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Purpose
Safety
The standard equipment required for carrying out an ecological study is detailed below.
Additional items required to measure abiotic factors will depend on the site selected and type
of study being undertaken.
Requirements per student or group of students
Notes
A quadrat
Either square or point quadrats can be used. Square frame quadrats with
subdivisions are useful. The standard square frame quadrat is
50cm 3 50cm, but smaller and larger ones can be used.
At least 50.
A clipboard
A clear plastic bag large enough to get clipboard
and hand with pencil in
Ranging poles
A clinometer
Access to a compass
Jam jars
Pooter
Sweep net
Tullgreen and/or Baermann funnel
Thermometer
Whirling hygrometer
Light meter
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
151
Student 1 of 8
Purpose
To carry out a study on the ecology of a habitat.
Observing patterns
Have you ever walked into a wood and noticed that the vegetation changes as you enter?
Why do the bluebells only occur under the trees? Or have you been clambering over a rocky
shore and spotted that the seaweeds grow in distinctive bands and that you only find mussels
where the tide is far out? What causes these patterns in plant and animal distribution? When
ecologists study habitats, they try to account for plant and animal distribution, correlating
them to the abiotic and biotic factors that are affecting the habitat.
Abiotic means non-living and examples of abiotic factors include light intensity, slope,
humidity, wind exposure, edaphic (soil) characteristics such as pH and soil moisture, and
many more. Biotic means living and examples of biotic factors include competition, grazing
and predation. All species of plants and animals you encounter in the wild are well adapted
to the set of conditions encountered in their usual habitat. If they werent they would either
grow somewhere else or become extinct!
Studying patterns
Look around your local habitats and spot any patterns in distribution and abundance of
organisms. You dont need to go far; you might notice something in your school grounds or
the local park. You might have a look at the distribution of plants in trampled areas of the
sports field or grass paths; are there any patterns?
Once you have identified a pattern, think about why it might have come about. Describe the
pattern and use appropriate biological ideas to suggest an explanation. Now you need to plan
a fieldwork investigation to test your idea.
When planning any investigation you need to:
decide what data you are going to collect
select suitable apparatus and methods
ensure you are going to collect valid and reliable data
decide how you will analyse it once collected
complete a risk assessment and decide on steps to avoid or minimise the harmful effects
of any hazards
conduct a trial to inform your planning.
Read the following section, which briefly mentions some of the techniques that you could
use. There is more detail in Student Practical support sheet Ecological sampling (page 26).
You can also look at the British Ecological Society (BES) website education pages the
students 161 section contains detailed information about sampling techniques. Your teacher
may also give you details of websites that can be used to help you prepare your plan.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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2 of 8 Student
Procedure
1 Plan how you are going to collect reliable and valid data that will test your hypothesis. You
need to make the following decisions.
The most appropriate sampling method to use (e.g. random or systematic sampling).
The position and length of any transect to use (Figure 1). You need to make sure your
transect extends far enough to sample all the possible zones.
The size and number of quadrats to use, and their positioning.
The species of plants and animals you are to record you should focus on those
which will enable you to test the hypothesis under investigation. (You may need to find
out more about the species concerned using secondary sources).
The method to use for measuring abundance.
The abiotic factor(s) you are going to record. Although you may be investigating the
correlation between, for example, soil moisture and the distribution of plant species,
there may be other factors that could affect the distribution of organisms. It is not
possible to control these variables but you can measure them and take them into
account when analysing your results.
The appropriate method for measuring the abiotic factor(s).
How the data will be analysed.
How to avoid or minimise any risks when completing the fieldwork.
A pilot study in advance of the main data collection will help you make these decisions.
peg marked 20
0 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
origin
peg marked
0
0.5 m 0.5 m
quadrat
no. 6
transect
continues
tape case
Figure 1 One way of laying out a tape measure for a transect study. Quadrats are laid down at regular
intervals along the tape and the abundance of species within each quadrat is recorded.
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Student 3 of 8
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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4 of 8 Student
Random sampling
Frequently, ecologists notice a distinct pattern that may be related to one or more factors at
two sites. For example, the vegetation in one field may be very different to that in another
field, or the species found under oak trees may be different to those under ash trees, or
the species upstream and downstream of an outflow pipe discharging into a river may
seem to differ. To make valid comparisons, samples need to be taken from both sites. If the
investigator chooses where to sample, the sample will be subjective. Random sampling allows
an unbiased sample to be taken.
Using a grid
position of 0.25 m2
quadrat using random
numbers 2, 4
Tape measure 2
In a habitat, such as a meadow or heathland, tape measures put on the ground at right-angles
to each other can be used to mark out a sampling area (Figure 1). Using a pair of random
numbers you can locate a position within the sampling area to collect your data. The random
numbers can be pulled from a set of numbers in a hat, come from random number tables, or
be generated by a calculator or computer. The two numbers are used as coordinates to locate
a sampling position within the area. The first random number gives the position on the first
tape and the second random number gives the position on the second tape.
Tape measure 1
Figure 1 Using measuring tapes to define a sample area.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
26
Student 5 of 8
Ecological sampling
If you are sampling fixed objects within an area, for example the area of Pleurococcus (an
alga) on the shaded side of trees in a wood or the number of woodlice under rocks, you
could number all the trees or rocks and then use random numbers to select which trees or
rocks to sample.
This sampling idea is also used when measuring the number of cells in a culture. The culture
is mixed to give a reasonably uniform distribution of cells and then a known volume is
placed on a haemocytometer (a special cavity slide with a ruled grid in the centre). You then
count the number of cells that occur in, say, 25 squares of the grid. Because you know the
dimensions of the grid squares and the depth of the liquid above the square, you can work
out the volume of culture in each square, and then calculate a mean number of cells per cm3
of the culture.
Systematic sampling
Random sampling may not always be appropriate. If conditions change across a habitat, for
example across a rocky shore or in a sloping meadow that becomes more boggy towards one
side, then systematic sampling along a transect allows the changes to be studied. A transect
is effectively a line laid out across the habitat, usually using a tape measure, along which
samples are taken. The sample points may be at regular intervals, say every 2m across a field,
or they may be positioned in relation to some morphological feature, such as on the ridges
and in the hollows in a sand dune system.
Sampling techniques
Quadrats
Quadrats are used for sampling plant communities and slow moving or stationary animals,
for example many of those found on rocky shores. There are two types of quadrat: a frame
quadrat and a point quadrat.
A frame quadrat is usually square; the most commonly used is 50cm by 50cm (0.25m2)
and may be subdivided into 25 smaller squares, each 10cm by 10cm. The abundance of
organisms within the quadrat is estimated (see the section Methods of measuring abundance
and Figure 3). Quadrats may be placed across the site to be sampled using random or
systematic sampling methods. Throwing quadrats is not random and can be dangerous.
It is important to sample enough quadrats to be representative of the site, but why do 1000
quadrats if 10 will give almost as accurate a result? To find out the optimum number of
quadrats required, record the number of species in each quadrat and plot the cumulative
results against number of quadrats until sampling additional quadrats does not substantially
increase the number of species recorded.
A point quadrat frame (Figure 2) enables pins to be lowered onto the vegetation below.
Each species touched is recorded as a hit. The percentage cover for a particular species is
calculated using the equation:
hits
3 100
% cover 5 ____________
hits 1 misses
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
27
6 of 8 Student
Ecological sampling
knitting needle
holes
metal spike
(such as a
tent peg)
inserted in
ground
multiple hit
Figure 2 A point quadrat frame. Each plant species touched by the needle is recorded.
Frequency
Frequency is the number or percentage of sampling units in which a particular species
occurs. This avoids having to count the number of individuals. If clover was recorded in 10
of the 25 squares that make up a 0.25m2 quadrat frame, the percentage frequency would be
40%. You need to be consistent when determining presence or absence in a sampling unit.
For example, you might decide that only plants rooted in the square are counted, or you
might decide that any plant or animal in the quadrat is counted including any that touch or
overhang the quadrat.
Percentage cover
This is the percentage of the ground covered by a species within the sampling unit. Count
the number of squares within the quadrat that the plant completely covers, then count those
that are only partly covered and estimate the total number of full squares that would be
completely covered by that species.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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Student 7 of 8
Ecological sampling
25 W bulb
soil sample
16 mesh flour sieve
jam jar
sunk into
soil
ground slopes
away from trap
for drainage
bait of meat
or ripe fruit
polythene
funnel
Pooter for collecting insects
glass collecting tube
clear plastic
tube
Organisms move
away from the heat
and light, falling
into the jar.
glass mouthpiece
80% alcohol
gauze covering
tube opening
air sucked
through
mouthpiece
60 W bulb
Sweep net
This net is swept through
low-growing vegetation,
collecting any animals
in the mesh net.
rod for
supporting bag
soil sample in
muslin bag
water
glass funnel
rubber
tubing
clip
beaker
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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8 of 8 Student
Ecological sampling
Aspect
Use a compass.
Temperature
Use a thermometer or temperature probe, but be aware that the time of day can influence the
values obtained, as will cloud cover. The thermometer or probe should be placed in the same
position each time a measurement is made to allow valid comparison of measurements.
Light
Use a light meter. Light readings can vary widely with time of day and cloud cover. It is
better to take all measurements over a short period or take regular readings over extended
periods using a datalogger.
Oxygen concentration
In aquatic systems, oxygen probes can be used to measure oxygen concentration.
Humidity
Relative humidity can be measured using a whirling hygrometer. It needs to be spun
for 60 seconds just above the vegetation before readings are taken from the wet and dry
thermometer and used to determine the humidity from a calibration scale.
Conductivity
The ability of a water sample to carry an electric current gives a measure of the dissolved
mineral salts. The conductivity of pure water is zero; increasing ion concentration raises the
conductivity.
Soil water
A sample of soil is dried at 110C until there is no further loss in mass. The % soil moisture
can be calculated using the equation:
mass of fresh soil 2 mass of dry soil
3 100
% soil moisture 5 ________________________________
pH
Universal Indicator or a pH meter can be used to test pH after mixing a soil sample with water.
If using Universal indicator in the field, it is best to use a proper soil testing kit that contains some
long glass tubes, with lines engraved on the sides, to show levels for adding soil and chemicals.
First, 1cm3 of soil is shaken with distilled water before adding one spatula of barium sulphate
(low hazard). This helps to flocculate (settle) the clay fraction, which is important as clay particles
are very small and will otherwise cloud the water for days. Then 1cm3 of pH indicator solution is
added and the pH recorded after the contents of the tubes have been allowed to settle.
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Teacher/Lecturer 1 of 3
Purpose
Safety
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
2 of 3 Teacher/Lecturer
a woodland margin passing from a field or other example of grazed or mown grassland,
through brambles into a wood. The key gradient is likely to be light intensity but it may
not be the only one.
a sand dune system from the shore across dunes into grassland and scrub as you go further
inland. In this case the key factors could be soil moisture, soil stability and organic matter,
although succession is also involved here.
a rocky shore from the low tide mark to the top of the beach. The key factor is the
proportion of time a part of the shore is left exposed to the air and to desiccation when
the tide is out.
a geological boundary between two types of rock, such as between limestone and millstone
grit.
Some examples where two sites can be compared:
grazed and ungrazed grassland
mowed and unmowed grassland
trampled and untrampled grassland
fertilised and unfertilised lawns
shaded and sunny sites
fast- and slow-flowing streams
chalk and sandy soil sites
understories of beech and oak woodlands.
The two sites could be compared by random sampling.
Teacher/Lecturer 3 of 3
Class organisation
Students can work in groups if they are not using this investigation for their A2 coursework.
The smaller the group the more ownership by individuals, whilst the larger the group the
more quadrats can be recorded and the bigger and statistically more meaningful the picture
that can be produced. A good group size is six working as three pairs. It helps if the whole
group can have access to a computer as soon as possible after collecting the data and to a
full set of equipment during data collection. Waiting to borrow equipment from other groups
wastes a lot of time and loses momentum.
Pace is also important it is possible to be too slow and nit-picking about accuracy, in
which case the students become bored and lose sight of the bigger picture. It is also possible
for the students to feel it is somehow efficient to get the job done as quickly as possible,
subsequently data are produced that are so inaccurate and incomplete that they do not
produce reliable results.
Useful references
Jones C. (1998) Fieldwork sampling animals. Biological Sciences Review, 10(4), 2325.
Jones C. (1998) Fieldwork sampling plants. Biological Sciences Review, 10(5), 68.
Williams G. (1987) Techniques and fieldwork in ecology. London: HarperCollins.
Field Studies Council identification sheets and booklets are very good.
These can be obtained from: FSC Publications, Preston Montford, Montford Bridge,
Shrewsbury SY4 1HW.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
1 of 1 Technician
For detailed guidance on the care and breeding of brine shrimps see the publication Brine
Shrimp Ecology by Michael Dockery and Stephen Tomkins, available from: Homerton Brine
Shrimp Project, Dept of Biological Sciences, Homerton College, Cambridge CB2 2PH
Tel: 01223 507175.
Price from Blades Biological, 48.26 (2009) including post and packing, brine shrimp eggs
and innoculum.
For more details see the teacher area of the British Ecological Society website.
General note
This practical takes place over several days. The students set up the beakers with eggs to
hatch on the first day. On subsequent days they count the number of eggs that have hatched.
Requirements per student or group of students
Notes
Available from pet shops. There are approximately 24 000 egg cysts
per gram so only tiny quantities are required. Brine shrimps will
breed and produce cysts that can be collected. They adhere to the
sides of an aquarium tank.
Stirring rod
Access to refrigerator
Access to water baths or incubators (one for each
temperature to be investigated)
A lamp or light from one side. For counting larvae on the second
day.
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Student 1 of 2
Brine shrimps
Brine shrimps are small, saltwater crustaceans; the adults are about 8mm in length. They
are relatively easy to keep in the laboratory and will produce dormant egg cysts that hatch to
produce young shrimp larvae.
first
antenna
male
(blue/green)
female
(brownish red)
1 mm
eggs
second
antenna
Drawings to show features of brine shrimps
Procedure
You will need:
Brine shrimp egg cysts
2g sea salt for each treatment
100cm3 de-chlorinated water for each
treatment
40cm3 beaker of salt water
100cm3 beakers (one for each temperature
to be tested)
Water baths or incubators (one for each
temperature to be investigated)
Stirring rod
Magnifying glass
Pair of forceps
Fine glass pipette
Bright light
Access to refrigerator
Sheet of A4 white paper
Sheet of graph paper 3cm 3 4cm
31
2 of 2 Student
of brine shrimps
6 Wet the piece of graph paper using a few drops of salt water. Dab the paper onto the
white sheet to pick up approximately 40 eggs. This will look like a tiny shake of pepper.
Use a magnifying glass to count the eggs. Cut the graph paper so that there are exactly
40 eggs.
7 Put the paper with the 40 eggs into the beaker (eggs-side down). After 3 minutes, use
a pair of forceps to gently remove the paper, making sure that all the egg cysts have
washed off into the water.
8 Repeat steps 2 to 7 for all the temperatures that are to be investigated.
9 If possible replicate the treatments.
10 Incubate the beakers at the appropriate temperatures, controlling exposure to light as far
as possible.
11 The next day count the number of hatched larvae in each of the beakers. To do this,
place a bright light next to the beaker. Any larvae will swim towards the light. Using a
fine glass pipette catch the brine shrimps and place them in a small beaker of salt water.
(It may be easier if the pipette is reversed with the tip inserted into the teat, providing
a wider bore to take up the shrimp.) Repeat the counting daily for several days. Brine
shrimps are very delicate and care must be taken when handling them. Finally, discuss
with your teacher the best method for disposing of the brine shrimps.
12 Record the number of larvae that have successfully hatched at each temperature.
13 Write up your experiment making sure your report includes:
a discussion of any health and safety precautions taken
comments on the ethical issues arising from the use of living organisms
results presented in the most appropriate way
an explanation of any patterns in the data using evidence from the data and your own
biological knowledge
comments on how valid your conclusion is
comments on how you ensured that the results obtained in this experiment were valid
and reliable
suggestions for how you could have made your results more reliable.
To find out more about brine shrimps, visit the British Ecological Society website.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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1 of 1 Teacher/Lecturer
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
10
Student 1 of 1
Purpose
Safety
Procedure
You may have the opportunity to complete experimental work using restriction enzymes and
gel electrophoresis or you may use the simulation of this.
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33
Teacher/Lecturer 1 of 2
Purpose
Safety
Restriction enzymes
The use of restriction enzymes to cut DNA and electrophoresis to separate the resulting
fragments is possible using equipment available from NCBE (National Centre for
Biotechnology Education) and Bio-Rad. Protocols for these practicals as pdf files can be
downloaded from their websites.
NCBE produce an electrophoresis base unit and a lambda DNA kit. Together these contain all
necessary gel electrophoresis apparatus, dried lambda DNA, three dried restriction enzymes
and both student and technical guides. The kit includes microsyringes and gel tanks but not
batteries needed as a power supply. The kit for eight electrophoresis stations (16 runs) cost
52 in 2009. The lambda protocol module, for use with eight students, cost 90 in 2009.
Have a look at the technical guides by downloading the pdf files from the NCBE website.
The NCBE publication Illuminating DNA also contains protocols for experiments using
restriction enzymes and electrophoresis. This publication can also be viewed on the NCBE
website.
NCBE has developed a genetic screening simulation, Natures dice. In this practical
simulation, the inheritance of a single gene that is involved in a particular genetic trait is
investigated. The kit contains 24 DNA samples donated by a large family who are affected by
the genetic condition.
Each student is given one or more of these DNA samples and they have to detect which
allele is present. They cut the DNA with a restriction enzyme and examine the resulting
DNA fragments by electrophoresis. The genetic data obtained is then combined with the
family tree to look at how the genetic trait is inherited.
This activity would provide an excellent alternative to the standard gel electrophoresis
practical suggested above. Further information about the kit and cost of materials is on the
website listed in the weblinks section for this activity.
Bio-Rad produces a Restriction Digestion and analysis of lambda DNA kit. This contains
dried lambda DNA, three restriction enzymes, buffers, microtubes and both Student and
Teachers guides. It does not include the micropipette, electrophoresis cell or power supply.
The kit for eight stations (eight runs) cost 86 in 2009; the cell and power supply needed to run
the gels cost an additional 4001 but can be borrowed from some ITT centres (see below).
You can have a look at the protocol by downloading the pdf file from the Bio-Rad website.
Their website contains more detail and alternative kits. Go to the site and then click the Life
Science education icon.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
11
2 of 2 Teacher/Lecturer
Pfizer and Bio-Rad have donated biotechnology equipment to 31 ITT centres around the
country as part of a National Year of Science project. Each centre holds equipment that
can be used to conduct biotechnology practicals for 25 students at a time. They have all
the equipment necessary for completing DNA fingerprinting, bacterial transformation
(genetically altering bacteria with a bioluminescent jellyfish gene), purification of a useful
protein, and the polymerase chain reaction (PCR).
It is envisaged that this equipment will be used for hands-on practical training of student
science teachers. There are also plans for loan schemes to be established to enable
local schools to borrow the equipment. To find out more about this project contact the
BioEducation Project Manager at Bio-Rad Laboratories (details can be found on the BioRad website).
Both organisations produce replacement equipment for their kits and also supply the
components separately.
Note that some of the Bio-Rad manuals accompanying their kits (unless recently revised)
may not carry full and appropriate health and safety guidance applicable in the UK (they
were written for a US audience). Where the instructions conflict with good practice that is
standard in the UK, additional precautions should be taken.
It is most likely that gel electrophoresis equipment using batteries or a low-voltage supply is
used. This is safe if the voltage is less than 40V DC. If equipment is used that is operated by
a power supply at a greater voltage, it is essential that the equipment is designed so that it
is impossible to make skin contact with the electrodes or electrolyte when an electric current
is flowing. This is usually achieved by a suitable design of lid for the gel tank which only
permits a current to flow when the lid is in place.
Also, although it is very unlikely to be needed, ethidium bromide, which is very toxic, is not
normally recommended for use in schools.
For general CLEAPSS guidance on electrophoresis, schools should consult section 11.1.7
of the Laboratory Handbook.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
12
1 of 1 Student
Purpose
Safety
Practical PCR
Scientists in forensics laboratories carry out the polymerase chain reaction (PCR) using
a machine called an automated thermal cycler. This is a programmable heating unit in
which the DNA to be amplified is incubated in a buffer solution with thermo-stable DNA
polymerase, primers and deoxyribonucleotides. The unit maintains the cyclical sequence of
temperatures for the PCR process.
Your school or college may be lucky enough to possess a thermal cycler but it is possible to
carry out PCR without them, using three separate thermostatically controlled water baths.
You simply have to move the DNA sample from bath to bath and complete 30 cycles! You
need a stopwatch, good teamwork and some sort of protection from the steam coming off
the hottest bath.
Having amplified the short tandem repeat sequences within your DNA sample, you will
then separate out the fragments using gel electrophoresis (see Practical 6.1). Comparing
the position of the bands on the gels to a standard or reference you will be able to draw
conclusions about the DNA sample you started with.
You will follow a practical protocol supplied by the company that produces the equipment
and reagents your school or college has purchased.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
34
Teacher/Lecturer 1 of 2
Purpose
Safety
PCR
It is possible to carry out practical PCR using equipment available from a number of
suppliers including NCBE (National Centre for Biotechnology Education), Bio-Rad
and Edvotek Europe. Although all organisations supply automated PCR thermal cyclers,
they are not absolutely necessary. Instead, PCR can be undertaken using three separate
thermostatically controlled water baths, although care must be taken with the hottest as a risk
of scalding exists. Note that during Science Year (September 2001July 2002) many schools,
colleges and initial teacher training institutions received free PCR equipment.
Student and teacher/technician protocols for the PCR practicals can be downloaded as pdf
files from the suppliers websites.
Equipment can be purchased either in class sets or individually, with consumables also
available in class-sized batches. All three organisations listed offer training courses, frequently
in association with other institutions like botanic gardens or science centres, giving teachers
and technicians the opportunity to carry out the practicals themselves.
There are social and ethical considerations to take into account when using DNA derived
from students for PCR. Although the suppliers should have chosen STR sequences that have
no biological significance, it may be socially and ethically less challenging to use plant DNA
as the sample. But working with ones own DNA can be uniquely motivating!
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
13
2 of 2 Teacher/Lecturer
Contact details and examples of the protocols offered by NCBE, Bio-Rad and Edvotek
Europe:
Organisation
NCBE
National Centre for Biotechnology Education
Science and Technology Centre
The University of Reading
Whiteknights
Reading,
RG6 6BZ
www.ncbe.reading.ac.uk
0118 987 3743
NCBE@reading.ac.uk
Bio-Rad
Bio-Rad Laboratories Ltd.
Bio-Rad House
Maxted Road
Hemel Hempstead
Hertfordshire
HP2 7DX
www.biorad.com/ Follow the links to
Life Science education, then About Biotechnology
Explorer
Freephone: 0800 181134
Edvotek Europe
The Biotechnology Education Company
PO Box 280
Hertford
SG13 9DG
www.edvotek.co.uk
Follow links to Experiments and then Polymerase
chain reaction
ukinfo@edvotek.com
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
14
Technician 1 of 1
Purpose
To investigate the effect of different antibiotics on bacteria.
This experiment could be done with antiseptics rather than antibiotics. Paper discs produced
with a hole punch are autoclaved, then dipped in a range of antiseptics. CLEAPSS guidance
on microbiology is in section 15.2 of the Laboratory Handbook. Sterilisation guidance is in
section 15.12.
Requirements per student or group of students
Notes
Bunsen burner
Bench spray of disinfectant
Soap or handwash
Paper towels
Marker pen for marking Petri dishes
Autoclaved after placing in cotton wool stoppered boiling
tube.
Forceps (metal)
A Mast ring or separate antibiotic discs
Adhesive tape
Space to keep dishes at room temperature
Eye protection
Not essential.
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153
Student 1 of 2
Purpose
Safety
Introduction
When a bacterial infection is diagnosed it is useful to be able to tell to which antibiotics it is
most susceptible. In some cases this information is known, but in other cases tests need to
be carried out to find out which antibiotic will be most effective. In this activity you will be
testing the effectiveness of several types of antibiotics on bacteria.
The standard method of doing this is to put discs of chromatography blotting paper soaked
in the various antibiotics onto an agar plate that has been inoculated with the bacteria.
Alternatively a Mast ring (a ring of paper with several arms, each treated with a different
antibiotic) can be used.
Procedure
You will need:
Agar plate seeded with a known bacterium
Bunsen burner
Bench spray of disinfectant, 1% Virkon or
equivalent
Soap or handwash
Paper towels
Marker pen
Autoclaved forceps
Mast ring or antibiotic-impregnated paper
discs
Adhesive tape
Eye protection
1 Wash your hands with the soap or handwash. Spray the working area thoroughly with the
disinfectant spray. Leave for at least 10 minutes, then wipe with a paper towel.
2 Work very close to a lit Bunsen burner. Prepare an agar plate seeded with bacteria. This
may have already been done for you. If not, follow the instructions in the section Pouring
agar plates in Practical 4.3 Edexcel AS Biology. Label the Petri dish on the base at the
edge with your name, the date and the type of bacterium it is inoculated with.
3 Flame the forceps and then use them to pick up an antibiotic disc or Mast ring. Raise the
lid of the Petri dish and place the Mast ring firmly in the centre of the agar; if individual
discs are used they will need to be spaced evenly around the dish.
4 Tape the dish securely with two pieces of adhesive tape (but do not seal it completely),
then keep it upside down at room temperature for 48 hours.
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2 of 2 Student
5 Wash your hands with soap or handwash and clean the bench again using the Virkon
spray.
6 After incubation, look carefully at the plate but do not open it. Where bacteria have
grown the plate will look opaque, but where the antibiotics have inhibited growth, clear
zones called inhibition zones will be seen. Measure the diameter of the inhibition zones
in millimetres and use this information to decide which antibiotic is most effective at
inhibiting the growth of the bacterium.
7 Collect data from other members of the class who used the other bacterial cultures.
8 Write a brief report of the results, comparing the different antibiotics and the effects on
the different bacterial cultures.
Questions
1 Are the inhibition zones circular? If not, what is a sensible measuring strategy?
2 What factors determine the diameter of the inhibition zones?
3 If class data are shared:
a what is the overall spread of the data
b do all individual results show the same trends if not, why not, and how could this
variability be represented on your graphs?
4 If you were working in a hospital laboratory, and you had just carried out this test on
bacteria isolated from sick patients, would you always choose the antibiotic that gave the
biggest inhibition zone? Are there any other factors you would need to consider?
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Teacher/Lecturer 1 of 1
Purpose
Safety
Answers
1 Choice of strategy may vary, but it must be clear and easy to carry out and produce
reliable results.
2 The rate of diffusion of the antibiotic will be influenced by the size of molecule, its
concentration and the potency of the antibiotic. If the antibiotic is effective at lower
concentrations the circle will be larger (all other things being equal). Gram-positive and
Gram-negative bacteria respond differently to antibiotics.
3 Responses will depend on the data, but should show a clear understanding of the
concepts of accuracy, validity and/or reliability when explaining variation. Suggestions
for the presentation of variation in graphs may also vary but need to identify anomalous
results clearly so that conclusion are obviously based on reliable results.
4 You may have to consider whether the patient is allergic to any of the antibiotics. For
example, allergy to penicillin is not uncommon. You may consider the state of the patients
immune system. In patients with a weakened immune system you would not want to
use a bacteriostatic antibiotic, that is, one that stops bacterial reproduction but does not
kill the bacteria. Some antibiotics can be used together and produce a larger effect when
combined than if administered separately. This is known as synergism.
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1 of 1 Technician
Purpose
To demonstrate the uptake of oxygen in respiration.
To measure the rate at which an organism respires.
Notes
Respirometer
Dropping pipette
Respirometers
A respirometer is shown on the Student sheet. Many schools and colleges have at least one
of the U-tube respirometers (Figure 7.1.4 on page 131 of the A2 textbook). These can be
used but can be a lot more fiddly, and the connections often leak. If the apparatus works,
the respiring organisms use up the oxygen and give off CO2. The CO2 is absorbed by the
soda lime. This means there is less air in the test tube so the liquid gets sucked towards the
tube with the organisms in it. If it does not work there is usually an air leak somewhere, or
possibly the organisms are too cold, or dead!
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Student 1 of 2
Purpose
Safety
Respirometers
Respirometers range from relatively simple pieces of equipment used in school science
labs with seeds or invertebrates, to elaborate devices the size of a room used to measure
respiration rates in humans living near-normal lives over a period of several days. In this
practical you will be using a very simple respirometer, while considering the advantages of
some of the slightly more complex ones.
Procedure
You will need:
Respirometer (see diagram
below)
5g of an actively respiring
organism
Soda lime
Coloured liquid
Dropping pipette
Permanent OHT marker pen
syringe
scale
three-way tap
glass tubing
coloured liquid
small organisms
gauze
soda lime
A simple respirometer
2 Place 5g of maggots or peas into the test tube and replace the bung.
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2 of 2 Student
3 Introduce a drop of marker fluid into the pipette or glass tube using a dropping pipette.
Open the connection (three-way tap) to the syringe and move the fluid to a convenient
place on the pipette (i.e. towards the end of the scale that is furthest from the test tube).
4 Mark the starting position of the fluid on the pipette tube with a permanent OHT pen.
5 Isolate the respirometer by closing the connection to the syringe and the atmosphere and
immediately start the stopclock. Mark the position of the fluid on the pipette at 1 minute
intervals for 5 minutes.
6 At the end of 5 minutes open the connection to the outside air.
7 Measure the distance travelled by the liquid during each minute (the distance from one
mark to the next on your pipette).
If your tube does not have volumes marked onto it you will need to convert the distance
moved into volume of oxygen used. (Remember the volume used 5 pr2 3 distance
moved, where r 5 the radius of the hole in the pipette.)
Questions
1 Why did the liquid move? Explain in detail what happens to the oxygen molecules, the
carbon dioxide molecules and the pressure in the tube.
2 It would have been better to set up a second, control tube that did not contain living
organisms but had everything else the same.
a What could cause a movement of the liquid in the control tube towards the respirometer?
b What could cause a movement of the liquid in the control tube away from the
respirometer?
c What could you do to correct your estimate of oxygen uptake if the liquid in the
control had moved too?
Extension
3 The diagram below and Figure 7.24 on page 148 of A2 Biology show two other types of
respirometer. What advantages and disadvantages do these have compared to the one
you are using?
soda
lime
drop of
liquid
wire
mesh
organism to
be studied
capillary
tube
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1 of 2 Teacher/Lecturer
Purpose
Safety
respirometer open
to syringe
respirometer open
to atmosphere
Tap positions for a three-way tap (viewed from the side)
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respirometer
isolated
Teacher/Lecturer 2 of 2
Answers
1 Simple answer: Oxygen molecules are absorbed by the organism and used in respiration. The
same number of carbon dioxide molecules are released but these are absorbed by the soda lime.
This reduces the pressure inside the test tube (fewer molecules 5 lower pressure). Atmospheric
pressure pushes the liquid along the tube, until the pressure in and outside the tube is equal.
Detailed respiration review answer: As above but should include reference to the role of oxygen
as the final electron acceptor, and the fact that it eventually combines with hydrogen to make
water. The carbon dioxide comes from the carbon dioxide released in the link reaction and the
Krebs cycle as the carbohydrate is broken down.
2 a A drop in temperature inside the tube, or an increase in atmospheric pressure.
b An increase in temperature inside the tube, or a decrease in atmospheric pressure.
c The distance moved by the liquid in the control in each minute towards the organisms
should be subtracted from the distance that the liquid in the experimental respirometer
moved. Movement of the liquid in the control away from the organisms should be added to
the distance in the experimental tube.
3 Diagram showing a very simple respirometer:
Disadvantages does not allow you to reset; it needs a control tube used alongside it; no scale so
measurements likely to be less accurate.
Advantages very simple to set up; minimal number of connections makes a good seal easier to
obtain.
1 cm3 syringe
experimental tube
screw clip
three-way tap
small organisms
gauze
KOH solution
absorbs carbon
dioxide
KOH solution
manometer tube
containing fluid
U-tube respirometer
Advantages does not need to have an additional control as the second tube balances out the
effects of changes in temperature or atmospheric pressure; the syringe allows you to move the
liquid in the U to reset the apparatus.
Disadvantages tendency for the connections to leak in elderly school/college models (making
the equipment useless); expense.
4 The experimental design should include either a U-tube respirometer or controls; a known mass
of maggots; a range of temperatures (between 5C and 40C); a suitable increment between
temperatures (say, 5C); repeated measurements (say, at least three at each temperature).
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Technician 1 of 2
Purpose
To investigate lung volumes and rate of breathing.
Safety
The safety guidelines in Sections 14.5 and 14.5 .1 in the CLEAPSS Laboratory Handbook
should be followed when using a spirometer.
This investigation is potentially hazardous. Students should be closely supervised at
all times when using a spirometer. If the students are allowed to breathe through the
spirometer for too long they can lose consciousness from lack of oxygen. Limiting the
time spent breathing through the spirometer and carefully observing each student should
prevent problems. When using oxygen and absorbing CO2: maximum time 5 minutes; in
any other situation: maximum time 1 minute. If a student becomes less alert or has any
feeling of suffocation they should stop immediately. Since the levels of CO2 are kept low by
the soda lime, the students wont be aware that they are running out of oxygen until it is
potentially too late.
A trained member of staff should use an oxygen cylinder to fill the spirometer.
Use eye protection when handling soda lime. Soda lime is corrosive. Do not handle directly:
use a spatula. Use a type of soda lime that changes colour when it is saturated, and replace
it when it changes colour. Use 510 mesh particle size. Follow CLEAPSS guidelines on
removing dust for spirometer use. A layer of polymer wool can be put at the inflow and
outflow of the soda lime canister chamber to prevent dust getting into the chamber. Ensure
that the soda lime canister is fitted so that air is breathed out through the canister.
The subject will feel some resistance to breathing when using a spirometer. Because of this
they should not use the spirometer while exercising. To investigate the effect of exercise,
readings should be taken immediately after exercise. If resistance to breathing suddenly
increases it may be due to valves in the spirometer sticking. If this occurs the valves may
need to be replaced.
Do not use oil, grease or glycerine on any part of the spirometer tubing as these may make
explosive compounds with oxygen. Water with a little liquid detergent can be used to aid
connection of tubes, etc. Keep all flames away from the spirometer.
It is essential to follow good hygiene practice with regard to cleaning and disinfecting the
mouthpiece and removing condensation and saliva from the tubes.
Care should also be taken to choose the subject, avoiding any students with asthma or
other breathing or circulatory problems.
(Asthmatics may use a spirometer if they are otherwise in good health.)
Read the manufacturers instructions carefully before using a spirometer.
In this activity students learn how a spirometer works and how to interpret the spirometer
trace that is produced. Alternatively a datalogger can be used with a traditional water-filled
spirometer or with an airflow spirometer. A range of different types of sensors can be used
and some alternatives are described on the Teacher sheet.
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2 of 2 Technician
Investigating breathing
A spirometer can be used to investigate the effect of exercise on breathing rate and lung
volumes. If you have not got access to a spirometer, measurement of vital capacity and tidal
volume can be carried out using breath volume bags. These are a fraction of the cost of a
spirometer. However, they cannot be used for measuring oxygen consumption.
Requirements per student or group of students
Notes
Spirometer
Kymograph
Kymograph paper
Safety
See notes on page 1.
Eye protection
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Student 1 of 4
Purpose
Safety
Using a spirometer
The apparatus shown below is a spirometer. Spirometers allow us to study both breathing
and respiration. In this activity you will learn how a spirometer works and how to interpret
the spirometer trace that is produced.
A spirometer
The general principle behind a spirometer is simple. It is effectively a tank of water with
an air-filled chamber suspended in the water. It is set up so that adding air to the chamber
makes the lid of the chamber rise in the water, and removing air makes it fall. Movements
of the chamber are recorded using either a kymograph (pen writing on a rotating drum), a
chart recorder, computer or datalogger.
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2 of 4 Student
Investigating breathing
Tubes run from the chamber to a mouthpiece and back again. Breathing in and out through
the tubes makes the lid of the chamber fall and rise. The volume of air the person inhales and
exhales can be calculated from the distance the lid moves.
The apparatus can be calibrated so that the movement of the lid corresponds to a given
volume. A canister containing soda lime is inserted between the mouthpiece and the floating
chamber. This absorbs the CO2 that the subject exhales. In which direction will the pen move
when the subject inhales?
Procedure
You will need:
Spirometer
Kymograph, chart recorder or computer
Soda lime (for the spirometer canister)
Disinfectant solution
Eye protection
Calibration
In order to interpret the spirometer trace you need to know what both the vertical and the
horizontal scales represent.
40
Student 3 of 4
Investigating breathing
Volume 02/dm3
6 Switch on the recording apparatus and at the end of an exhaled breath turn the tap so
that the mouthpiece is connected to the spirometer chamber. The trace will move down as
the person breathes in. After breathing normally the subject should take as deep a breath
as possible and then exhale as much air as possible before returning to normal breathing.
tidal
volume
vital
capacity
Time/minutes
A sketch of a trace showing normal breathing and one forced breath in and out
A diagram of a spirometer trace is shown above. In this example the subject has breathed in
and out normally three times, then taken as deep a breath in as possible, then forced the air
from their lungs. Several pieces of information about the subjects breathing can be read off
this kind of trace, or worked out from it.
The tidal volume is the volume of air breathed in and out in one breath at rest. The tidal
volume for most adults is only about 0.5dm3.
Vital capacity is the maximum volume of air that can be breathed in or out of the lungs in
one forced breath.
Breathing rate is the number of breaths taken per minute.
Minute ventilation is the volume of air breathed into (and out of) the lungs in one minute.
Minute ventilation 5 tidal volume 3 rate of breathing (measured in number of breaths
per minute).
Some air (about 1dm3) always remains in the lungs as residual air and cannot be breathed
out. Residual air prevents the walls of the bronchioles and alveoli from sticking together. Any
air breathed in mixes with this residual air.
41
4 of 4 Student
Investigating breathing
chamber than was breathed in. If we breathe into and out of the spirometer for (say) 1
minute, a steady fall in the spirometer trace can be seen. The gradient of the fall is a measure
of the rate of oxygen absorption by the blood, and so is a measure of the rate of respiration
by the body.
1 Using the trace produced in class, or one provided by your teacher/lecturer, find the
following values:
a tidal volume
b vital capacity
c breathing rate
d minute ventilation.
2 Use the trace produced in class, or one provided by your teacher/lecturer, to work out the
rate of oxygen consumption in someone at rest.
3 What differences would you expect if the subject had been exercising before a trace was
taken?
4 Describe how you could use the apparatus to measure changes in breathing and
respiration rates due to exercise. (Note that the apparatus you have used may not be
suitable for use during exercise, and that measurements need to be taken immediately
after exercise has stopped. Discuss with your teacher which is the best method for
use with your apparatus.) State what exercise would be appropriate, and any hazards
involved. Sketch the shape of the trace you would expect before and after exercise.
Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.
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1 of 4 Teacher/Lecturer
Purpose
To investigate lung volumes and rate of breathing.
Spirometers
A copy of a spirometer trace is provided at the end of these notes, for use when there is no
access to a spirometer or as extra data to interpret.
The practical describes the use of a spirometer with a kymograph. Alternatively a datalogger
can be used with a traditional water-filled spirometer or with an airflow spirometer. A range
of different types of sensors can be used and some alternatives are described below using a
spirometer with a datalogger.
If you have not got access to a spirometer, measurement of vital capacity and tidal volume
can be carried out using breath volume bags. These are a fraction of the cost of a spirometer.
However, they cannot be used for measuring oxygen consumption.
Safety
Use eye protection when handling soda lime. Soda lime is corrosive. Do not handle directly: use a spatula.
A spirometer should only be used with supervision. Students with breathing or circulation (heart) problems
or suffer from epilepsy should not use the spirometer. Read the manufacturers instructions and safety notes
before using the equipment. CLEAPSS guidance is in section 14.5.1 of the Laboratory handbook.
If the students are allowed to breathe through the spirometer for too long they can lose consciousness
from lack of oxygen. Limiting the time spent breathing through the spirometer and carefully observing each
student should prevent problems. When using oxygen and absorbing CO2: maximum time 5 minutes; in any
other situation: maximum time 1 minute. If a student becomes less alert or has any feeling of suffocation
they should stop immediately. Since the levels of CO2 are kept low by the soda lime, the students wont be
aware that they are running out of oxygen until it is potentially too late.
Stop using the spirometer at once if the student experiences any unusual breathing problems or feels dizzy
or uncomfortable. (Asthmatics may use a spirometer if they are otherwise in good health.)
A trained member of staff should use an oxygen cylinder to fill the spirometer. If only a few breaths are to be
measured, then atmospheric air is acceptable instead.
The subject will feel some resistance to breathing when using a spirometer. Because of this they should
not use the spirometer while exercising. To investigate the effect of exercise, readings should be taken
immediately after exercise. If resistance to breathing suddenly increases it may be due to valves in the
spirometer sticking. If this occurs the valves may need to be replaced.
After use, the tubing from the spirometer should be removed and placed in 70% ethanol, to disinfect the
internal ribbed surfaces where microorganisms might remain.
18
Teacher/Lecturer 2 of 4
Investigating breathing
pully
motion sensor
counterweight to
tension cord
cord
distance varies
moving lid
moving lid
water
Carbon dioxide and oxygen levels in the spirometer lid can be datalogged. Monitoring these
levels gives another safety check that the CO2 absorber is working and that O2 levels are not
falling dangerously low.
Alternatively, many of the datalogging companies now produce an airflow spirometer which
measures volume by integrating the flow data over time. These devices are very lightweight,
do not need carbon dioxide absorbers and do not need cylinders of medical oxygen (they
use the oxygen from the atmosphere).
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3 of 4 Teacher/Lecturer
Investigating breathing
Answers
1 The values here are for the trace provided at the end of these notes. Obviously if a
students own trace is used the answers may be different.
a Tidal volume between 0.5 and 0.8dm3. Encourage students to take an average over
several breaths.
b Vital capacity 5 2.55dm3 averaged over the two breaths.
c Breathing rate 5 between 18 and 20 breaths per minute (depending on the section of
the graph used).
d Minute ventilation 5 19 breaths per minute 0.65dm3 5 12dm3 min1.
2 The rate of oxygen consumption determined from the trace at the end of these notes is
approximately 1.0dm3 min1 for the first 30 seconds.
3 If the subject had been exercising, the rate of oxygen consumption would be higher, so
the slope would be steeper. The trace would also show that the subject was breathing
faster and more deeply.
4 This question allows students to think through the practicalities of using a spirometer
in an investigation. They should think about the type of exercise: it is easier to use a
spirometer immediately after running up and down stairs than after swimming or
paragliding. They may also consider individual variation and sample sizes.
They should also think about the limits imposed by the amount of oxygen available in the
tank. Comments on the effect of the spirometer on the subject are also worth encouraging
a spirometer often causes breathing to become slightly more laboured particularly with
older models that have smaller diameter tubing.
The graph should show that breaths become deeper and more frequent after exercise,
with a greater rate of oxygen consumption.
After exercise
Volume 02/dm3
Before exercise
Time/minutes
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Teacher/Lecturer 4 of 4
Investigating breathing
1 cm
Volume 02(dm3)
5
Kymograph speed:
64 mm min1
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Technician 1 of 1
Purpose
To investigate habituation of snails to a stimulus.
Notes
Board
Stop watch
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Student 1 of 2
Purpose
To investigate habituation of snails to a stimulus.
Touching snails
Lots of people, at some time in their childhood, will have touched a snail in the garden and
noticed that it withdraws its eye stalks into its body. For such a slow-moving animal this
seems a very quick response, this suggests it is an important response for protection and
survival. A snail only withdraws into its shell when it is either inactive or threatened. When
touched, it withdraws to avoid danger. Do snails become habituated to the stimulus, ceasing
to withdraw with repeated stimulation? In this investigation you will collect data to find out if
habituation to a touch stimulus does occur in these organisms.
Safety
Wash your hands thoroughly after touching the snails once all the equipment has been put
ready for disinfection.
Take care that the stimulus causes no harm to the snails.
Procedure
You will need:
One giant African land snail (or a garden snail if not available)
One dampened cotton wool bud
Suitable clean, firm surface for the snails (e.g. a plastic chopping board)
Stopwatch
1 Collect one giant African land snail, and place it on a clean, firm surface. Allow the snail
to get used to its new surroundings for a few minutes until it has fully emerged from its
shell.
2 Dampen a cotton wool bud with water.
3 Firmly touch the snail between the eye stalks with the dampened cotton wool bud and
immediately start the stopwatch. Measure the length of time between the touch and the
snail being fully emerged from its shell once again, with its eye stalks fully extended.
4 Repeat the procedure in step 3 for a total of 10 touches, timing how long the snail takes
to re-emerge each time.
5 Record your results in a suitable table.
6 Present your results in an appropriate graph.
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2 of 2 Student
Questions
1 Write a hypothesis which this experiment will test.
2 Using your graph, state if you think there is a positive, negative, or no, correlation
between the number of stimulations and the time for eye stalk withdrawal.
3 Explain any patterns or trends in your data, supporting your ideas with evidence from
the data and your biological knowledge of habituation. Relate your findings to your
hypothesis.
4 Suggest a reason why snails may become habituated to a prodding stimulus in the wild.
5 Evaluate the procedure used for this experiment.
6 This experiment has been shown to be less successful if the snails are handled regularly
prior to the experiment. Suggest why handling prior to the experiment could affect the
results of the experiment.
Going further
7 Write a null hypothesis that this experiment will test.
8 Complete a Spearmans rank rs correlation test to determine if there is a statistically
significant correlation between the variables. A table with the headings below will help.
Number of times
the snail has
been stimulated
Rank stimulation
Time/seconds
Rank time
Difference/D
D2
9 Use a table of critical values to accept or reject your null hypothesis. If your calculated
Spearman rank value (rs) is greater than the critical value, then the null hypothesis is
rejected. If your calculated rs value is less than the critical value, then the null hypothesis
is accepted.
10 Write a statistical conclusion for your experimental data. Make sure you include:
your calculated value of rs
the number of pairs of data
the significance level
the critical value
whether the null hypothesis is being accepted or rejected.
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1 of 1 Teacher/Lecturer
Purpose
Safety
To investigate habituation of
snails to a stimulus.
Answers
1 As the number of stimuli increase, the time taken for the snail to re-emerge will decrease
a negative correlation.
2 Students present the results as a scatter graph, with the number of stimuli on the x-axis, and
the time taken to re-emerge on the y-axis. Students should make a simple statement, from
their results, as to whether there appears to be a positive, a negative, or no, correlation.
3 There is a negative correlation as the number of stimuli increase the time taken for the
snail to re-emerge decreases. Students should make a reference to the data. With repeated
stimulation, Ca21 channels in the presynaptic membrane become less responsive. Less
Ca21 crosses the membrane into the presynaptic (sensory) neurone. As a result less
neurotransmitter is released into the synaptic cleft. This means that an action potential
across the postsynaptic membrane is less likely. Fewer action potentials are produced in
the postsynaptic motor neurone so less of a response is observed.
4 The snail learns that the stimulus is not causing harm, and so it is withdrawing
unnecessarily. This effect may be used in its natural habitat when faced with repetitive
stimuli, such as vegetation touching the head/eye stalks.
5 Relevant comments would include: the need for replication using snails of approximately
the same size and age; the need to control of the size and position of the stimulation; the
need to control other variables that may affect the response, e.g. drying out of the snail;
difficulties in determining when the snail has fully extended (measuring the eye stalk
length prior to stimulation may overcome this problem); effect of handling the snail prior
to the experiment.
6 If the snails have been handled too much prior to the experiment, they may have already
become habituated to this type of stimulus so further stimulations will not change the response.
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