NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Unit 3
Activities
[REVISED ADVANCED HIGHER]
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Acknowledgements
The publisher gratefully acknowledges permission to use the following sources: image of haemoglobin
from http://commons.wikimedia.org.uk/wiki/File:1GZX_Haemoglobin.png and image of a nucleosome
from http://commons.wikimedia.org/wiki/File:Nucleosome_structure.png both © Richard Wheeler
(Zephyris); image of beta sheets from http://commons.wikimedia.org/wiki/File:PDB_1jy6_EBI.jpg ©
http://www.ebi.ac.uk; image of kinases from http://commons.wikimedia.org/wiki/File:Ch4_kinases.jpg ©
National Institute of General Medical Sciences; image of DNA X-ray from
http://commons.wikimedia.org/wiki/File:ABDNAxrgpj.jpg, ‘Physical Chemistry of Food’, vol. 2, van
Nostrand Reinhold: New York, 1994, I.C. Baianu et al; image of a protein primary structure from
http://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg and image of DNA Exons from
http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif both © The National Human Genome
Research Institute; image of electrophoresis from http://commons.wikimedia.org/wiki/File:SDSPAGE_Electrophoresis.png © Bensaccount at en.wikipedia; image no 3418 of African sleeping sickness
from http://phil.cdc.gov/phil/details.asp © CDC/Alexander J. da Silva, PhD/Melanie Moser; image no
11820 of Giemsa-stained light photomicrograph revealed the presence of a Trypanosoma brucei parasite,
which was found in a blood smear from http://phil.cdc.gov/phil/details.asp © CDC/Blaine Mathison;
image from Toxicology in Vitro 18 (2004) 1–12, Workshop report, The humane collection of fetal bovine
serum and possibilities for serum-free cell and tissue culture, reprinted from Toxicology in Vitro 18, Vol
1-12, Workshop report, The humane collection of fetal bovine serum and possibilities for serum-free cell
and tissue culture by J. van der Valk,D. Mellor,R. Brands,R. Fischer,F. Gruber,G. Gstraunthaler,L.
Hellebrekers,J. Hyllner,F.H. Jonker,P. Prieto,M. Thalen,V. Baumans, 2004 with permission from Elsevier
http://www.journals.elsevier.com/toxicology-in-vitro/; image from article, Conservation, Variability and
the Modeling of Active Protein Kinases
http://www.plosone.org/article/slideshow.action?uri=info:doi/10.1371/journal.pone.0000982&imageURI
=info:doi/10.1371/journal.pone.0000982.g001 © 2007 Conservation, Variability and the Modeling of
Active Protein Kinases by James D. R. Knight, Bin Qian, David Baker, Rashmi Kothary; image from
article Proteomics of Trypanosoma evansi Infection in Rodents from
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.000979 © 2010 Proteomics of
Trypanosoma evansi Infection in Rodents by Nainita Roy, Rishi Kumar Nageshan, Rani Pallavi, Harshini
Chakravarthy, Syama Chandran, Rajender Kumar, Ashok Kumar Gupta, Raj Kumar Singh, Suresh
Chandra Yadav, Utpal Tatu; image of Signal transduction from
http://commons.wikimedia.org/wiki/File:Signal_transduction_v1.png © Roadnottaken at the English
language Wikipedia
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UNIT 3 (AH, BIOLOGY)
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Contents
Activity 1: Scientific method study design
4
Activity 2: Scientific method investigation proposal
11
Activity 3: Phage and the scientific process
16
Activity 4: Reading a scientific research paper
25
Activity 5: Scientific literature and communication
37
Activity 6: Scientific ethics A
40
Activity 7: Scientific ethics B
41
Activity 8: Pilot study, peer review and plagiarism
43
Activity 9: Gel electrophoresis pilot study
45
Activity 10: Enzyme kinetics pilot study
50
Activity 11: Controls, sampling and ensuring reliability
54
Activity 12: Sampling using transect
59
Activity 13: Evaluating experimental design
63
Activity 14: Evaluating data analysis
66
UNIT 3 (AH, BIOLOGY)
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3
ACTIVITY 1
Activity 1: Scientific method study design
Scientific research is a highly methodical process and requires meticulous
planning and the development of a clear hypothesis in order to determine a
conclusion. As part of your Advanced Higher Biology assessment you will
carryout an independent investigation. The hardest part of any investigation is
the planning and determination of the independent and depe ndent variables as
well as the confounding variables that must be controlled in order to ensure
reliable and valid data. In this activity you will be analysing a series of mock
investigations to determine the reliability and validity of the hypothesis and
conclusions made.
1.
2.
3.
4.
5.
Three scenarios relating to investigations are outlined below.
For each scenario read the description carefully.
Think about reliability, accuracy, repeatability, replications,
confounding variables and controls.
Make notes on aspects of each investigation in response to the
questions provided. You may wish to use Google Docs to manipulate
and annotate the text as a class to develop your understanding
further.
There may be details of each of the investigations that you are
unsure of but these should not prevent you from evaluating the
validity of the methods as described.
These notes will be used as the basis of a tutorial where you will discuss
the reliability and validity of results in relation to your Advanced Higher
Biology investigation. This will then allow you to start thinking about
your own investigation and the variables that will interact with the
independent variable and thus alter the dependent variable.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 1
1.
A learner is interested in the effects of herbal remedies on bacterial
growth and tested three herbal remedies, rosemary oil, sage oil and
lavender oil, on the growth of the bacteria Escherichia coli. Using an
aseptic technique the learner spread E. coli on agar petri dishes and
transferred chromatography paper soaked in each of the oils to each
dish. Each petri dish contained four discs of standard size of one oil
type, and three dishes were produced for each oil type. The bacteria
were incubated overnight at 30°C. The next day the zone of inhibition
of growth of E. coli was recorded around each disc. Four measurements
from each disc were recorded and the average zone of inhibition
calculated. The whole experiment was then replicated. The learner’s
results showed that the greatest zone of average inhibition occurred
around lavender oil within the repeats and between the replicate
experiments, and it was concluded that lavender oil has the greatest
antibacterial effect.
What variables were controlled?
What variables were not controlled?
Is the learner’s conclusion valid?
Space for notes
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ACTIVITY 1
2.
A learner is interested in comparing the catalase content of potatoes
during the chitting process (sprouting the tubers before planting). The
learner’s hypothesis is that as potatoes grow new shoots and grow roots
the hydrogen peroxide concentration will increase as metabolic
processes, respiration and photosynthesis increase. As a result, there
will be a parallel increase in the catalase concentration in the potato to
break down the toxic hydrogen peroxide produced. The learner took a
bag of 20 potatoes of the same species and placed them in chitting trays
on a windowsill in a greenhouse. Over a period of a month potatoes
were sampled at set time intervals, taking 10 samples in total and
repeating each sample twice. Using a core borer the learner removed a
set volume of potato, homogenised it in buffered isotonic solution and
took a known volume of this sample and exposed it to hydrogen
peroxide of a known concentration and volume. The initial pressure
change in a sealed environment as a result of the oxygen liberated in the
reaction was measured. The results showed that the initial pressure
change in the sealed environment as a result of the oxygen liberated was
greater the longer the potato was left to chit. The learner concluded that
chitting increases the concentration of catalase in potatoes as a result of
the increased metabolic activity of the potato during the chitting
process.
What variables were controlled?
What variables were not controlled?
Does chitting affect the concentration of catalase in this species of
potato?
Space for notes
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 1
3.
A learner is interested in investigating the effect of light intensity on
the growth of roots and shoots in garlic ( Allium sativum), measured in
terms of length and biomass. The learner also wants to investigate the
effect of light intensity on the mitotic index in the root apical meristem
of garlic and relate this to the growth observed. The learner set up five
cardboard boxes with lids. Four boxes had rectangular sections cut out
and replaced with neutral density filters of known absorbance, one of
which transmitted 100% light. One box was left intact to create a dark
environment. A light source of 700 lux was used. Thirty gar lic cloves
were prepared and suspended across 100 ml beakers containing tap
water to stimulate root development. Two cloves were used in each
beaker and three beakers were placed in each environment 20 cm from
the light source. The garlic was left to grow for 12 days. Water was
replaced in each beaker daily to prevent fungal growth and reduce the
effects of evaporation. The shoot and root lengths were measured at
regular intervals and the dry biomass was recorded at the end of the
experiment. The mitotic index was calculated at the end of 9 days by
removing one healthy root tip from each clove, exposing it to acetic
orcein (stain) and counting the cells using a binocular microscope. The
experiment was repeated and replicated. The experimental results
showed that average root length and average shoot length (after 9 and
12 days, respectively) were both greatest for garlic plants grown in total
darkness. In addition, garlic plants grown in total darkness had, on
average, the greatest biomass relative to the ini tial mass of the clove.
The experimental results also showed that light had no effect on the
mitotic index, while neither root length nor root biomass was related to
the mitotic index.
What variables were controlled?
What variables were not controlled?
Can the learner conclude that light has no effect on the mitotic
index?
Space for notes
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ACTIVITY 1
Answers
1.
What variables were controlled?
Disc size.
Temperature of incubation.
Time of incubation.
Repeats done.
Replicates done.
Aseptic technique used for plating.
What variables were not controlled?
No control using water-soaked discs instead of oil.
No control done without discs to show no contamination or inhibition
occurred.
Discs not prepared aseptically.
Length of time discs exposed to oil before pl ating.
Different diffusion rates of oils will affect rate of movement through
the agar.
Is the learner’s conclusion valid?
No as no control has been done for comparison and diffusion rates have
not been accounted for.
2.
What variables were controlled?
Volume of potato used using a core borer.
Hydrogen peroxide concentration and volume.
Isotonic buffered solution during homogenisation to minimise cellular
effects on catalase.
Experiment repeated
What variables were not controlled?
No control using water instead of hydrogen peroxide and water instead
of potato extract.
Light intensity for chitting.
Temperature for chitting.
Humidity for chitting.
Temperature of reaction.
Experiment not replicated.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 1
Does chitting affect the concentration of catalase in this species of
potato?
The conclusion is not valid because:
No control set up to determine whether the pressure change is as a
result of the interaction between hydrogen peroxide and catalase.
Light intensity has not been controlled in the greenhouse t herefore the
rate of photosynthesis will vary over the duration of the experiment.
Temperature is not controlled in the greenhouse therefore over the
duration of the experiment temperature would vary greatly and may
affect the results.
Humidity is not controlled in the greenhouse and thus potatoes may
have dried out over the duration of the experiment and thus increased
the relative concentration of hydrogen peroxide in the potato.
Temperature is not controlled during the reaction in a thermostatically
controlled water bath, dramatically affecting the potential rate of
reaction.
Only repeats were done at known time intervals, no replicates using
separate potatoes of the same species were done.
3.
What variables were controlled?
Light intensity from light source.
Distance from light source.
Growth environment in box.
Water volume in beaker.
Number of cloves per beaker.
Control done in full light and dark.
Time.
Experiment repeated and replicated.
Method for mitotic index.
What variables were not controlled?
Temperature in box, affecting rate of reactions and growth.
Initial mass/size of cloves, affecting energy source available for initial
growth.
Random selection of cloves not used, resulting in an experimenter
effect.
Sample size small, reducing the validity of any conclusion drawn.
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ACTIVITY 1
Can the learner conclude that light has no effect on the mitotic
index?
The learner will need to qualify the variables they did not control and
justify their conclusions in line with this. Temperature changes during
the experiment may not have varied greatly and thus would have little
effect on growth rate. The initial mass of cloves may have a significant
effect on initial growth rate because of the energy stores available. The
learner would need to increase sample size an d randomise the selection
of cloves to minimise experimenter effect and any potential anomalies.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 2
Activity 2: Scientific method investigation proposal
Your name:
Practitioner’s name:
Investigation proposal
Use the numbered points below to outline your investigation. This should be
presented in electronic format to your class practitioner.
1.
2.
3.
4.
Area of interest:
Observation:
Question:
Background reading and understanding (insert references here – these
should be a minimum of three different sources and formats, eg book,
journal, websites).
References should be set out as follows:
Books
Author(s) (surname followed by initials) (year of publication) Title.
Publisher, place of publication, page number(s).
eg Wright R (2005) Environmental Science: toward a sustainable future.
Pearson Prentice Hall, New Jersey, p 446.
Journals/periodicals
Author(s) (surname followed by initials) (year of publication) Title of article.
Name of Journal, Volume number (part number if appropriate), page
number(s).
eg Bowsher C (2007) Designer starches. Biological Sciences Review, 19 (3),
18–20.
Websites
As many of the following items as are available must be given: author, date,
title, publisher, the URL and the date you accessed the material (because the
site may be updated between the time the writer uses it and the point at which
a reader refers to it).
eg The Mammal Society (2006) Position statement: badgers and bovine
tuberculosis. URL: http://www.abdn.ac.uk/mammal/badgers_tb.shtml.
Visited: August 2007.
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ACTIVITY 2
5.
6.
7.
8.
9.
10.
Independent variable:
How will you change the independent variable?
Dependent variable:
How will you measure the dependent variable?
Title, including operationalised independent and dependent variable:
Confounding variables: fill in the table below.
Confounding variables
eg Temperature
Control of confounding variables
eg Use thermostatically controlled
water bath set to 25C
Add further rows to table as required.
11.
Control(s): fill in the table below.
Control(s)
Reason for control
Add further rows to table as required.
12.
13.
14.
Aim:
Hypothesis:
Potential risk: fill in the table below.
Type of risk
How is the risk minimised?
Add further rows to table as required.
The following points will be completed after this initial proposal has been
discussed with your class practitioner. You will need to complete a more
detailed risk assessment before you begin.
15.
16.
17.
18.
Method:
Equipment:
Pilot study:
Limitations of equipment and procedure:
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 2
Example
Your name: A Biologist
Practitioner’s name: A Practitioner
Investigation proposal
Use the numbered points below to outline your investigation. This should be
presented in electronic format to your class practitioner.
1.
2.
3.
4.
Area of interest: Microbiology
Observation:
Pasteurised milk and UHT milk
Question:
How does pasteurisation affect bacteria?
Background reading and understanding (insert references here – these
should be a minimum of three different sources and formats, eg book,
journal, websites).
Books
Taylor DJ, Green NPO and Stout GW (2008) Biological Science 1 and 2, 3rd
edn. Cambridge University Press, Cambridge, p 518.
Journals/periodicals
Grant IR, Hitchings EI, McCartney A, Ferguson F and Rowe MT (2002)
Effect of commercial-scale high-temperature, short-time pasteurization on the
viability of Mycobacterium paratuberculosis in naturally infected cows' milk.
Applied Environmental Microbiology, 68 (2), 602–607.
Websites
The Dairy Council (2011) Varieties of Milk. URL:
http://www.milk.co.uk/page.aspx?intPageID=43. Visited: September 2011.
Now record your background understanding based on your reading of the
above highlighted sources. You will select your own sources. Remember to
think about the reliability of the source and avoid plagiarism.
5.
6.
7.
Independent variable:
Temperature of pasteurisation and holding time of pasteurisation.
How will you change the independent variable?
Pasteurise at set temperatures in thermostatically controlled
environments for set times.
Dependent variable:
Growth of bacteria.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 2
8.
9.
10.
How will you measure the dependent variable?
Viable counts of E. coli and M. luteus after pasteurisation.
Title, including operationalised independent and dependent variable:
Investigation into the effect of different holding times and temperature s
during pasteurisation on the growth of the bacteria E. coli and M.
luteus.
Confounding variables: fill in the table below.
Confounding variables
Temperature
Contamination with undesirable
bacteria and fungi
Growth medium in petri dishes and in
pasteurisation
Measuring mass of broth and agar
constituents
Volume measurements for broths and
agar
Starter culture
Growth vessel for pasteurisation
Growth vessel for bacterial serial
dilutions after pasteurisation
Temperature during pasteurisation
Temperature of broth before
inoculation
Temperature during incubation
Time in incubator
Counting number of bacteria
Control of confounding variables
Use thermostatically controlled water
bath set to 25°C
Good aseptic technique and autoclave
all equipment and growth media
Use standard nutrient broth with all
bacteria
Use balance: minimum 2 decimal
places
Use sterile pipettes for small volumes
Sourced from supplier and maintained
under aseptic conditions
Use universal bottles
Petri dishes
Thermostatically controlled water
bath at set temperature
Set up blank universal bottle and
measure internal temperature until it
is equal to the temperature of the
water bath
Thermostatically controlled at 30C
in incubator
24 hours
Use serial dilutions at each timeframe
and do viable count of colonies
present
First establish initial number of
bacteria present in growth media to
determine change
Add further rows to table as required.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 2
11.
Control(s): fill in the table below.
Control(s)
No pasteurisation of M. luteus and E.
coli
Blank with no M. luteus or E. coli
Reason for control
To compare to actual experiment to
determine effect
To show that the aseptic technique is
good and no external contamination
of plates has occurred during the
process
Add further rows to table as required.
12.
13.
14.
Aim:
To determine the effect that different holding times and temperatures
have during pasteurisation on the death rates of M. luteus and E. coli.
Hypothesis:
Reducing the temperature at which pasteurisation takes place will
significantly increase the holding time needed for pasteurisation to take
place.
Potential risk: fill in the table below.
Type of risk
Bacteria used
How is the risk minimised?
Use the aseptic technique at all times,
disposing of all materials in Virkon or
autoclave
Wash hands
Use microbiological spills kit if a
spill/breakage occurs
Carefully record all plates used and
unused, and temperatures of
incubation
Add further rows to table as required.
The following points will be completed after your initial proposal has been
discussed with your class practitioner. You will need to complete a more
detailed risk assessment before you begin.
15.
16.
17.
18.
Method:
Equipment:
Pilot study:
Limitations of equipment and procedure:
UNIT 3 (AH, BIOLOGY)
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15
ACTIVITY 3
Activity 3: Phage and the scientific process
Introduction
This activity involves a series of lessons that focus on aspe cts of scientific
process, namely (from part 1(a) of Unit 3)
In science, refinement of ideas is the norm, and scientific knowledge can
be thought of as the current best explanation, wh ich may then be updated
after evaluation of further experimental evidence.
Failure to find an effect (ie a negative result) is a valid finding, as long as
an experiment is well designed. Conflicting data or conclusions can be
resolved through careful evaluation or can lead to further, more creative,
experimentation.
The focus of this activity is the bacteriophage. The activity involves several
stages (practical and discussion) that could be used independently. It is
designed to link the concepts of Unit 3 with knowledge concepts from both
Unit 1 and Unit 2.
Outline of learning activity
1.
Setting the scene – videoclip introducing resistance and phage therapy.
2.
Discussion of PLOS article – developing new antibiotics and
alternatives.
3.
Protein binding in phage assembly and attachment – videoclips.
4.
T4 b bacteriophage plaque assay – an advanced microbiology practical.
5.
Phage display – a discussion of this creative use of phage.
16
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
1.
Setting the scene
Watch the video clip (13:11 minutes):
http://www.youtube.com/watch?v=JG6dnOligeM&feature=related
This clip introduces the problem of bacterial resistance to antibiotics and the
possibility of using phage therapy as an a lternative. It also links bathing in
the River Ganges to the discovery of bacteriophages. The development of
phage therapy was interrupted in the West by the development of antibiotics.
The clip describes the development of phage therapy in the former Sovi et
Union and an interest in developing this treatment in the (less regulated)
veterinary market before attempting to develop human phage treatments.
Discussion of the clip could focus on whether medical science could develop
along such different routes in different parts of the world today. It would be
appropriate at this point to ensure that learners understand the basic cycle of
the bacteriophage. It would also be appropriate to check that there is good
understanding of the evolution of resistant strains of bacteria and the
horizontal transmission of resistance genes through bacterial conjugation.
2.
Discussion of the PLOS Biology article: ‘New Antibiotics—
Resistance is Futile’
Powledge TM (2004) New Antibiotics—Resistance Is Futile. PLoS Biol 2(2):
e53. doi:10.1371/journal.pbio.0020053
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.
0020053
This is a review article written in 2004. It is suggested that different
individuals in the class are tasked with reading the different sections. The
class practitioner can then guide learners through the following suggested
discussion questions.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
Suggested discussion questions
Introductory paragraphs
Q: How do these paragraphs illustrate that knowledge has to be updated in
science?
Q: In this context in particular why is it important for scientists to report
failure to find an effect (negative results)?
Tried and true – and tired?
Q: What is the usual way that antibiotics have been discovered?
Q: How might this screening be carried out in the laboratory?
Improving on nature
Q: What is the focus of this section?
Q: How might antibiotics be modified?
Finding new targets
Q: What is the focus of this section?
Q: What techniques or knowledge are required?
Phage therapy
Q: Is it a good analogy to describe phage-infected cells as zombies?
Q: Are there any potential drawbacks of phage therapy?
Q: How might phage enzymes be used?
18
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
Suggested discussion questions and answers
Introductory paragraphs
Q: How do these paragraphs illustrate that knowledge has to be updated in
science?
A: As resistance accelerates, new treatments have to be developed to keep
ahead of the pathogen.
Note that Streptococcus pneumoniae is now treated by vaccination, a
development not predicted by this article.
Q: In this context in particular why is it important for scientists to report
failure to find an effect (negative results)?
A: In the field of medicine it is as essential to know which treatments do not
work as to know which treatments do work.
Tried and true – and tired?
Q: What is the usual way that antibiotics have been discovered?
A: Natural compounds from fungi. Screening of chemical libraries.
Q: How might this screening be carried out in the laboratory?
A: Clearance zones measured on lawn plates of test bacteria (Kirby –Bauer
testing).
Improving on nature
Q: What is the focus of this section?
A: That existing antibiotics could be modified to increase t heir effectiveness.
Q: How might antibiotics be modified?
A: Through modifying the metabolic pathways in the organisms that produce
the antibiotics.
Finding new targets
Q: What is the focus of this section?
A: Designing antibiotics that hit the most vul nerable target in the bacterium –
rational drug design.
Q: What techniques or knowledge are required?
A: Genome analysis and looking for key genes that control the cell cycle.
Using that information to develop ligands that bind to the proteins that are
transcribed by these genes.
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ACTIVITY 3
Phage therapy
Q: Is it a good analogy to describe phage-infected cells as zombies?
A: No, science is always more strange than science fiction! Zombies are
supposedly the dead coming back to life whereas a phage -infected cell is not
yet dead in reality, but it no longer has any control over its metabolism.
Q: Are there any potential drawbacks of phage therapy?
A: Resistance will probably develop, although it has not done so yet.
Regulations may be too tight to allow phage medicin es, although they can
probably be developed for agriculture or veterinary uses.
Q: How might phage enzymes be used?
A: Phage can lyse host cells enzymatically. These enzymes are specific to the
host bacteria and can be used in treatments to destroy the ba cterial species.
3.
Protein binding in phage attachment and assembly
It is assumed that practitioners will have resources from current Higher
Biology suitable for quickly teaching the basics of the cycle of a
bacteriophage. Watching these video clips will help learners to understand the
importance of protein binding in both attachment to host and assembly of
phages.
T4 Bacteriophage introduction (1:51 minutes)
http://www.youtube.com/watch?v=ehbZpo8oXSs&feature=related
T4 targeting host cell (0:30 minutes)
http://www.youtube.com/watch?v=yzTwm_zvlCo&feature=related
T4 Bacteriophage assembly: a clip showing all of the proteins that have to be
synthesized by the host cell to form a new capsid (5:31 minutes)
http://www.youtube.com/watch?v=Ofd_lgEymto
Lambda phage replication (1:26 minutes)
http://www.youtube.com/watch?v=cqCC4EEmM3Q
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
4.
T4 b bacteriophage plaque assay
This is an advanced microbiological technique and is suitable for learners
who have already mastered the skills of aseptic technique and safe
manipulation of microorganisms. It would also be appropriate for the
classroom practitioner to be supported by level 3 trained technical staff. It is
suggested that practitioners trial this procedure before use with a class. An
outline protocol is included on the next page.
The procedure can be carried out as a simple demonstration of the parasitic
nature of the bacteriophage. Plaques (gaps) in the bacterial lawn indicate that
the bacteria are being destroyed by bacteriophage.
Alternatively, the procedure can be carried out in a manner that allows an
estimate of the density of bacteriophage to be calculated using serial dilution.
Another alternative is to develop the protocol to investigate another variable.
A suitable suggestion would be to vary temperature to investigate the effect
of temperature on binding.
Alternatively, virus–host specificity can be demonstrated by showing that
plaques do not form with this bacteriophage and other types of bacteria. This
procedure, which can be used to identify bacteria, is known as phage typing.
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ACTIVITY 3
Plaque assay estimation of T4 b bacteriophage concentration
Aim
To estimate the number of plaque-forming units (PFUs) of bacteriophage
present in a viral stock using E. coli b.
Background
Bacteriophages are viruses that infect bacteria.
T4 b is a lysogenic phage that infects E. coli b.
If a lawn of bacteria is infected with bacteriophage infected areas show up as
gaps known as plaques.
The bacterial cells in the plaques have been destroyed.
The number of plaques is proportional to the concentration of the virus or the
success of binding of virus to the bacteria.
Materials
3 nutrient agar plates (it is better if they are about 3 days old as fresher plates
may be too wet)
10 ml sterile pipette
2 ml sterile pipette
Stock virus bacteriophage T4 b (eg supplied by Philip Harris)
24 hour broth culture of E. coli
Sterile 1% saline
Sterile Universal bottles
100 l autopipette and sterile disposable tips
Glass spreader
Ethanol
Multioxidising disinfectant
1% bleach
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
Method
Prepare serial dilutions of virus to 10-3, 10-4 and 10-5 in Universal bottles
with sterile saline.
Pipette 0.5 ml of E. coli onto each petri dish and spread with glass spreader.
Flame the glass spreader before and after use.
Leave for 10 minutes to absorb onto agar.
Pipette 0.5 ml of each dilution of virus onto separate plates and spread using
glass spreader.
Leave for 10 minutes to absorb onto agar.
Incubate at 30°C inverted for 24 hours.
Count plaques on lawn.
Adjust for dilution factor and initial pipetting volume and express as PFU per
ml.
Safety
All waste disposed of to disinfectant or autoclave.
All manipulations should use aseptic technique.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 3
6.
Phage display – a creative use of phage technology in drug
design
Learners should visit the following webpage:
http://www.dyax.com/research/phage-display-discovery-tool.html
Work through the following presentation:
Discuss the use of phage display in identif ying specific ligand molecules that
may be of use in medicinal drug development. Areas to consider are:
1.
A phage library is a collection of many millions of different genetically
modified phage particles.
2.
The genetic variation in these phages is expressed as differences in their
outer protein coats.
3.
These differences alter the ability of the phage to bind to target
molecules.
4.
The phages that bind to a target molecule can be easily gathered.
5.
The phages that bind contain the genetic information to make the
ligands that will bind to the target molecule.
6.
Once a ligand is known, its use in potential therapy can be investigated.
7.
Therapies are often based on the protein–protein interactions of ligands
changing the shape, and therefore behaviour, of the target molecule.
24
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
Activity 4: Reading a scientific research paper
The basic structure of a scientific research paper
Title: This tells you the topic of the authors’ investigation and the overall
outcome. A research paper is usually the work of a team of scientists.
Abstract: In this section the scientists briefly summarise their investigation.
Introduction: In this section the scientists describe the background to their
research. They will say what is already known about the topic they are
investigating, and make references to the experiments that have established
this.
Materials and Methods: In this section the scientists describe in detail how
they carried out their experiments. Enough detail should be included for other
research groups to replicate the study.
Results: In this section the scientists present the results they obtained. These
may be displayed in the form of tables of data and/or graphs.
Discussion: In this section the scientists make conclusions from their re sults
and discuss their significance. They describe how their conclusions fit in with
the knowledge discussed in the introduction. The scientists may also evaluate
their investigation and suggest ideas for further experiments.
References: In this section the scientists provide details of all the previous
work that they used in order to carry out their investigation and write their
paper. References usually follow either the Harvard or the Vancouver system.
Acknowledgements: In this section the scientists thank other people and
funding boards that have made contributions to their work.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
How to read a scientific research paper for comprehension
General tips
- Annotate your copy of the paper as much as possible with any comments,
queries or thoughts you have.
- Highlight unfamiliar scientific terms and find out what they mean.
- You may need to consult other sources of information to help you
familiarise yourself with what is being discussed.
- The authors will often use abbreviations in their writing and symbols on
their figures. You may find it useful to make a key that explains what these
terms represent.
- At the paper’s heart will usually be the simple question of ‘What is the
effect of X on Y?’ You need to work out what X and Y are, how they were
manipulated and measured, and what the effect was.
The abstract
- Read this first to find out what the authors’ main questions, experiments
and conclusions were.
The introduction
- Read as much as you need in order to understand the background.
The methods
- Read this to familiarise yourself with the experimental procedures the
authors used. You will need to identify the independent and dependent
variables and how they were manipulated and measured.
- Try drawing a flow chart to show how the experiment was done.
The results
- Look at the figures (graphs) first. These often convey most of the results.
Make sure you know what any abbreviations mean. Try writing a short
summary of each figure to describe what it shows.
- If you want more details read the authors’ description of their results. If
statistics are used, focus on what they imply, not how they were produced.
- If a measured difference between two groups is reported to be statistically
significant then this means that the probability of the difference being
found if there was actually no difference between the two groups is low
(less than 0.05). The authors will be reasonably confident that the
difference they measured (in the dependent variable) has been caused by
their differing treatment of the two groups (the independent va riable).
The discussion
- Read this to find out what conclusions the authors drew.
- How do the conclusions relate to the original experimental aims?
- How do the conclusions relate to the evidence presented?
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UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 4
Analysis of a scientific research paper
Introduction
In this activity you will read a primary research paper. This is a report written
by scientists in which they describe original research that they have done,
what conclusions they have made and how their findings fit into the current
understanding of the topic. Reports of this nature are published in scientific
journals.
The paper you will read is taken from a scientific journal called Science.
Science is an American publication that publishes original research from
across all the scientific disciplines. Science tends to only publish research
that is of particular significance and therefore it has a very high impact
factor. This means that the papers that are in Science are cited (referenced)
many times in other papers, usually because of the importance or their
findings.
Before answering the questions below, read through the document ‘The basic
structure of a scientific research paper’. This will help you to get an idea for
the style in which papers are written and how the authors present their
‘story’.
The structure of papers published in high -impact journals such as Science is
slightly different. The introduction, results and discussion are not separated
from each other by subheadings but are written in continuous paragraphs.
Only the most important features of the method are described, the fine details
are included in the references, which are available as supplementary
information (accessible on the journal’s website) or described in other
research papers published elsewhere. The focus of the paper is very much on
the findings and their importance for the advancement of the field.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
Paper analysis
Requirement of human renal water channel aquaporin -2 for vasopressindependent concentration of urine
This paper describes some major findings that have increased our
understanding of the way in which the hydrophilic signalling molecule ADH
regulates the water permeability of the collecting tubules in the kidney.
Use the document ‘How to read a scientific research paper’ to help you read
through the whole paper and make notes, then answer the questions below.
Questions
1.
Sketch a diagram of a nephron and collecting duct to show the locations
of the channel proteins described in the introduction.
2.
By what name is the hormone ADH referred to in the paper?
3.
(a)
How does ADH act to increase the concentration of urine?
(b)
Describe the location of AQP2 when inserted into the membrane
of an inner medullary collecting duct cell and the direction of the
water concentration gradient at that point.
(c)
People who have the condition nephrogenic diabetes insipidus (NDI)
are insensitive to ADH. What could cause ADH to be ineffective?
4.
The authors assigned the gene for AQP2 to human chr omosome 12
using a gene probe. They then sequenced the gene and obtained the
amino acid sequence of the AQP2 protein.
The gene probe was made out of complementary DNA (cDNA) for the
rat AQP2 gene. cDNA is double-stranded DNA that is made using an
mRNA template. One of the enzymes required to make cDNA is reverse
transcriptase, which catalyses the transcription of a single -stranded
DNA molecule from an mRNA template.
28
(a)
Which other enzyme would be required to make cDNA?
(b)
How many amino acids long is the human AQP2 protein?
(c)
Use Figure 1 to show that the similarity between the human AQP2
protein and the rat AQP2 protein is 89.7%.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
5.
The authors then sequenced parts of the AQP2 gene of a male patient
with NDI and compared the sequences to two indepe ndent control
individuals. They found that their patient had two heterozygous point
mutations. One of the point mutations resulted in the substitution of
proline for serine at one position in the amino acid sequence of the
AQP2 protein.
(a)
What could the term ‘independent’ mean in this context?
(b)
Use the information in Figures 1 and 2 to work out why the two
point mutations to the AQP2 gene are designated R187C and
S216P.
(c)
Use the diagrams of proline and serine to suggest how the
replacement of serine by proline could distort the shape of one of
the AQP2 protein’s alpha-helices.
Proline
6.
Serine
It was found that the patient had inherited an AQP2 allele with the
mutation from their father and an AQP2 allele with the mutation from
their mother.
Why is the individual described as a compound heterozygote for the
AQP2 gene?
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
7.
In the next part of the investigation egg cells from the model organism
Xenopus laevis (a frog species) were used. The egg cells were injected
with RNA carrying the sequence for the wild-type AQP2 protein and
RNA with the two mutant sequences. The water permeability of the
injected cells was measured. The results are displayed in Table 1.
(a)
What was this experiment designed to check?
(b)
What control was used and what was its purpose?
(c)
Draw an appropriate graph to present the data given in Table 1.
(d)
The authors concluded that the co-injection of a mutant RNA
sequence with the wild-type sequence had no effect on the activity
of the wild-type protein. What evidence from Table 1 and your
graph supports this? What other conclusions can be made from the
data?
(e)
How do the data in Table 1, the pattern of inheritance displayed in
Figure 3A and the information in the third paragraph of the paper
support the conclusion that the patient’s NDI is an autosomal
recessive condition?
8.
Summarise how the authors’ overall findings suggest that AQP2 is the
water channel regulated by the action of ADH.
30
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
Paper analysis answers
Requirement of human renal water channel aquaporin-2 for vasopressindependent concentration of urine
Learners will find that they require background knowledge to interpret some
parts of the paper. They should be encouraged to try to work out what they
will need to know to understand the concept an d how they will go about
finding this out. They should also be encouraged to think carefully about
which pieces of information in the paper will allow them to answer the
questions that are asked.
Answers to questions
1.
Sketch a diagram of a nephron and collecting duct to show the locations
of the channel proteins described in the introduction.
Aquaporin 1 is found in the membranes of cells in the proximal tubule
and the descending thin limb of the loop of Henle. Aquaporin 2 is found
in the membrane of cells in the collecting ducts. Principal cells and
inner medullary collecting duct cells are two of the cell types found in
the collecting ducts.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
2.
By what name is the hormone ADH referred to in the paper?
Arginine vasopressin (AVP) or just vasopressin.
3.
(a)
How does ADH act to increase the concentration of urine?
ADH binds to the V2 receptor (found on the basolateral
membrane of collecting duct cells)  Cell signalling pathways 
Water channels inserted into apical membrane of cells.
(b)
Describe the location of AQP2 when inserted into the membrane
of an inner medullary collecting duct cell and the direction of the
water concentration gradient at that point.
AQP2 is inserted into the apical membrane. The term ‘apical
membrane’ applies to the cells that make up the lining of tubes.
The apical membrane is on the side of the cell that faces the
lumen (the inside of the collecting duct in the case of the kidney).
The water concentration is higher in the lumen of the collecting
duct than in the luminal cells.
(c)
People who have the condition nephrogenic diabetes insipidus
(NDI) are insensitive to ADH. What could cause ADH to be
ineffective?
– Being homozygous for mutant alleles of the gene encoding the
V2 receptor (or simply possessing the mutant allele of the gene
if male as gene is X-linked).
– A mutation to any gene coding for a protein involved in the
intracellular signalling pathway that links the binding of ADH
to its receptor to the insertion of the water channel into the
cell membrane.
– A mutation to the gene coding for the water channel regulated
by ADH.
32
UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 4
4.
The authors assigned the gene for AQP2 to human chromosome 12
using a gene probe. They then sequenced the gene and obtained the
amino acid sequence of the AQP2 protein.
The gene probe was made out of complementary DNA (cDNA) for the
rat AQP2 gene. cDNA is double-stranded DNA that is made using an
mRNA template. One of the enzymes required to make cDNA is reverse
transcriptase, which catalyses the transcription of a single -stranded
DNA molecule from an mRNA template.
(a)
Which other enzyme would be required to make cDNA?
DNA polymerase
(b)
How many amino acids long is the human AQP2 protein?
271
(c)
Use Figure 1 to show that the similarity between the human AQP2
protein and the rat AQP2 protein is 89.7%.
Total number of amino acids in human and rat AQP2 proteins =
271.
Number of amino acids by which the two proteins differ = 28.
Percentage similarity = (243/271) × 100 = 89.7% (1dp)
5.
The authors then sequenced parts of the AQP2 gene of a male patient
with NDI and compared the sequences to two independent control
individuals. They found that their patient had two heterozygous point
mutations. One of the point mutations resulted in the substitution of
proline for serine at one position in the amino acid sequence of the
AQP2 protein.
(a)
What could the term ‘independent’ mean in this context?
Not related
(b)
Use the information in Figures 1 and 2 to work out why the two
point mutations to the AQP2 gene are designated R187C and
S216P.
The first letter is the symbol for the amino acid that has been
replaced. The number is the position of the amino acid in the
chain. The last letter is symbol for the amino acid that is now
present at this location as a result of the mutation.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
(c)
Use the diagrams of proline and serine to suggest how the
replacement of serine by proline could distort the shape of one of
the AQP2 protein’s alpha-helices.
Proline lacks an amino-group hydrogen so cannot form a
hydrogen bond at this location. The size of its R group may also
prevent the alpha-helix from taking a regular shape.
6.
It was found that the patient had inherited an AQP2 allele with the
mutation from their father and an AQP2 allele with the mutation from
their mother.
Why is the individual described as a compound heterozygote for the
AQP2 gene?
They possess two different mutant alleles of the AQP2 gene.
7.
In the next part of the investigation egg cells from the model organism
Xenopus laevis (a frog species) were used. The egg cells were injected
with RNA carrying the sequence for the wild-type AQP2 protein and
RNA with the two mutant sequences. The water permeability of the
injected cells was measured. The results are displayed in Table 1.
(a)
What was this experiment designed to check?
That the mutant AQP2 alleles identified resulted in non -functional
AQP2 water channels.
(b)
What control was used and what was its purpose?
Injecting the egg cells with water. Done to check that any
measured changes in the water permeabilities of the ce lls were
caused by the effect of the injected RNA rather than injection per
se.
34
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 4
(c)
Draw an appropriate graph to present the data given in Table 1.
(d)
The authors concluded that the co-injection of a mutant RNA
sequence with the wild-type sequence had no effect on the activity
of the wild-type protein. What evidence from Table 1 and your
graph supports this? What other conclusions can be made from the
data?
The SEM error bars for condition 1 (wild-type AQP2 RNA
injected) and conditions 5 and 6 (mutant and wild-type AQP2
RNA injected) all overlap.
This suggests that the difference between the results for these
conditions is not significant.
The error bars for condition 1 (wild-type AQP2 RNA injected) and
condition 2 (water injected) do not overl ap. This suggests that
wild-type AQP2 RNA significantly increases the water
permeability of the egg cells, ie it results in the production of a
functional AQP2 water channel.
UNIT 3 (AH, BIOLOGY)
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35
ACTIVITY 4
The error bars for conditions 3 (R187C RNA injected) and 4
(S216P RNA injected) overlap with each other and those of
condition 2 (water injected). This suggests that both types of
mutant RNA have no effect on the water permeability of the egg
cells, ie they do not result in a functional AQP2 water channel
being produced.
(e)
How do the data in Table 1, the pattern of inheritance displayed in
Figure 3A and the information in the third paragraph of the paper
support the conclusion that the patient’s NDI is an autosomal
recessive condition?
In the third paragraph the authors state t hat they assigned the
AQP2 gene to chromosome 12, so the condition will not be sex
linked.
A comparison of conditions 1, 5 and 6 in Table 1 shows that wild type RNA can still produce a functional AQP2 protein in the
presence of mutant RNA. This suggests that a person who was
heterozygous for the wild-type AQP2 allele would not have NDI.
The pedigree diagram in Figure 3A shows that neither of the
parents is affected by NDI, despite carrying a mutant allele of the
AQP2 gene. The same applies to the patient’s sister.
8.
Summarise how the authors’ overall findings suggest that AQP2 is the
water channel regulated by the action of ADH.
ADH is not effective in people with NDI. The patient in the study had
NDI, but had functional ADH receptors. The patient had mu tations to
both of their alleles for the AQP2 gene. Expression of the mutant alleles
in frog egg cells did not have a significant effect on their water
permeability compared to a control, whereas expression of the wild type allele greatly increased the cells’ water permeability. This
suggests that the patient’s ADH insensitivity arises from the hormone’s
inability to cause functional AQP2 water channels to be inserted into
the apical membrane of the cells in collecting ducts of the kidney.
http://www.sciencemag.org/content/264/5155/92.abstract
36
UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 5
Activity 5: Scientific literature and communication
The following papers can form the basis for class d iscussion based around the
area of scientific literature and communication.
Case 1: The media’s MMR hoax
Learners should read the following articles:
The media’s MMR hoax by Ben Goldacre (Bad Science)
http://www.badscience.net/2008/08/the-medias-mmr-hoax/
Wakefield’s article linking MMR vaccine and autism was fraudulent
BMJ 2011; 342; doi: 10.1136/bmj.c7452 (Published 5 January 2011)
Cite this as BMJ 2011; 342: c7452
http://www.bmj.com/content/342/bmj.c7452
Discussion should centre on the following points:
Scientific reputation
The difference between pre-publication communication and peer-review
The requirement for scientific literacy within the media
The outcome of a vaccination scare
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 5
Scientific reputation:
The MMR story illustrates how a scientific reputation can be lost after being
judged by others to have used a fraudulent approach to science. In particular
the BMJ editorial makes statements about the integrity of the data collection
and reporting. It should be noted that the BMJ is being sued by Andrew
Wakefield in Texas as a result of this editorial.
The difference between pre-publication communication and peer-review:
The data of Krigsman is queried in the article by Ben Goldacre. He points out
that this has been reported at conferences but not published in a peer reviewed form that allows evaluation of methods, results and analysis.
The requirement for scientific literacy within the media :
The media’s reporting of MMR is severely criticised. In particular, the media
did not clearly point out the difference between correlation and causation, the
lack of reliability of a small study or the difference between a hypothesis and
a conclusion. The use of non-scientifically trained journalists is worrying, as
is the apparent ranking of opinion above evidence derived from scientifically
valid data, not to mention the cherry-picking of results that appeared to
support the media’s story.
The outcome of a vaccination scare:
The reduced rates of measles vaccination through lowered uptake of MMR
led to an increase in the rates of measles in the UK. Discuss the nature of
herd immunity and the conflict between social responsibility and individual
freedom.
38
UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 5
Case 2: HIV denial in the internet era
Learners should read the following paper from PLOS Medicine:
Smith TC, Novella SP (2007) HIV Denial in the Internet Era. PLoS Med 4(8):
e256.;doi:10.1371/journal.pmed.0040256
http://www.plosmedicine.org/article/info%3Adoi%2F10.1371%2Fjournal.pme
d.0040256
Misinformation can spread rapidly via the internet. Learner s should be asked
to consider how the strategies employed by t hose who deny HIV might be
appealing to those browsing the internet. Learners might enjoy discussing
their own difficulties in remembering to use the scientific approach of
reasoned evaluation of hard evidence when faced with emotive or persuasive
non-scientific communication.
The group should discuss what they think an appropriate response from the
scientific community should be when confronted by denial of HIV, evolution
or climate change. During this discussion it would be right to emphasise that,
in a scientific context, opinion should be informed by the evaluation of
evidence (data). A scientific debate should centre around issues such as
validity of measures, accuracy, appropriate controls, reliability of data, etc.
UNIT 3 (AH, BIOLOGY)
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39
ACTIVITY 6
Activity 6: Scientific ethics A
The ethical framework for using animal models in research
The class should be given the link to the following article and asked to read
it, make notes and form opinions for a class discussion.
Ferdowsian HR, Beck N (2011) Ethical and Scientific Considerations
Regarding Animal Testing and Research. PLoS ONE 6(9): e24059.
doi:10.1371/journal.pone.0024059
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal .pone.002
4059
In class, possibly after the PowerPoint on scientific ethics, ask learners to
outline what they think the guidelines should be for the use of animal models
in research.
Try to help the class achieve a balanced consensus view rather than al lowing
polarised views to take root and be held without willingness to compromise.
To help do this discuss the most extreme positions (such as no regulation at
all or no animal testing at all) and get learners to identify the pros and cons
of implementing either of these extreme ethical frameworks. From these
positions encourage learners to use the information in the article provided to
try to establish some evidence-based criteria for the legislative or regulatory
framework for animal testing.
As a second activity, discuss the use of animal models in school science, eg
woodlice, daphnia, etc. Discuss what guidelines should operate within a
school biology department.
For the latter, it is worth referring to the IoB publication Materials of Living
Origin in Schools.
40
UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 7
Activity 7: Scientific ethics B
In animal studies, the concepts of replacement, reduction and refinement are
used to avoid, reduce or minimise harm to animals.
The value or quality of science investigations must be justifiable in terms of
the benefits of its outcome, including the pursuit of scientific knowledge. The
risk to and safety of subject species, individuals, investigators and the
environment must be taken into account. As a result, many areas of scientific
research are highly regulated and licensed by governments. Legislation limits
the potential for the misuse of studies and data.
1.
Read the paper on ‘The humane collection of fetal bovine serum and
possibilities for serum-free cell and tissue culture’. The major elements
of the text have been highlighted for reference in the accompanying
pdf, but it is recommended that you read the text in its entirety so that
you can develop your own view on the use of FBS in vitro cell
culturing.
2.
Your views will form the basis of a discussion on the ethical and moral
decisions surrounding the use of FBS in vitro.
3.
You may annotate the paper and you may decide to place the paper in
Google docs so that you can annotate it further as a class bef ore your
tutorial.
4.
The tutorial will focus on discussing the need for regulation and
legislation in science and the need for scientists to question their
methods.
5.
What are the bigger questions that this paper raises with regards to
biological research?
6.
Are there situations that justify the harm of an organism for our greater
success?
7.
What happens when human tissue/cells/volunteers are also
manipulated?
UNIT 3 (AH, BIOLOGY)
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41
ACTIVITY 7
Use the links below to aid your own thinking and form a reasoned debate.
1.
Cancer research HELA cells and Henrietta Lacks
The Immortal Life of Henrietta Lacks
http://rebeccaskloot.com/faq/
2.
The Human Tissue Act 2004
http://www.nuffieldbioethics.org/human -tissue/human-tissue-humantissue-uk-developments
3.
Bioethics debate
http://bioethicsbytes.wordpress.com/
http://bioethicsbytes.wordpress.com/2008/11/20/to -opt-in-or-opt-outorgan-donation-in-the-uk/
4.
University of Leicester: Model Organisms in Biomedical Research
(video clip)
http://www.youtube.com/watch?feature=player_embedded&v=Jj5QlYlE
66w#!
42
UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 8
Activity 8: Pilot study, peer review and plagiarism
This activity is designed to focus on the following parts of the arrangements
documents:
1 Scientific principles and process
(b) Scientific literature and communication
(c) Scientific ethics
2 Experimentation
(a) Pilot study
Part 1: Pilot study (2–3 periods)
The suggested sequence of activities involves the selection of an area of
investigative experimental work that is both reasonably familiar to learners
yet allows some scope for individual variation in methodology or selection
and control of variables. Suitable class research topics could be:
 the influence of drugs such as ethanol on Daphnia heart rate
 the influence of factors such as humidity on woodlouse behaviour
 the influence of factors on the activity of catalase.
There is scope to select activities that would fit within either Unit 1 or Unit 2
as appropriate. The activity works best if all members of the class are
working on different individual variations of the same basic experiment.
The key idea is that the practical should have enough familiarity t o learners
that they should not need to pilot the activity in order to become familiar with
the basic method. Instead, their pilot study should involve the individual
selection of methods of varying the independent variable, measuring the
dependent variable and controlling any important confounding variables.
By the end of the pilot study phase each learner should have written up their
pilot study under the following headings:
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 8
Pilot study write-up
 Title
 Aim
 Method
 Table of results
Part 2: Peer review (1–2 periods)
For the peer review phase of the activity learners are paired or arranged in
groups so that they are able to swap their pilot study write -ups. As peer
reviewers learners spend 10 minutes or so reading through the write -up they
have been given. They should check the write-up for clarity, detail and
precision. They should write a list of questions or further details that they
require from the original authors in order to carry out the experiment
according to the instructions given. Once clarific ation has been sought, a
brief plenary is helpful to encourage learners to reflect on the requirement for
full details within scientific writing.
Following this discussion learners then attempt to follow the method that they
have been peer reviewing. This is an attempt to demonstrate replication by
another research team. If there is time it is interesting to consider how closely
matched the original author’s and peer reviewer’s data are, and if they differ
discuss reasons why.
Part 3: Plagiarism checking
After discussion of the different findings within the class, for homework ask
the learners to write a short review article (ie an essay) on the class research
topic. Each learner should aim to summarise the findings of the different
researchers in the class as well as bringing in some information from other
sources. This review should be submitted in electronic form.
In class, once the short review articles have been submitted, arrange for the
class to check their own work for plagiarism. This can be do ne using an
online plagiarism checker linked to Google or another search engine. In this
case small sections of text can be checked at any one time. Alternatively,
your school or college may have access to a more substantial plagiarism
checker.
This will be a good starting point to move on to the topic of scientific ethics
and plagiarism in particular.
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ACTIVITY 9
Activity 9: Gel electrophoresis pilot study
Content area: Experimentation
(a)
Pilot study
Integral to the development of an investigation, a pilot stud y is used to help
plan procedures, assess validity and check techniques. This allows evaluation
and modification of experimental design. A pilot study can be used to
develop a new protocol or to enable an investigator to become proficient in
using an established protocol.
The aim of this activity is to gain experience of following a multistep
protocol for protein electrophoresis, which is an experimental technique used
in the study of proteomics.
Background
What is proteomics?
Proteomics has evolved from genomics. After the successful mapping of the
genomes of many different organisms, variations in proteins now allow a
further level of evolutionary mapping to take place. Whereas genomics
focuses on DNA in the nucleus of cells, proteomics looks at the pr otein
composition of a cell. The word proteome is derived from a combination of
protein and genome. Proteome is defined in the Oxford dictionary as being
‘the entire complement of proteins that is or can be expressed by a cell, tissue
or organism’. The field of proteomics is becoming increasingly important in
research as many diseases operate by causing some degree of disruption to
specific proteins.
Demonstration of protein electrophoresis in the laboratory
For classroom use and in order to gain experi ence of following a multistep
protocol, the BIORAD Biotechnology Explorer™ Comparative Proteomics
Kit is a useful tool to demonstrate the technique of protein electrophoresis.
To summarise, learners use SDS-PAGE (sodium dodecylsulphatepolyacrylamide gel electrophoresis) to separate and analyse the protein
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 9
profiles of muscle tissue from a variety of fish species. Proteins are usually
separated using polyacrylamide gels as their small pore size means they have
a high resolving power. The process separate s the proteins according to their
molecular weight. To facilitate this, the proteins must first be processed into
a linear form. This is done prior to the electrophoresis by denaturing the
proteins. A strongly negatively charged detergent SDS (sodium dodec yl
sulphate) is added which binds to and coats the proteins and effectively gives
them a uniform charge along their entire length, masking the different
charged groups of the amino acids in the chain. With the different charged
groups along the different protein no longer a variable, the molecular mass of
the protein is the main variable that affects their migration rate through the
gel during electrophoresis. By comparing the protein profiles of different fish
species, learners can test the hypothesis that protein profiles are indicators of
genetic and evolutionary relatedness.
The principles behind this activity are:
 it allows learners to investigate evolution in the laboratory through the
generation of data that can be used to construct cladograms (phylogenetic
trees) of the fish species analysed
 it allows learners to study muscle protein structure and function
 to gain skills in carrying out a multistep protocol as a pilot study
 if wished, this technique can be used as an open-ended tool for further
investigative study.
The basic process involved in SDS-PAGE is shown below:
http://commons.wikimedia.org/wiki/File:SDS -PAGE_Electrophoresis.png
Atttributed to Bensaccount at en.wikipedia
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ACTIVITY 9
Lesson 1: Preparatory Lesson (45 minutes)
Access to the internet will be required for this activity.
Students should already be familiar with the fact that the basic contractile
components of muscle cells are the myofibrils that are bund led into muscle
fibres. Each myofibril consists of a linear series of contractile units called
sarcomeres that are made up of actin and myosin protein filaments. Although
actin and myosin are the main components involved in muscle contraction,
there are numerous other muscle proteins also required. Actin and myosin are
highly conserved across all animal species but other muscle proteins show
more variability. These variations reflect the evolutionary history of the
species and adaptations to live in different environments.
A suggested approach to this pre-lab activity is given in the BIORAD
Biotechnology Explorer™ Comparative Proteomics Kit 1 manual . To
summarise, the lesson is best started with a brainstorming session on fish and
their environments. This could cover the different environmental factors that
are thought likely to influence fish movement and muscle development.
Taking cost and availability into account, five different fish species should be
selected for investigation. (It should be noted that less than a gram of each
fresh raw fish sample is required to complete this investigation. Many fish
counters, particularly smaller local shops will give these free of charge or for
a minimal cost. It should also be noted that previously frozen raw fish works
just as well as fresh. Label your fish samples clearly before freezing to reduce
the possibility of error.)
Students should access an internet database (http://www.fishbase.org/) – this
contains information on the biology of most of the world’s fish species.
Students should research the fish species that they are planning to use in the
gel separation process. The BIORAD Biotechnology Explorer™ Comparative
Proteomics Kit manual contains a specific student sect ion which contains
Fish Data Sheets and focused questions to allow students to target their
research on the chosen fish species.
The lesson should conclude with the students predicting and justifying which
two fish out of the five are likely to have the most similar protein profiles.
Similarly, students should predict and justify which two fish out of the five
chosen will have the fewest muscle proteins in common and have very
differing protein profiles.
There is an alternative pre-lab activity on the evolution and classification of
fish given in the manual that could be done as a homework exercise or as
additional work.
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ACTIVITY 9
Lesson 2: Protein Extraction from Muscle (45 minutes)
There is a detailed protocol set out in the BIORAD Biotechnology Explorer™
Comparative Proteomics Kit manual with both instructions for teachers and
students to follow. In this lesson, students prepare the protein extracts from
the five fish muscle samples. This lesson can be immediately followed by
Lesson 3, if time permits. The allocation of time for each of these lessons is a
guideline only. When planning lessons, the inexperience of students to this
new technique should be taken into account , particularly if the focus of the
activity is that of a pilot test intended to allow stude nts to gain skills in
mastering a multistep procedure.
Lesson 3: Electrophoresis – Gel loading, Running and Staining
(45 minutes)
In this lesson, students separate the proteins in their fish muscle extracts
using SDS-PAGE and then stain their gels to visualise the proteins. It is
advisable to wear gloves if handling gels.
Lesson 4: Analysis and Interpretation of Results
(45 minutes and homework)
Completed gels can be dried (instructions for this are given in the proteomics
manual) or alternatively photographed, scanned or photocopied to retain a
record of results.
Analysis of results – in order to draw meaningful conclusions or inferences
about the similarity or difference in protein profiles of the fish samples,
detailed gel analysis is required. An example of a gel is detailed in the
manual and clear instructions about how to analyse their gel is given to
students. Students need to create a standard curve and record the distances
migrated in millimetres by the prestained standard proteins used o f known
molecular weight. These standards should measure molecular weights of 37,
25, 20, 15 and 10 kilodaltons (kD). This is an exercise in student precision as
accuracy to 0.5 mm is required. Results are then plotted onto two -cycle semilogarithmic graph paper to construct a curve if the protein molecular mass
against the distance migrated.
For each of the five fish samples that have been analysed, students can
determine the molecular masses by measuring the distance each band has
migrated from the base of its well on the gel. Using the standard curve,
students can work out the molecular mass of the proteins found in each fish
sample. Comparing each fish profile individually with the other fish profiles
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ACTIVITY 9
allows students to work out if the fish have any bands in common. Finally,
using this information, students are instructed how to construct a cladogram
(phylogenetic tree) based on their results.
The aim of this learning activity is to gain experience using a multi -step
protocol but it also reinforces work covered in Unit-2 of the AH Biology
course relating to evolution and rate of evolution.
Useful information
Literature and price list are available online at www.explorer.bio-rad.com.
It is recommended that practitioners access the life science education section
from the top toolbar on the homepage of www.explorer.bio-rad.com and
select classroom kits from the pull-down catalog index box. Please note that
literature is free to download but practitioners have to register on the site. An
overview of the kit will appear on the catalogue page.
Overview of key features of kit
 Investigation can be completed in three 45-minute lab sessions.
 Each kit contains sufficient materials for eight learner workstations (two to
four learners per workstation).
Not included in the kit
With the exception of basic laboratory supplies, there is a requirement for
fixed-volume (5 and 10 µl) and adjustable micropipettes (2 –20 µl), plus fish
samples (five to eight types from store, river, lake or ocean), 1 g each per
workstation, and power supplies.
Suggested reference sites
Human Proteome Organisation
http://www.hupo.org/research/hpp/
American Medical Association
http://www.ama-assn.org/ama/pub/physician-resources/medicalscience/genetics-molecular-medicine/current-topics/proteomics.page
Clinical and Biomedical Research at the University of Leeds
http://www.proteomics.leeds.ac.uk/
General proteomic information
http://en.wikipedia.org/wiki/Proteomics
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ACTIVITY 10
Activity 10: Enzyme kinetics pilot study
Introduction
Neutrase® is a commercial enzyme prepared by Novozyme. It is a bacterial
protease produced by a selected strain of Bacillus amyloliquefaciens.
Commercially it is used to break down proteins to soluble peptides and it is
routinely used in DNA extraction to lower the load of contaminating protein.
‘Neutrase® is a neutral protease with an optimum activity around pH 5.5 –7.5
and 45–55°C. It is a metallo-proteinase, requiring zinc ions for its activity.
Consequently, it is stabilised by the presence of calcium ions and inhibited by
EDTA. The stability of Neutrase® at a certain temperature is influenced by
the type and concentration of the proteins present. Neutrase® can be
inactivated by heat treatment, eg 2 minutes at 85°C.’
National Centre for Biotechnology Education (NCBE)
In addition to the above information Neutrase® is demonstrably active at
around 20°C. (Nuffield)
Milk powder such as Marvel® provides a good bench substrate for Neutrase®
and it can be demonstrated that at 1% Neutrase® will cause a 2% suspension
of Marvel to clear rapidly. The clearance is due to the digestion of casein in
milk from a relatively insoluble suspension to soluble peptides.
The course of this reaction can be followed by measuring the time taken for
the milk suspension to clear by eye or more precisely by using colorometric
measurements of transmission without requiring expensive colorometric
substrates.
Colorometric/spectrophotometric methods should be considered using data
logging. Suitable equipment is available from the following suppliers.
Mystrica (software PC only)
The Mystrica colorimeter, http://www.mystrica.com
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 10
PASCO (software i.e. Data Studio PC/MAC)
http://www.pasco.com/family/datastudio/index.cfm
http://www.pasco.com/prodCatalog/PS/PS-2121_pasport-colorimetersensor/#resources_widget_3_slider_1
General references
NCBE
Neutrase®:
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/ENZYMES/ Neutrase®.h
tml
Safety:
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/ENZYMES/PDF/Enzym
eSafety.pdf
Price:
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/PDF/NCBEpricelist.pdf
Nuffield
http://www.nuffieldfoundation.org/practical -biology/quantitative-food-testprotein-content-powdered-milk
The task
To undertake a pilot study with suitable negative and positive controls in
order to follow the effect of Neutrase® on milk pro tein.
A progress curve should be plotted to determine the effect of changing
substrate concentration on enzyme activity.
1.
As a pilot study learners should be given stock Neutrase® at 100%
(NCBE stock liquid extract) and Marvel at 2% in distilled water. Th ey
should add the enzyme to give an end concentration of 1% and observe
what happens.
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ACTIVITY 10
2.
The following questions should then be posed:
Q: How do you know the reaction has ceased?
A: The solution should clear.
Q: What would be a valid negative control?
A: By heat-treating a sample of enzyme at 85°C and adding this as a
control treatment. This is this better than simply adding no enzyme
as it does not alter the total volume.
Q: If we were to look at the effect of substrate concentration on
Neutrase®, what would be a suitable positive control?
A: The reaction allowed to proceed under optimal conditions.
Q: Are replicates required?
A: Yes, always.
Discuss how many and how constructed, eg a true replicate is not
simply a repeat of the experiment using the same batch of reagents.
Q: How can the rate of the reaction be determined and when is the
reaction linear?
A: This is a difficult one as clearing is subjective. This discussion
should lead into one where a qualitative result such as the initial
pilot should not be seen as sufficiently rigorous enough.
Rate implies measurements against a time base and recording of a
change in the dependant variable, casein in milk powder, at various
time points. This is best made colorimetrically with data logging
over time.
The use of a blue end filter or setting works well for this activity but
further discussion is needed about how a valid blank can be created
to determine the clearance of Marvel over time by looking at
increased transmission.
Note: Pure water would be a poor blank. Why?
Even when all substrate has been digested the dissolved peptides will
still give background absorption over water. How can this be
overcome? (Pre-digesting a blank may be one solution where a
completely digested Marvel sample is used to show 100%
transmission. Discuss the validity of this approach.)
 At the end of this discussion learners should be encouraged to construct a
second pilot using colorimeters to measure the effect of changing substrate
concentration on the progress of enzyme activity using the above model.
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ACTIVITY 10
Further work
It should be possible to determine Michaelis –Menten kinetics for Neutrase®
and derive values for K m and V max using this basic system.
As Neutrase® is a metaloprotease the effect of EDTA or Zn + on activity may
provide a useful topic for an Advance Higher investigation.
In addition, this model for Neutrase® should allow the study of the inhibition
of enzymes by a range of molecules. This model is attractive because of the
ease of handling of the basic system and its adaptability. Basic reagents are
cheap, although investment in colorimeters would be required to get the most
out of the system. The Mystrica colorimeter is good in this regard as it can be
used as a stand-alone tool or be connected to a comput er for data logging.
Various useful experiments are also available at the Mystrica website above.
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 11
Activity 11: Controls, sampling and ensuring
reliability
Content area: Controls, sampling and ensuring reliability
Title: Investigating heart rate in Daphnia
Practitioner’s guide
Daphnia or water fleas are small crustaceans commonly found in lakes and
ponds, and widely available in pet shops as fish food.
In the laboratory, the transparency of the Daphnia exoskeleton allows the
heartbeat to be easily counted under a light microscope at low power or under
binocular magnification. The ease with which the heart rate can be measured
makes this an ideal organism for investigation of factors that may affect heart
rate, such as:





effect of ethanol
effect of caffeine
change of water temperature
change of water salinity
effect of painkillers (paracetamol, ibuprofen, aspirin etc).
Obviously, the effect of changing these conditions on an invertebrate
crustacean cannot be used to predict the effect of chemicals on larger
vertebrate organisms but it is useful as an indicator of how these factors can
affect metabolic processes.
The experimental design of this practical means it is an ideal example of the
need for controls and to consider aspects of sampling. The n atural variation
of this organism is such that it affects sample size and consideration must be
taken to ensure that sufficient measurements are made so that the overall
results can be considered reliable.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 11
Learners must consider the following:
 Size of Daphnia used from the stock culture. Daphnia, like all species,
show natural variation in size and this may have an effect on heart rate and
also the ability of the learner to count heart rate accurately.
 Brooding Daphnia. Usually in a stock culture there are brooding Daphnia,
which are easily recognisable due to the presence of the developing young
visible inside the transparent exoskeleton. Temperature and chemicals may
well have different effects on brooding Daphnia and it would be wise to
consider this when selecting a sample.
 Age of stock culture and the storage conditions of Daphnia.
 How competent do learners feel about being able to reliably and accurately
count the heart rate of Daphnia? Perhaps learners should consider a short
pilot experiment before taking results to ensure that experimenter error
does not affect the reliability of results.
 Because of the natural variation in the population of Daphnia learners
need to consider that they may need a larger sample size due to the
population being variable.
 Learners need to consider time constraints, the need for replicates and
most importantly the fact that the experiment should be repeated as a
whole to check the reliability of the results.
Technician’s guide
Apparatus required
 Culture of water fleas – available from pet shops as fish food (often pet
shops only get one delivery of these per week so it would be sensible to
check with local supplier).
 Cotton wool
 Cavity microscope slides and coverslips
 Plastic droppers (with the end cut off to draw up Daphnia)
 Stopclocks
 Distilled water
 Microscopes low power light or binocular
For temperature investigation
 Ice box (0°C)
 Water baths at 20°C, 30°C and 40°C (adding ice to water taken from 20°C
can produce 10°C)
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ACTIVITY 11
For chemical investigations
Ethanol is one of the chemicals whose use by learners is specified by
CLEAPSS as per guidelines given below:
http://www.cleapss.org.uk/attachments/articl e/0/SSS60.pdf?Secondary/Scienc
e/Student%20Safety%20Sheets/
The concentrations of ethanol used are very low. Suggested ethanol
concentrations of 1%, 2%, 5% and 10% could be used.
Paracetamol and aspirin – both come in soluble form.
1.0% solutions can be made up by dissolving two 500 mg tablets in 100 ml of
distilled water.
0.1% solutions can be made up by dissolving two 500 mg tablets in 1000 ml
of distilled water.
Ibuprofen – does not come in soluble form, but ibuprofen water -based gels
can be used as they are water soluble.
1% solution – 2 g of gel dissolved in 100 ml of distilled water.
0.1% solution – 2 g of gel dissolved in 100 ml of distilled water.
This activity has been designed to be a two -lesson activity:
Lesson 1: Design of experiment and pilot study measuring the heart rate of
Daphnia in water.
Lesson 2: Looking at the effect of one of the factors on the heart rate of
Daphnia.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 11
Learner guide
Background information
Daphnia or water fleas are small crustaceans commonly found in lakes an d
ponds, and widely available in pet shops as fish food.
In the laboratory, the transparency of the Daphnia exoskeleton allows the
heartbeat to be easily counted under a light microscope at low power or under
binocular magnification. The ease at which the heart rate can be measured
makes this an ideal organism for investigation of factors which may affect
heart rate, such as:





effect of ethanol
effect of caffeine
change of water temperature
change of water salinity
effect of painkillers (paracetamol, ibupr ofen, aspirin etc).
Only one of these factors will be investigated and your practitioner will
outline which factor the class as a whole will be studying.
Safety
Good laboratory practice is expected at all times. Please remember that
Daphnia are living organisms and as such should be treated with care. None
of the design elements of this experiment is intended to cause the death of the
organism and after they have been measured, Daphnia should be returned to
the storage container.
Lesson 1
It is very important that time is spent designing your experiment and using all
the information you have learned about use of controls, managing
confounding variables, sample size and ensuring the reliability of your
results. You must consider sample size, replication o f your results and also
repeating your experiment to ensure reliability of overall results.
Once you have planned your experiment, you need to complete a pilot test to
allow you to become familiar with a new technique and to become competent
in measuring the heart rate of Daphnia.
Observing the heart rate of Daphnia
 Take a few strands of cotton wool and place them onto the cavity of a
cavity microscope slide.
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ACTIVITY 11
 Using a plastic dropper (with the end cut off) draw up one of the Daphnia
from your designated sample population and transfer it onto the cavity
slide with a few drops of the solution.
 The cotton wool will restrict the movement of the Daphnia and allow you
to focus the slide on the microscope at low power and look for the heart
beating through the transparent exoskeleton.
 Once you are comfortable that you can see and count the heart rate of the
Daphnia, it is important that you ensure you can count reliably and
accurately.
 The heart rate of the Daphnia can be about 300 beats per minute (bmp) and
this may increase depending on what factor you decide to investigate. Can
you count that fast in a minute and be sure of your results?
Recording results
There are several possible methods of recording your results, all of which
need to be done with a timer to tell you when you have reached a minute or
30 seconds:
 Use of a clicker that you push down on the top and it registers the total
number of Daphnia heart beats you have recorded.
 With a pen, put a dot onto a piece of paper for every heart beat you count.
It is best if you do this in a pattern that allows you to count the dots easily
afterwards.
 Set up a calculator such that pressing the ‘=’ button adds one onto the total
on the screen.
Lesson 2
Using the skills you have practised in Lesson 1, go on to i nvestigate the
effect of one of the factors on the heart rate of Daphnia.
Analysis of results
There is likely to be considerable variation in the data collected because of
the variability of the Daphnia and the reliability of the measurement
technique used.
Class results for the heart beat of the sample population of Daphnia should be
gathered. Mean results and the standard deviation should be calculated.
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UNIT 3 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY 12
Activity 12: Sampling using transect
Introduction
Observational studies are key to investigating ecological changes. One classic
approach is the line transect, where the distribution of species is recorded
along a fixed sample line. The transect may be placed to intersect an area that
shows variance in species distribution. Moving on the line and measuring
biotic and abiotic factors can make an inference about how and why species
diversity is being affected.
An issue with this is that there is no way to directly control any independent
variable. Instead a correlation of species distribution with similar patterns of
other biotic or abiotic factors is attempted.
Trampling is the name given to the effect of animal or man -made movement
on terrestrial ecosystems. This can be due to footfall or mechanised transport.
In this simple approach we will design a series of sampling methods to
investigate the following observations for a representative section of a strip
of land crossing a meadow between two gates.
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ACTIVITY 12
Aerial view of a track going
through a meadow
Dense grass
Grass/buttercup
Daisy/clover
15 m
Bare ground
9m
The initial observation suggests that vegetation occurs in belts running in the
direction of the path.
1.
From the diagram and the introduction come up with a hypothesis for
the change in species diversity.
2.
How would you test your hypothesis?
3.
What confounding variables may exist in relation to plant distribution?
4.
Can these variables be controlled or factored into your study?
5.
How can we measure the density and diversity of species across the
path?
6.
What possible factors may cause a change in plant distribution?
7.
How can these factors be measured and to what degree of ac curacy and
reliability?
8.
How would you display and analyse your results?
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ACTIVITY 12
Practitioner’s notes
The aim of this exercise is to get learners to think about the problems of
sampling in the field. Some suggestions are as follows:
The initial observation suggests that vegetation occurs in zones radiating
from the path’s centre outwards.
1.
From the diagram and the introduction come up with a hypothesis for
the change in species diversity.
(a) One hypothesis could be that increased soil compaction reduces
species diversity.
(i)
Commercial penetrometers can be purchased but these can
also be made by dropping a weighted spike inside a captive
pipe. The depth to which the spike enters the soil is measure
of how compacted the soil is.
2.
How would you test your hypothesis?
(a) Measurements of soil compaction across the belt could be
compared with plant distribution and diversity.
3.
What confounding variables may exist in relation to plant distribution?
(a) Any of the factors mentioned in part 7 that are not a component of
trampling.
4.
Can these variables be controlled or factored into your study?
(a) It is essential that these factors are measured and compared
across the transect to see if they show any correlation with plant
density.
5.
How can we measure the density and diversity of species across the
path?
(a) A belt transect crossing the path across the whole cross -sectional
area would be appropriate. Abundance of all of the test organisms
described would be measured using 0.25 m 2 quadrats. This is an
example of systematic sampling.
(b) 18 samples should be taken across a 9 m line stretched equally
across the width of the diagram area.
(c) Several belt transects should be taken across the target to
establish reliability.
6.
What possible factors may cause a change in plant distribution?
(a) Soil pH, soil moisture, soil compaction, light intensity,
competition and degree of trampling are all potential variables.
Direct damage to plants as a result of trampling.
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ACTIVITY 12
7.
How can these factors be measured and to what degree of accuracy and
reliability?
(a) Appropriate measuring instruments should be used for abiotic
factors and multiple readings taken and averaged per quadrat.
(b) It is possible to theorise about the effects of competition but they
are difficult to measure. Likewise a numerical value for trampling
is difficult to define and would require direct observation of the
target area to estimate footfall or traffic density.
8.
How would you display and analyse your results?
(a) Graphs of all measured factors should be constructed to display
the variation of all measured variables and factors across the
belt.
(b) Correlation of change of plant type and density with the
independent variable should be looked for.
(i)
It is essential that all potential confounding variables do not
negate any correlation of the dependant and independent
variables.
(ii) In addition any removal or inhibition of a species may
negate its competitive effect on another species. Learners
should be asked to think about the changed distribution of
plants in light of this possibility.
Further activities
The opportunity to carry out belt and line transects in the field should be
encouraged.
Various links and ideas related to trampling can be found on the Field Studies
Council website: http://www.field-studiescouncil.org/urbaneco/urbaneco/grassland/trampling.htm.
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UNIT 3 (AH, BIOLOGY)
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ACTIVITY 13
Activity 13: Evaluating experimental design
Evaluating experimental design and data
Have appropriate control conditions or control groups been included in the
experiment?
Have all the confounding variables been effectively controlled?
Who was tested/observed/measured?
Were the experiments done in a petri dish, a non -human animal or a living
human?
How big was the sample size? Was it big enough to draw reliable
conclusions?
What are the raw numbers?
Are any data, details or labels missing or unclear?
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ACTIVITY 13
Practitioner’s notes
This activity was inspired by the information presented in the book Bad
Science by Ben Goldacre. The aim is for learners to become more ef fective at
critically evaluating scientific claims. They will be made aware of the type of
questions that they should be asking to decide whether an experimental
design is suitable for generating valid and reliable conclusions.
Part 1: Critical evaluation of another scientist’s experimental designs
In this activity learners will view four PowerPoint slides describing an
investigation into the effectiveness of a fictional drug at preventing colds.
They will also be given the slides in handout form.
The practitioner should act out the role of a research scientist for the drug
company and make the presentation as sincerely as possible, with the aim of
convincing the learners of the validity of the findings. A vote could be taken
in the class after the presentation to find out how many people would use the
drug based on the information they have received so far.
The learners must then take the role of reviewers and try to pick as many
holes in the presentation as they can. They should generate a list of quest ions
that they would want to ask the research scientist about the work. It is worth
dividing this part of the activity into two phases: in the first learners are
given time to come up with evaluative questions themselves and in the second
learners can be given the guidance document on the types of questions that
could be asked and asked to tailor these to the specific details of the
experiments described in the presentation.
The practitioner can then take the learners through the annotated version of
the PowerPoint presentation and give them the opportunity to raise their
questions and criticisms of the experimental designs that are described. They
may have some that are not covered here!
Part 2: Generating a dubious description of an experimental design
In this activity learners should work individually or in small groups to
produce a computer-based PowerPoint presentation with a similar structure to
the one they have just seen, which they will then give to their peers. The aim
of the presenter is to get away with as many invalid/unreliable claims as
possible based on the experimental design they describe in the presentation.
The aim of the viewers is to identify the elements of the experimental design
that would compromise its validity and reliability.
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ACTIVITY 13
Learner notes
Aim: To become more familiar with the types of questions that must be asked
to evaluate the validity and reliability of an experimental design.
Part 1: Critical evaluation of another scientist’s experimental designs
You will be shown a presentation in which a scientific investigation is
described. Your task is to take the role of a peer reviewer and identify critical
questions that would need to be asked of the method and experimental design
in order to determine whether the conclusions th at are presented are valid and
reliable.
Part 2: Generating a dubious description of an experimental design
In this activity you need to produce a presentation that has a similar structure
to the one you have already seen to convince someone to use anoth er fictional
product. You need to briefly describe an experimental design, the results and
the conclusions you have made. You will then present this to your peers.
Your aim is to get away with making as many invalid/unreliable claims as
you can by being deliberately vague about the design of your experiment and
the method you have followed. When you watch the other presentations your
aim is to pick out as many points as possible that could compromise the
validity and reliability of the experimental design a nd generate questions to
ask the presenter to clarify their descriptions.
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ACTIVITY 14
Activity 14: Evaluating data analysis
Evaluating the reliability of an experimental design using
descriptive statistics
When you make repeated measurements of a variable in a biological
investigation it is very unlikely that you will consistently obtain exactly the
same values. This may be due to an inability or failure to consistently control
confounding variables, a small amount of measurement error and inherent
variation between measurements. Biological systems are complex: even a
single cell may have thousands of variables associated with it that vary
during an experiment. As a result of this there will always be some variation
in your results.
To get an idea of how varied your data are, and therefore the reliability of
your experimental method the data can be analysed using descriptive
statistics. Descriptive statistics can provide a measure of the variability of a
data set. This can be used to compare the variability of d ata sets, eg to make a
comparison between replicates. The variability can be presented as a number
or displayed on a graph of the data as an error bar.
The simplest measure of the variability of a data set is the range. This is the
largest value minus the smallest value. This is easy to calculate, but is
distorted by extreme values so is generally not used.
The standard deviation is one of the most commonly used measures of data
variability. It can be thought of as how much each measurement differs on
average from the mean of the data set. The larger the standard deviation of a
data set the more variable the data.
How to calculate the standard deviation of a data set manually
The values in a set of data can be given the symbols x 1 , x 2 and x 3 , where x 1 is
the smallest value. A general value in the data set is given the symbol x i . The
number of values in the data set is given the symbol n.
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ACTIVITY 14
1.
2.
3.
4.
5.
6.
Calculate the mean ( x ) of the values in the data set: (∑x)/n.
Calculate how much each value deviates from the mean: x i – x .
Square each of the deviations: (x i – x ) 2 .
2
Calculate the
 sum of the squared deviations: ∑( x i – x ) .
Divide the sum of the squared deviations by the number
of values minus

2
1: (∑(x i – x ) )/(n – 1). This
 value is known as the variance of the data
set.

Calculate square root of the answer to 5: √[∑( x i – x ) 2 ]/(n – 1). This is
the
 standard deviation (S X or SD) of the data set.
How to calculate the standard deviation of adata set using Excel
1.
Enter each of the values in the data set into a row of cells.
2.
Click on an empty cell and enter the formula =STDEV(
3.
4.
Highlight all the cells containing the data set values.
Type ) and press Enter.
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ACTIVITY 14
Example
An investigation was carried out to establish the effect of ethanol on the
activity of the enzyme catalase. Both the experiment and the control were
repeated nine times.
Experiment results (enzyme + substrate + ethanol)
Reaction number
Initial rate of reaction
(units/second)
1.
1
2
3
4
5
6
7
8
9
10
15
19
16
14
16
20
18
17
17
18
Calculate the mean:
(15 + 19 + 16 + 14 + 16 + 20 + 18 + 17 + 17 + 18)/10
= 17 units/second
2.
Calculate the deviation of each value from the mean:
Reaction number
Initial rate of reaction
(units per second)
Deviation from mean
3.
5.
6.
2
3
4
5
6
7
8
9
10
15
–2
19
2
16
–1
14
–3
16
–1
20
3
18
1
17
0
17
0
18
1
Calculate the squared deviation of each value from the mean:
Reaction number
Initial rate of
reaction (units per
second)
Deviation from
mean
Squared deviation
from mean
4.
1
1
2
3
4
5
6
7
8
9
10
15
19
16
14
16
20
18
17
17
18
–2
2
–1
–3
–1
3
1
0
0
1
4
4
1
9
1
9
1
0
0
1
Calculate the sum of the squared deviations:
4 + 4 + 1 + 9 + 1 + 1 + 1 = 30
Divide the sum of the squared deviations by 9: 30/9 = 3.3 (this value is
known as the variance of the data set).
Take the square root of the answer to 5: √ 3.3 = 1.83.
The standard deviation of the experimental results is 1.83.
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ACTIVITY 14
Thinking point: Why would the total of the deviations not be an effective
measure of the variability of a data set?
Control results (enzyme + substrate + distilled water)
Reaction number
Initial rate of reaction
(units per second)
1
2
3
4
5
6
7
8
9
10
18
13
16
8
13
11
14
10
13
14
Calculation of standard deviation
Mean = 13
Reaction number
Initial rate of reaction
(units per second)
Deviation from mean
Squared deviation
from mean
1
18
2
13
3
16
4
8
5
13
6
11
7
14
8
10
9
13
10
14
Sum
130
5
25
0
0
3
9
–5
25
0
0
–2
4
1
1
–3
9
0
0
1
1
74
Variance = 8.2 , standard deviation = 2.87.
Comparing the standard deviations of the two data sets tells us that the values
obtained for the control condition are over twice as variable as those obtained
from the experimental condition.
The variability of the data for the two conditions can be shown on a graph:
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ACTIVITY 14
The height of the bars is the mean of each data set. The vertical lines above
and below each bar are called error bars.
On this graph the length of each error bar above and below the mean is the
standard deviation of the data set.
Error bars can be plotted using other measures of data variability apart from
the standard deviation so it is very important to always include a key with the
graph to state what the error bars represent.
How to plot standard deviation error bars on Excel charts
1.
Double click on the bars (or points if a line graph).
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ACTIVITY 14
2.
Highlight error bars from the menu and select their appearance. If you
have plotted a line graph you should select the Y error bars. Select
Custom and click on Specify Value.
3.
Insert the standard deviation values for eac h condition/data set between
the brackets, with commas to separate them. Enter the same values in
the same order as the bars appear on the chart in both boxes.
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ACTIVITY 14
Task 1
An investigation was carried out to find out the effect of incubation
temperature on the viability of yeast cells grown in liquid culture. The
following results were obtained:
Incubation temperature of 20°C
Yeast cell
cultures
incubated at
20°C
Percentage
viability of
cells
1
2
3
4
5
91
92
95
90
92
Incubation temperature of 25°C
Yeast cell
cultures
incubated at
25°C
Percentage
viability of
cells
1
2
3
4
5
97
93
95
92
93
A.
Calculate the standard deviation of each data set (either manually or
using Excel).
B.
Plot the data as a graph with error bars to show the variability within
each data set (either by hand or using Excel).
C.
Compare the variability of each data set.
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Task 2
An investigation was carried out to find out the effects of creatine
supplements on the racing performance of greyhounds. The following results
were obtained:
Control condition (greyhounds given a placebo)
Greyhounds
given
placebo
Time taken
to run 400
m (s)
1
2
3
4
5
6
7
8
9
10
28.6
25.6
29.1
28.3
24.9
29.3
27.7
29.1
27.4
27.9
Experimental treatment condition (greyhounds given creatine
supplement)
Greyhounds
given
creatine
supplement
Time taken
to run 400
m (s)
1
2
3
4
5
6
7
8
9
10
24.2
25.2
24.6
24.1
23.6
25.7
25.6
26.9
24.9
25.2
A.
Calculate the standard deviation of each data set (either manually or
using Excel).
B.
Plot the data as a graph with error bars to show the variability within
each data set (either by hand or using Excel).
C.
Compare the variability of each data set.
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ACTIVITY 14
Determining whether or not the results are significantly different
In biological investigations you will often make a comparis on between the
results obtained from a control group and a treatment (or experimental) group
in which you have systematically altered one specific variable.
Both data sets can be expected to show variation. It is common for biological
variables to show a particular pattern of variation called a normal
distribution. In a normal distribution the data are symmetrically distributed
around the mean of the data set.
In the graph below the blue line represents the measurements taken from the
control condition and red line the measurements taken from the treatment
group.
Thinking point: Can you identify the mean of each data set? Mark them on
the graph.
For the two data sets graphed above the two means have different values.
This could have arisen by chance or it could be a systematic effect caused by
the experimental manipulation or treatment that one of the groups received.
Essentially we want to know whether the variation between the groups is
large relative to the variation within groups.
Research scientists often start an investigation by taking the position that
there should be no difference between the results of the experiment and the
control, ie the treatment has no effect. This is called the null hypothesis (H 0 ).
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Once the results for each condition have been obtained they can be analysed
to establish whether or not they are significantly different from each other. In
scientific writing the term ‘significant’ has specific connotations so should be
used with caution.
A difference is statistically significant if there is a very low probability of
it occurring by chance if there really is no systematic difference between
the two groups. The cut-off probability most commonly used in research
science is 5%. This is called the 0.05 significance level.
If a difference is found to be statistically significant then the null hypothesis
can be rejected. Providing the experiment has been designed to effectively
control the confounding variables then it can be concluded that the single
factor by which the data sets differ is likely to be causing the difference
between the results.
So how do we find out whether or not the results are significantly
different?
The starting point is to consider the difference between the means of the two
data sets. The greater the difference the more likely the results are
significantly different.
However, the means of each data set can be expected to show a certain
amount of variability. If you replicated the investigation, it is unlikely that
you would get exactly the same mean for each group.
The range of values in which the true mean is most likely to occur (the
population mean) is called the standard error of the mean (SEM).
How to calculate the standard error of the mean of a data set:
Divide the standard deviation of the data set by the square root of the number
of measurements in the data set (sample size).
SEM = SD/√n
The SEM values can be plotted as error bars on a graph to give a visual
indicator of the likelihood that the means are significantly different from each
other (see example below). If the error bars overlap it is unlikely that the
difference between the two data sets is statistically significant. If the error
bars do not overlap it is likely that the difference between the means is not
due to chance alone. To find out whether this is the case it is necessary to
carry out a statistical test. There are many different statistical tests and the
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ACTIVITY 14
one that is chosen depends on what type of data you have and what question
you want to answer about it. In this acti vity a t test will be used.
The t test
You can use the t test described in this section to check for a significant
difference between two data sets as long as you can be sure that your
measurements come from an underlying population that is normally
distributed. You cannot use the t test if your data does not consist of
quantitative measurements.
In a t test the mean and the standard error of the mean of the data sets are
used to calculate a number called a t statistic. The greater the value of the t
statistic the more likely the difference between the results is significant.
Doing a t test: calculating the t statistic
The formula is:
t’ = ( x 1 – x 2 )/√[(s 1 2 /n 1 ) + (s 2 2 /n 2 )]

where x 1 and x 2 are the means of the two data sets, s 1 2 and s 2 2 are the
variances
of each data set (see previous section), and n 1 and n 2 are the

number of measurements (sample size) in each data set. The data set with the
larger mean should be designated as data set 1.
Once you have calculated a t statistic it is necessary to check a table of values
to find out whether the number is larger than a certain critical t value. If the t
statistic is larger than the critical t value then the results are significantly
different. The critical t value depends on number of measurements in each
data set (the sample sizes). For statistical reasons this number is modified to
generate a number called the degrees of freedom (df). For the t test described
here the df is (n 1 – 1) or (n 2 – 1), whichever gives the smaller value.
A table of critical t values for 1–30 df at the 0.05 significance level is given
on the last page of this document. To find out whether a calculated t statistic
is significant it is necessary to compare it to the critical t value associated
with the df of the results.
Thinking point: What does the pattern of values in the t distribution table
tell you about the effect of sample size on the outcome of a t test?
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ACTIVITY 14
Example
For the enzyme investigation described in the previous example the null
hypothesis is that treatment of catalase with ethanol has no effect on the
initial reaction rate it produces.
To accept or reject the null hypothesis it is necessary to find out whether the
results from the two conditions are significantly different f rom each other.
The first step in this process is to calculate the standard error of the mean
(SEM) for each condition.
- Experiment data set: SEM = 1.83/√10 = 0.577 (using non -rounded SD
value)
- Control data set: SEM = 2.87/√10 = 0.907 (using non -rounded SD value)
The SEM values can be plotted as error bars on a graph of the results. These
give a measure of the variability of the each of the mean values. If the error
bars for the two conditions overlap it is unlikely that the two means are
significantly different.
On the graph shown below the SEM error bars do not overlap. It is likely, but
not certain, that the two means are significantly different from each other.
To find out whether there is a statistically significant difference between the
control and experiment results it is necessary to do a statistical test. In this
case a t test is most appropriate.
t’ = (17 – 13)/√[(8/10) + (3.3/10)]
t’ = 3.72 (9 degrees of freedom)
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ACTIVITY 14
The statistical table shows that at a significance level of 0.05 the critical t
value for 9 degrees of freedom is 2.262.
As 3.72 > 2.262 the probability of observing such different means if the null
hypothesis were true is 5% or less.
The null hypothesis can therefore be rejected as false; the results of the two
groups are significantly different from each other, as was suggested by the
fact that the SEM error bars did not overlap.
Providing that all the relevant confounding variables have been adequately
controlled in the investigation, it can be concluded that the cause of the
difference between the initial rate of reaction measured in the experiment and
in the control is likely to be the presence of ethanol rather than chance alone.
Task 3 (use the data from Task 1)
A.
B.
C.
D.
E.
F.
G.
H.
I.
J.
What would the null hypothesis be for this investigation?
Calculate the standard error of the mean for the two conditions.
Draw a graph with the variability of the means for each condition
shown as error bars.
Comment on the implications of the degree of overlap between the error
bars.
Carry out a t test to find the t statistic for the data.
How many degrees of freedom are there?
What is the critical t value for this number of degrees of freedom at the
0.05 significance level?
Does your t statistic exceed the critical t value?
What does your answer to H imply about the null hypothesis?
Write a conclusion to the investigation based on the results and the
result of the statistical test.
Task 4 (use the data from Task 2)
A.
B.
C.
D.
E.
F.
G.
78
What would the null hypothesis be for this investigation?
Calculate the standard error of the mean for the two conditions.
Draw a graph with the variability of the means for each condition
shown as error bars.
Comment on the implications of the degree of overlap between the error
bars.
Carry out a t test to find the t statistic for the data.
How many degrees of freedom are there?
What is the critical t value for this number of degrees of freedom at the
0.05 significance level?
UNIT 3 (AH, BIOLOGY)
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ACTIVITY 14
H.
I.
J.
Does your t statistic exceed the critical t value?
What does your answer to H imply about the null hypothesis?
Write a conclusion to the investigation based on the results and the
result of the statistical test.
Using and interpreting error bars correctly
In this activity you have met two types of error bars, those that show the
standard deviation (SD) of the data set and those that show the standard error
of the mean (SEM) of each data.
It is perfectly acceptable to use either type of error bar on a graph as they can
both be used to make comparisons between the data sets. However, care must
be taken in exactly how you use the error bars to evaluate the data.
- SD error bars are used for describing and comparing the variability of the
measurements within each data set.
- SEM error bars are used for inferring whether the measurements in each
data set are significantly different to each other. It is important to
remember that the degree of overlap between the error bars of each data
set indicates the likelihood of the results being significant different. To
find out if they actually are significantly different you must carry out a
statistical test.
If you include error bars on a graph it is very important that you always
include a key to state what kind of variability they represent.
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ACTIVITY 14
Values for the t distribution
If the value obtained from the t test is greater than the critical value of t
given below (for the correct number of degrees of freedom associated with
the data) then this indicates that the difference between your sample means is
statistically significant at the 0.05 level. This means that if the null
hypothesis were true, the probability of obtaining the observed difference
between the means is no larger than 5%, ie it is unlikely that the difference
between the means is due to chance alone, so the null hypothesis can
therefore be rejected.
Number of degrees of freedom (df)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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Critical t value for
p(t ≥ t critical ) = 0.05
12.706
4.303
3.182
2.776
2.571
2.447
2.365
2.306
2.262
2.228
2.201
2.179
2.160
2.145
2.131
2.120
2.110
2.101
2.093
2.086
2.080
2.074
2.069
2.064
2.060
2.056
2.052
2.048
2.045
2.042
ACTIVITY 14
Critical t values for higher numbers of degrees of freedom and other
probabilities can be found at http://www.jeremymiles.co.uk/misc/tables/ttest.html. The critical t values given here (as in the table above) are for twotailed t tests, where the null hypothesis (and research hypothesis) is nondirectional.
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