black Fur, Ff” for the heterozygous condition. Label the third beaker “Gray Fur, ff”
for those squirrels with the homozygous recessive genotype. (See below):

Beakers:

Black fur FF Black fur Ff Gray fur ff

4. Start with 25 red and 25 green beans. Put the fifty beans (representing alleles) into the
plastic bag and shake it up (represents a mixing of alleles via reproduction between

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 MF011 – General Biology II

squirrels).
Scenario #1 – Hardy-Weinberg Equilibrium:

1. Without looking at the beans, select two at a time, and record the results on the data
form on the following page - next to "Generation 1." For instance, if you draw one red
and one green bean, place a mark in the chart under "Number of Ff individuals."
Continue drawing pairs of beans and recording the results in your chart until all beans
have been selected and sorted. (Please note that the total number of individuals will
be half the total number of beans because each squirrel requires two alleles.
2. For this simulation, count the F and f alleles (beans) that were placed in each of the
beakers for "black squirrels" in the first round and record the number in the chart in
the columns labelled "Number of F Alleles" and "Number of f Alleles." Repeat this
step for the “gray squirrels.” Total the number of F alleles and f alleles for the first
generation and record this number in the column labeled "Total Number of Alleles."
Below is a sample of how your results might look:

No of No of Total Gene Gene

Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1

3. Place the alleles of the squirrels (which have grown, survived and reached
reproductive age) back into the plastic bag and mate them (shake bag) again to
obtain the next generation.
4. Repeat steps “a” through “c” to obtain generations two through five. Try to make sure
everyone in your group has a chance to either select the beans or record the results.
5. Determine the gene frequency of F and f for each generation and record them in the
chart in the columns labeled "Gene Frequency F" and "Gene Frequency f." To find
the gene frequency of F, divide the number of F by the total, and to find the gene
frequency of f, divide the number of f by the total. Express results in decimal form.
The sum of the frequency of F and f should equal one for each generation.

DATA – SCENARIO #1 (HARDY-WEINBERG EQUILIBRIUM)

No of No of Total Gene Gene

Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1
2
3
4
5

Scenario #2 – Natural Selection:

1. As with the Hardy-Weinberg scenario, your group will start with 25 red and 25
green beans. Put the fifty beans (representing alleles) into the plastic bag and
shake it up (represents a mixing of alleles via reproduction between squirrels).
2. Select two beans (alleles) at a time from the bag without looking, and record the
results on the data form next to "Generation 1." For instance, if you draw one red
and one green bean, place a mark in the chart under "Number of Ff individuals."
Continue drawing pairs of beans and recording the results in your chart until all
beans have been selected and sorted. Place the "squirrels" into the appropriate

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 MF011 – General Biology II

dish: FF, Ff, or ff.

3. The FF and Ff squirrels are born with shiny black fur. Unlike the Hardy-Weinberg
situation above, squirrels with black fur living in a wooded environment stand out
against the dull gray/brown background more than their gray-furred relatives (see
photo on the first page for an example). This is especially true in the colder
months once deciduous trees have dropped their leaves, creating a landscape
full of grayish trees and a forest floor covered by brown, dried leaves. The shinny,
black-coated squirrels easily stand out in this environment, especially in large
forest tracts where red-tailed hawks abound.
These keen-eyed raptors spot the conspicuous black squirrels and swoop
down upon them often before they can escape. Therefore, the black variants are
less likely to reach reproductive age and pass on their genes. Place half the
beans from the FF and Ff containers aside before beginning the next round.
4. Once half of the beans have been removed from the homozygous dominant
and heterozygous beakers, you may now count the remaining F alleles
(beans) in each container. Do the same for the f alleles. Total the number of
F alleles and f alleles for the first generation and record this number in the
column labeled "Total Number of Alleles." Below is a sample of how your
results might look:

No of No of Total Gene Gene

Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1

5. Place the alleles of the surviving squirrels (which have grown and reached
reproductive age) back into the container and mate them again to get the next
generation.
6. Repeat steps “a” through “e” to obtain generations two through five. Make sure
everyone in your group has a chance to either select the beans or record the results.
7. Determine the gene frequency of F and f for each generation and record them
in the chart in the columns labeled "Gene Frequency F" and "Gene Frequency
f." To find the gene frequency of F, divide the number of F by the total, and to
find the gene frequency of f, divide the number of f by the total. Express results
in decimal form. The sum of the frequency of F and f should equal one for each
generation.

DATA – SCENARIO #2 (NATURAL SELECTION)

No of No of Total Gene Gene
Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1
2
3
4
5

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 MF011 – General Biology II

Scenario #3 – Genetic Drift:

1. Unlike the Hardy-Weinberg scenario, your group will start with 40 red and 10 green
beans. The cause of this imbalance is the result of the founder effect. A professor at
a college in Ohio studied the black squirrel variety in her laboratory and a few of her
graduate students accidentally released twenty black individuals onto the campus,
flooding the gene pool with the dominant allele. Put the fifty beans (representing
alleles) into the plastic bag and shake it up (represents a mixing of alleles via
reproduction between squirrels).
2. Without looking at the beans, select two at a time, and record the results on the data
form on the following page - next to "Generation 1." For instance, if you draw one
red and one green bean, place a mark in the chart under "Number of Ff individuals."
Continue drawing pairs of beans and recording the results in your chart until all
beans have been selected and sorted. (Please note that the total number of
individuals will be half the total number of beans because each squirrel requires two
alleles.)
3. For this simulation, count the F and f alleles (beans) that were placed in each of the
beakers for "black squirrels" in the first round and record the number in the chart in
the columns labeled "Number of F Alleles" and "Number of f Alleles." Repeat this
step for the “gray squirrels.” Total the number of F alleles and f alleles for the first
generation and record this number in the column labeled "Total Number of Alleles."
On the next page is a sample of how your results might look:

No of No of Total Gene Gene

Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1

4. Place the alleles of the squirrels (which have grown, survived and reached
reproductive age) back into the plastic bag and mate them (shake bag) again to
get the next generation.
5. Repeat steps “a” through “d” to obtain generations two through five. Try to make
sure everyone in your group has a chance to either select the beans or record the
results.
6. Determine the gene frequency of F and f for each generation and record them in
the chart in the columns labeled "Gene Frequency F" and "Gene Frequency f." To
find the gene frequency of F, divide the number of F by the total, and to find the
gene frequency of f, divide the number of f by the total. Express results in decimal
form. The sum of the frequency of F and f should equal one for each generation.

DATA – SCENARIO #3 (GENETIC DRIFT)

No of No of Total Gene Gene
Generatio No of FF No of Ff No of ff
F f no of frequency frequency
n individuals individuals individuals
alleles alleles alleles of F of f
1
2
3
4
5

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 MF011 – General Biology II

3.0 Questions

1. What conditions must be present for Hardy-Weinberg Equilibrium to be in effect?

2. Many deleterious genes are recessive and therefore are expressed only in the
homozygote. Many of them are also lethal and many of them have also been present
in the human gene pool for hundreds of thousands and possibly even millions of years.
How do you explain the persistence of these genes over such long periods of time in
the face of such intense selective pressure against them.
3. Explain why evolution cannot happen if the Hardy-Weinberg equilibrium is true in a
population

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 MF011 – General Biology II

Practical 6
DNA Extraction from Human Buccal Cells

1.0 Background Information

With the advent of gene technology, it is important to understand not only the
phenotype of the organism but also the genotype. Previously, you should have learnt the
analysis of genetic traits and the various ways where they can be transmitted from parents to
children (by phenotype analysis). Each chromosome is divided into different sections called
genes. Genes are the basis of inheritance where traits like hair colour and blood type are
controlled by the production of proteins by these genes. Genes contain coded instructions that
the body uses to assemble hundreds of different types of proteins that make an individual
unique!

These amazing trait controllers (genes) are made up of molecules called

deoxyribonucleic acid (DNA). DNA is a double-helical polymer bound together by hydrogen
bonds between complementary base pairing nucleotides (A to T, G to C). A particular gene is
a set of coded instructions made up of a particular order of nucleotides. The variation of which
allows the myriad of codes to exist in an organism for it to be unique. This is what controls the
genotype of an organism and henceforth, the extraction and isolation of an organisms DNA is
imperative, in order to allow further insight into the organism using different molecular-based
methods.

In this experiment, you will be taking a closer look at this DNA molecule. You will be
extracting your own DNA using buccal/cheek cells as the starting material.

2.1 Materials

Saline
15 ml centrifuge tube
Paper cup
Drinking water
Vortex
Centrifuge
10% SDS
Bromelain protease (50mg/mL)
Ice cold isopropanol
Graduated pipettes

2.2 Methods
IMPORTANT NOTE: Ensure that you have not eaten in the past 1 hour before conducting
this experiment (if you are the DNA donor). Ensure that gloves are worn at all times in
the experiment.

1. Swish you mouth with about 100 mL drinking water, for about 20 seconds, to remove
any food particles. Discard this wash into the sink.
2. Using a permanent marker pen, label your group name onto the paper cup and 15 mL
centrifuge tube containing 10mL saline.
3. Pour all the 10mL saline solution into your mouth and vigorously swish for 60s. Do not
discard the centrifuge tube.
4. Expel the saline mouthwash into the labelled paper cup.

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 MF011 – General Biology II

5. Carefully, pour the saline mouthwash from the paper cup, back into the 15 mL
centrifuge tube from step 2. Tightly cap the tube.
6. Pass the capped tubes to the laboratory technician in order to be centrifuged
(4500 rpm, 5 min).
7. Upon centrifuging, you should be able to see your buccal cell pellet (the whitish lower
solid layer at the bottom of the tube). Gently, pour away the supernatant (the liquid
upper layer).
8. Place the tube on ice.
9. Add 2 mL saline into the tube and vortex for 5-10 seconds.
10. Add 1 mL 10% (w/v) sodium dodecylsulphate (SDS) solution (active component in
detergents).
11. Gently tap the tubes several times (~8 times) to gently mix the contents. You may invert
the tube twice if needed.
12. On ice, add 2 – 3 drops of the lab supplied bromelain protease enzyme into the tube.
13. Gently tap the tubes several times (~8 times) to gently mix the contents. You may invert
the tube twice if needed.
14. Cap the tube and place it is a 50oC for 10 minutes.
15. With a clean pipette, gently pipette in 10 mL ice cold isopropanol (95% v/v) slowly into
the tube. Tip: Place the filled pipette with its tip against the inside wall of the test tube.
Slowly allow the isopropanol to dribble down the inside of the tube.
16. Cap and place the tube in a test tube rack at room temperature for 10 minutes. DO
NOT mix, shake, or bump the test tube during this period.
17. The isopropanol is lighter than the contents of the tube. When added according to the
directions, the isopropanol will form a clear layer ABOVE the suspension.
18. Observe the test tube for 5 minutes. The DNA will gradually separate from the
suspension and rise into the isopropanol layer. Describe the appearance of the DNA.
19. Take a photo as proof of your observation.
20. To remove the accumulated DNA from the tube, follow the directions for DNA spooling
as below:-
a. Gently insert the glass rod through the isopropanol layer into the
clumped/accumulated DNA.
b. Carefully, twirl the rod between your fingers, winding the DNA strands onto the
rod.
c. Slowly remove the rod. Describe the appearance of the spooled DNA.
d. Take a photo as proof of your observation.

3.0 Questions
1. Which one of the following do you think will contain DNA?
Bananas; concrete; fossils; meat; metal; spinach; strawberries.
Explain your reasoning.
2. What effect would the SDS have on the cell membranes and cold ethanol on DNA?
3. What type of enzyme would be needed to separate the DNA into smaller pieces?
4. Is the DNA extracted pure enough for further applications (i.e. PCR)?
5. If you were to repeat the experiment with an equal number of red blood cells, the
amount of DNA collected would either: increase / decrease / stay the same (choose
one). Explain your answer.

Adapted from:-
Bres, M., Weisshaar, A., 2008. Thinking about Biology: An Introductory Laboratory Manual. 3rd
Ed. Pearson Prentice Hall: New Jersey, USA. Pg. 333 - 338.
Teaching AS Biology Practical Skills. University of Cambridge: International Examination. Pg.
74 – 78.

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 MF011 – General Biology II

Practical 7
The Effect of Penicillin on Bacterial Growth

Learning Outcome
By the end of this practical you should be able to:
 Pour agar plates using aseptic technique
 Experience growing microorganisms
 Use the disc diffusion technique needed to undertake a microbiological investigation
 Use vernier callipers to measure clear zone diameters
 Assess the reliability of results

Background Information
Penicillin is an antibiotic that prevents the synthesis of mucopeptides in bacterial cell walls by
preventing the formation of peptide bonds; it is bactericidal. Staphylococcus epidermidis forms
small white colonies.

Penicillin is effective against Staphylococcus epidermidis. Discs containing penicillin are placed
on an agar plate containing Staphylococcus epidermidis. If the penicillin has been effective
there will be a clear zone around the disc. The size of the clear zone is a measure of the
effectiveness of the penicillin

Materials
• Bunsen burner, to enable good aseptic conditions
• 10cm3 bacterial culture in nutrient broth
• 10cm3 molten nutrient agar*
• 10cm3 sterile water*
• Sterile 90 mm Petri dish
• 1cm3 pipette*
• Filter paper discs* or prepared penicillin discs
• Forceps
• 10cm3 1% penicillin solution (students may carry out dilution of this to achieve
several concentrations for testing)
• 20cm3 absolute ethanol, to enable sterilisation of forceps
• 50cm3 bactericidal disinfectant for containment of used forceps and pipettes,
also to clean work surfaces.

Rules for working with microorganisms:

1 BEFORE STARTING WORK cover all cut or broken skin with a waterproof dressing.
2 WEAR tightly fitting disposable gloves when working with all live cultures.
3 WEAR a clean laboratory coat with all the fastenings closed.
4 BEFORE AND AFTER each working session wash the bench surface with bactericidal
disinfectant.
5 AFTER each working session dispose of gloves into the sterilin bag or other disposal
container provided. WASH your hands with bactericidal soap.
6 SWAB any spillages with bactericidal disinfectant. DISPOSE of any contaminated paper in
the sterilin bag or other disposal container provided.
7 NEVER place anything in your mouth whilst working with microorganisms. This includes
foods, liquids, gummed labels, pipettes etc.
8 ALL CULTURES should be labelled clearly with the following information:
Your name
Name of the organism
Type of nutrient medium used
Date
9 NO CULTURE should be left for more than one week. Incubators are checked daily and
outdated cultures will be removed for safe disposal.
10 WHEN you have completed your work with a culture the petri dish should be placed in a
sterilin bag or into a container of bactericidal disinfectant.

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 MF011 – General Biology II

Procedure
Preliminary study – Safety and Plate Pouring
After watching the demonstration on plate pouring and aseptic technique prepare a pour plate
as follows.
1 Read the safety rules for working with microorganisms.
(Step 2 is done by lab technician)
2 Use a sterile 1cm 3 pipette to place 1cm3 of Staphylococcus epidermidis (albus) into a 50
cm3 of nutrient agar aseptically.

Day One – Preparation of the Pour Plate

Draw lines on the base of the petri dish so that the base is split into 4 equal parts.
Label the sections 1 to 4.
1 Label the base of the petri dish with your name, name of the organism, type of nutrient agar
and the date.
2 Add 10cm3 of nutrient agar to the petri dish using aseptic technique and leave to set.
3 Using aseptic technique place an antibiotic disc on the surface of the agar using flamed
and cooled forceps. Note the concentration of antibiotic on the disc.
4 Seal the petri dish.
5 Incubate the petri dish at 25°C for 1 day.

Follow Up – Data Collection

1 Without opening the lid observe the presence of the clear zone around each disc and record
in the result.

25