Edexcel A2 Biology Practicals (Complete)

Technician 1 of 1
Practical 5.1 Looking for patterns and correlations
Purpose
Safety
To carry out a study on the ecology of

a habitat.
Teachers/lecturers must follow their LEA/school/college

policy and local rules for off-site visits, especially with
regard to identification of hazards and risk assessments.
Additional information is available in the DCSF guidance
Health and Safety of Pupils on Educational Visits on the
Teachernet website. The hazards will vary depending on
the site chosen. Risk assessments will identify the risk.
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 50m tape measure. If this is not available a

1020m tape measure can be used
A piece of rope with 0.5m intervals marked on it will also work.

Two will be needed if a grid layout is to be used.
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.
Small, numbered pegs
At least 50.
A clipboard
A clear plastic bag large enough to get clipboard
and hand with pencil in
To protect students notes if it rains.
Ranging poles
The geography department may have these already.
A clinometer
The geography department may have one already.
Small plastic vials or dishes
For holding invertebrates while identifying them on site.
A key to organisms likely to be found in the area
The Field Studies Council produces a range of excellent guides.
Access to a compass
To describe the aspect.
Access to an OS map of the area
To accurately pinpoint the site and for background knowledge.
Jam jars
For pitfall traps.
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
Practical 5.1 Looking for patterns
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.
23
2 of 8 Student
Practical 5.1 (cont.)
Looking for patterns
Completing a transect study

One of the easiest patterns to spot is zonation in the vegetation and animal distribution as
you go from one place to another the vegetation and animal distribution changes. A zonation
can often be explained by a gradual change (a gradient) in one or more physical or abiotic
factors. A transect is often used to study zonation in vegetation or non-mobile animal
distribution. A transect is a line along which systematic records can be made.
Comparing two sites

Frequently ecologists may notice a distinct pattern that does not show a gradual change and may
be related to one or more factors at the two sites. For example, the vegetation in one area of a field
may be very different from the rest of the field, or the species found upstream and downstream
of an outflow pipe discharging into a river may seem to differ. A transect may not be the best
method for this type of investigation; instead sampling of each area may be more appropriate.
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.
24
Student 3 of 8
2 Collect the data.

3 When you have collected your data, you must present it in an appropriate way to help
you identify any patterns in the data. For transect data you can draw kite diagrams by
hand or use a computer programme such as FieldWorks which may be available from
your teacher.
4 Analyse your data to reveal any patterns or significant differences, and explain the main
relationships between species and abiotic factors using scientific knowledge. Determine
if your original hypothesis was correct. If you have suitable data, you can calculate
correlation coefficients between your biotic and abiotic data. For example, you can see
if there is a significant positive or negative correlation between the factor you think is
responsible for the pattern and the distribution of the organisms you have recorded.
Remember that correlations do not prove cause and effect.
5 In your write-up interpret your results using biological principles and concepts. Support
any conclusions you make with results. Discuss the limitations of your results and
conclusions based upon them, and suggest modifications that you could make to the
procedure.
25
4 of 8 Student
Practical support sheet Ecological sampling
Why sample in ecology?

In an ideal world when investigating, say, the number of dandelions in two meadows, you
would count every single dandelion in each. The problem is that this might take forever
and become very, very boring. So, instead, you need to take a sample. You might estimate
the number of dandelions in each meadow by counting the number in several small areas
and then multiplying up to calculate a value for each meadow. The idea is to maximise the
usefulness of your data while minimising the effort required to collect them.
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.
26
Student 5 of 8
Practical support sheet (cont.)
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
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.
Methods of measuring abundance

Density
Count the number of individuals in several quadrats and take the mean to give number per
unit area, for example per metre squared (m22). In many plant species (e.g. grasses) it is very
difficult to distinguish individual plants, so measuring density is not possible.
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.
Estimating animal populations

Quadrats cannot be used for mobile animals as these dont stay in the quadrats. A variety
of different nets and traps need to be used. Animals that occur on the soil surface may be
sampled using a pitfall trap (Figure 3). Those in vegetation can be sampled using a pooter
directly or indirectly (after being knocked from the vegetation onto a white sheet). Insects
and other small invertebrates found in leaf litter can be collected using a Tullgren funnel.
Markrelease methods can also be used.
28
Student 7 of 8
Ecological sampling
Pitfall trap for sampling arthropods

flat stone prevents
rainfall filling trap
stick support
Tullgren funnel for collecting organisms

from the soil or leaf litter
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
air and arthropods

drawn into tube
cork or
rubber bung
specimen tube
where arthropods
collect
Organisms move
away from the heat
and light, falling
into the jar.
glass mouthpiece
80% alcohol
gauze covering
tube opening
air sucked
through
mouthpiece
Alternatively a muslin bag of soil

surrounded by water can be used
to collect living organisms. This
is a Baermann funnel.
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
Figure 3 Net and traps for sampling animals.
29
8 of 8 Student
Ecological sampling
Measuring abiotic factors when sampling the environment

Angle of slope
Use a clinometer.
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 ________________________________
mass of fresh soil
Soil organic matter

A dry soil sample of known mass is heated in a crucible for 15 minutes to burn off all the
organic matter. The mass is re-measured after the soil sample has cooled. The % soil organic
matter is calculated using the equation:
mass of dry soil 2 mass of burnt soil
3 100
% organic matter in soil 5 ________________________________
mass of dry soil
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.
30
Teacher/Lecturer 1 of 3
Practical 5.1 Looking for patterns
Purpose
Safety
To carry out a study on the ecology of

a habitat.
Teachers/lecturers must follow their LEA/school/college

policy and local rules for off-site visits, especially with
regard to identification of hazards and risk assessments.
Additional information is available in the DCSF guidance
Health and Safety of Pupils on Educational Visits on the
Teachernet website. The hazards will vary depending on
the site chosen. Risk assessments will identify the risk.
Notes on the procedure

Students need to carry out a study on the ecology of a habitat to produce valid and reliable
data (including the use of quadrats and transects to assess the abundance and distribution of
organisms and the measurement of abiotic factors). This activity outlines how to approach
a fieldwork investigation. It briefly mentions some techniques that might be used. It does
not attempt to provide detailed accounts of the techniques. Additional details are provided
on the Student Practical support sheet Ecological sampling (page 26) and there is a wealth
of information on the British Ecological Society student 161 education web pages about
sampling techniques. There are also examples of projects with data and virtual tours of
the rocky shore and sand dune habitats. The Field Studies Council website also has online
resources for students including information on different urban ecosystems.
The methods used will depend on the habitat and factors under investigation. Any suitable
methods could be used that give students the opportunity to have first-hand experience of
field data collection. The school/college grounds can be a valuable resource.
Other activities could be covered in a fieldwork context; for example, feeding relationships
and the transfer of energy through ecosystems, or succession, could be investigated through
a specific habitat.
Fieldwork can be used as students investigation for A2 coursework. Teachers/ lecturers and
students should ensure that the proposed investigation meets the assessment criteria.
When planning coursework for Unit 6 there is a full description of the requirements
illustrated with exemplars in the Tutor support material for Unit 6 on the Edexcel website.
Select a site carefully

The site selected should show some clear relationships.
Some examples where transects can be used:
a playing field or grassy area from an area of high trampling to an area of low trampling.
The Field Studies Council online resources include a case study on the distribution of
ribwort plantain and greater plantain in trampled and non-trampled areas.
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.
Identification dont panic!

Identification is not as big a problem as you may think. The only species students need focus
on are those which would support the hypothesis under investigation. Trying to identify each
species in a quadrat down to the tiniest piece of moss can be a poor use of time and can
actually prevent students from seeing the wood for the trees.
A transect at a woodland margin could be successfully done recording only tree cover, grass,
brambles, nettles, ivy, bare ground, wild garlic (with give-away smell) and bluebells, together
with good light meter readings and soil pH (often showing no pattern), especially if you start
to consider something like leaf surface area of brambles at the same time.
But the principles are:
select a site where students can cope with the species identification
before teaching students to identify another species, ask yourself What is the ecological
point of teaching this?
make use of expert help if available this could include a Field Study Centre.
The Field Studies Council produces excellent laminated cards to aid with field identification
in particular habitats.
The students will be relying on you for identification so careful preparation is important. You
may like to produce a record sheet that includes the species you want to focus on.
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.
FieldWorks: an invaluable IT resource

FieldWorks is available on CD and produced by Interpretive Solutions, Hallsannery Field
Centre, Bideford, Devon EX39 5HE. It benefits from having grown out of the learning
experiences of many students.
Students can enter their own data, and present these using kite diagrams or other formats. It
also contains information about species and habitats including rocky shores, sand dune, salt
marsh, moorland, woodlands and fresh water.
The data can be subjected to statistical analysis using correlation coefficients (Pearsons
parametric or Spearman rank non-parametric), particularly useful with transects. The
programme can also calculate species diversity indices and carry out t-tests and z-tests.
Other statistical packages are available for use in ecology from software companies and other
suppliers.
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.
1 of 1 Technician
Practical 5.2 The effect of temperature on the hatching success of

brine shrimps
Purpose
To investigate the effect of temperature on the hatching success of brine shrimps.
To develop certain experimental skills, namely considering the ethical issues arising from the
use of living organisms, presenting results, producing reliable results, identifying trends in data
and drawing valid conclusions.
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.
Notes
Brine shrimp egg cysts
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.
100cm3 beakers (one for each temperature to be tested)

100cm3 de-chlorinated water for each treatment
Tap water can be de-chlorinated by leaving it to stand for 48 hours.
40cm beaker of salt water

2g sea salt for each treatment
Students could be supplied with the salt water already prepared.
Stirring rod
Access to refrigerator
Access to water baths or incubators (one for each
temperature to be investigated)
A range of temperatures between 5 and 35C is recommended. The

optimum for hatching is 28C.
White A4 sheet of paper

Sheet of graph paper 3cm 4cm
Magnifying glass
Pair of forceps
Bright light
A lamp or light from one side. For counting larvae on the second
day.
Fine glass pipette
For counting larvae on the second day.
Small beaker of salt water
For counting larvae on the second day.
Large beaker or tank of salt water 1 substrate and

algae, set up well in advance. Details in the Dockery and
Tomkins book
To hold the brine shrimps after hatching. This tank can be

maintained for subsequent investigations of brine shrimps.
152
Student 1 of 2

brine shrimps
Purpose
To investigate the effect of temperature on the hatching success of brine shrimps.
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
1 Decide on a range of temperatures from 5C to 35C to be tested.

2 Place 2g of sea salt into a 100cm3 beaker.
3 Add 100cm3 of de-chlorinated water and stir until the salt completely dissolves.
4 Label the beaker with sea salt and the temperature at which it will be incubated.
5 Place a tiny pinch of egg cysts onto a large sheet of white paper.
31
2 of 2 Student
The effect of temperature on the hatching success
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.
32

brine shrimps
Purpose
To investigate the effect of temperature on the hatching success of brine shrimp.

If students are given basic information on maintaining brine shrimps this activity could be
planned before they are given the Student sheet. The need to keep conditions other than
temperature constant should be appreciated. Brine shrimps hatch in salt water that is 2 to
5% salt (optimum 2.8%) and pH 8.5. Oxygen must be present. Light is an added (but not
essential) factor for hatching. Datalogging could be used to check that the conditions within
each of the treatments are maintained at a constant level.
This experiment could be completed by small groups of students. If each group completed the
range of temperatures then the error between replicates could be investigated. Brine shrimps
will hatch in 24 hours at temperatures between 25 and 30C with an optimum of 28C.
This activity can be used to highlight experimental and investigative assessment objectives, in
particular the ethical issues arising from the use of living organisms (including the disposal
of the brine shrimps after the activity) and for the environment, and the need to identify both
dependent and independent variables and, where possible, control or allow for them. The
method suggested provides precise measurements. The need for valid, reliable results should be
considered and the random nature of error could be investigated.
The practical procedure and technical notes are based on an investigation that appears in
the British Ecological Society publication Brine Shrimp Ecology by Michael Dockery and
Stephen Tomkins. This book contains detailed information on the care and breeding of brine
shrimps. It is available from the British Ecological Society or 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, including the preparation of a salt water aquarium in which to keep the
brine shrimps, see the teacher area of the British Ecological Society website.
10
Student 1 of 1
Practical 6.1 DNA gel electrophoresis
Purpose
Safety
To use gel electrophoresis to separate DNA

fragments of different sizes.
If you undertake gel electrophoresis make sure

you are aware of the hazards and follow the
instructions of your teacher very carefully.
Procedure
You may have the opportunity to complete experimental work using restriction enzymes and
gel electrophoresis or you may use the simulation of this.
33
Practical 6.1 DNA gel electrophoresis
Purpose
Safety
To use gel electrophoresis to separate DNA

fragments of different sizes.
If gel electrophoresis is to be used a full risk

assessment should be obtained for the procedure
and carefully followed by both staff and students.
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.
11
DNA gel electrophoresis
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.
12
1 of 1 Student
Practical 6.2 DNA amplification using PCR
Purpose
Safety
To use PCR to amplify DNA.
If you undertake PCR make sure you are aware

of the hazards and follow the instructions of
your teacher very carefully.
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.
34
Practical 6.2 DNA amplification using PCR
Purpose
Safety
To use PCR to amplify DNA.
If PCR is to be used a full risk assessment

should be obtained for the procedure and
carefully followed by both staff and students.
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!
13
DNA amplification using PCR
Contact details and examples of the protocols offered by NCBE, Bio-Rad and Edvotek
Europe:
Organisation
Example of suitable protocols
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
Amplifying lambda DNA (from the Illuminating

DNA booklet)
Investigating plant evolution
Analysing mitochondrial DNA. This practical
requires a microcentrifuge and the protocol is not
downloadable from the NCBE website. It is based
on a protocol developed for a DNA workshop at the
Cold Spring Harbor Laboratory. It can be obtained
direct from NCBE.
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
Crime scene investigator PCR basics

PV92 PCR infomatics kit
GMO investigator kit (only useful if GM foodstuffs
are easily available)
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
332 Mitochondrial DNA analysis using PCR
14
Technician 1 of 1
Practical 6.3 Which antibiotic is most effective?
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.
Notes
Agar plates seeded with bacteria or equipment for their

preparation (i.e. access to one of two broth cultures of known
bacteria; Universal McCartney bottle of nutrient agar; sterile
Petri dish; sterile pipette)
Suitable bacteria are listed in various catalogues. Phillip Harris

use E. coli (K12 strain) and Staphylococcus albus in their kit.
The Student sheet with instructions for preparation of seeded
agar plates, Pouring agar plates, can be found in Edexcel AS
Biology Practical 4.3.
Safety
The bacterial culture could present a biohazard and should be
handled and disposed of in accordance with current guidelines on
microbiology in schools.
Disposal of agar plates and other used equipment, including
forceps, must be in accordance with current best practice.
Autoclaving is the preferred method of disposal over any chemical
disinfection method.
Bunsen burner
Bench spray of disinfectant
1% Virkon (preferred) or equivalent.
Soap or handwash
This does not need to be bactericidal.
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
Separate antibiotic discs are usually cheaper, but it is

impossible to obtain more than two types of antibiotics using
these.
Adhesive tape
Space to keep dishes at room temperature
This will be needed for at least 48 hours.
Eye protection
Not essential.
153
Student 1 of 2
Purpose
Safety
To investigate the effect of different

antibiotics on bacteria.
Wear eye protection.

The microorganisms are a potential biological hazard.
Use aseptic techniques when transferring the bacteria
to the Petri dishes. Clean the bench with antibacterial
disinfectant. Do NOT open the Petri dishes once they
have been incubated.
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.
35
2 of 2 Student
Which antibiotic is most effective?
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?
36
Purpose
Safety
To investigate the effect of

different antibiotics on bacteria.
Eye protection is not necessarily required during microbial

work, though it is good standard practice.
The microorganisms are a potential biological hazard. Use
aseptic techniques when transferring the bacteria to the Petri
dishes. Clean the bench with antibacterial disinfectant. Do
NOT open the Petri dishes once they have been incubated.

The aim of the practical is to show students a method of determining bacterial sensitivity to
different antibiotics and to give them practice at using aseptic techniques. This could be done
with antibiotic discs or the same technique could be used with antiseptics. The Student sheet
suggests using different bacterial species but the activity could be done using just one species.
However, using different species should illustrate that they are not all equally susceptible.
Although allergic response by patients is considered in the questions, where antibiotics are
already impregnated on to discs, the risk for students with allergic responses is not great.
Discs are not handled directly and no airborne dust is created by the discs.
The behaviour of students must be considered. If they might attempt to lift a piece of tape
from an incubated dish, the agar plates should be sealed around the circumference after
incubation but before being returned for observation. Also, to prevent agar plates being
removed from the lab, count all plates back in again before students are allowed to leave.
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.
15
1 of 1 Technician
Practical 7.1 Measuring the rate of oxygen uptake
Purpose
To demonstrate the uptake of oxygen in respiration.
To measure the rate at which an organism respires.
Notes
Respirometer
See the diagram on the Student sheet. If a pipette is used,

the scale shown will not be needed. Ideally the syringes are
attached with a three-way tap. If these are not available a
rubber tube and clip can be used. U-tube respirometers would
be even better, if there is a class set available. See also section
15.10 on respirometers in the CLEAPS Laboratory Handbook
for details of other (bulk) suppliers.
5g of an actively respiring organism
Use actively germinating peas, beans or other seeds, or

maggots, or woodlice.
Roughly a tablespoon of soda lime
To absorb the carbon dioxide.

Safety
Soda lime is corrosive, but much less of a hazard than solutions
of potassium or sodium hydroxide. Even so, eye protection is
needed when handling the soda lime. Do not handle directly: use
a spatula. Soda lime can sometimes be dusty. To avoid exposing
invertebrates to corrosive dust, take the soda lime outside and
pour from beaker to beaker to blow the dust away. Position
yourself so that you cannot inhale the dust during this activity.
About 2cm3 of coloured liquid
e.g. water and food colour or equivalent.
Dropping pipette
Or 1cm3 syringe plus hypodermic needle (in cover until in

use) if appropriate to students.
Permanent OHT marker, or chinagraph pencils
For marking the position of the coloured liquid.
Solvent to remove the marker

Small amount of cotton wool to wipe pipette
Stopclock
Eye protection
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!
154
Student 1 of 2
Purpose
Safety
To demonstrate the uptake of oxygen in

respiration.
To measure the rate at which an organism
respires.
Wear eye protection when handling soda lime.

Soda lime is corrosive. Do not handle directly:
use a spatula.
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
Solvent (to remove the marker)

Cotton wool
Stopclock
Eye protection
1 Assemble the apparatus as shown in the diagram below.
syringe
scale
three-way tap
glass tubing
1 cm3 pipette or glass tube
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.
37
2 of 2 Student
Measuring the rate of oxygen uptake
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.)
9 Record your results in a suitable table.

10 Calculate the mean rate of oxygen uptake during the 5 minutes.
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
A very simple respirometer
4 Design an experiment to investigate the effect of different temperatures on the rate of

oxygen uptake in maggots. Remember that the maggots will need time to acclimatise to
each new temperature.
38
Purpose
Safety
To demonstrate the uptake of oxygen in

respiration.
To measure the rate at which an organism
respires.
Wear eye protection when handling soda lime.

Soda lime is corrosive. Do not handle directly:
use a spatula.

Rate of respiration is not a learning outcome in the specification. The respirometer shown in
the diagram is a very simple one; more complex ones (e.g. U-tubes) can be used if available.
Question 1 can be used as a summary activity for student understanding of respiration if
students are asked to complete it in as much detail as possible and to state the source of
carbon dioxide as well as its fate.
The choice of what respiring organisms to put in the tubes is left to you. Germinating peas,
maggots or woodlice are commonly used.
If you are using pipettes there is no need to do any volume calculations; students just read
the change in volume off the scale on the pipettes. If you are using thick-walled capillary
tubing it is worth reminding students of the formula for working out the volume of oxygen
used, and explaining it again.
volume of oxygen used 5 pr2 3 distance moved
where r 5 the radius of the capillary tubing or pipette.
Three-way taps can cause confusion with some students. A diagram of how the three-way
tap works is shown in the diagram below. Putting up an OHT of this diagram during the
practical can help.
1
respirometer open
to syringe
respirometer open
to atmosphere
Tap positions for a three-way tap (viewed from the side)
16
respirometer
isolated
Measuring the rate of oxygen uptake
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).
17
Technician 1 of 2
Practical 7.2 Investigating breathing
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.
155
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.
Notes
Spirometer
If the spirometer has a pen it is worth checking that it is still

working they fail quite quickly. Always keep the ink reservoir
with its piece of wire in place. Some new spirometers can be
set up to feed the data into a computer. See notes on Teacher/
Lecturer sheet about using spirometers with dataloggers.
Safety
See notes on page 1.
Kymograph
See CLEAPSS Laboratory Handbook Section 15.1 for advice on

speeds, etc.
A chart recorder, computer or datalogger can be used in place
of a kymograph.
Kymograph paper
Be aware that it matters which way up you attach the paper.

If it is the right way up the pen moves smoothly over the join
in the paper. If it is the wrong way up it catches on the join.
Soda lime for the spirometer canister
Safety
See notes on page 1.
Disinfectant solution, ethanol or fresh 0.1% hypochlorite

followed by rinsing with water
For rinsing mouthpiece and breathing tubes after use.

To minimise an unpleasant taste on the mouthpiece, use
Milton made up at the manufacturers dose. However, this
takes 30 minutes to achieve disinfection.
Eye protection
156
Student 1 of 4
Purpose
Safety
To investigate lung volumes

and rate of breathing.
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. If you have
breathing or circulation (heart) problems or suffer from epilepsy
you should not use the spirometer. Read the manufacturers
instructions and safety notes before using the equipment.
Stop using the spirometer at once if you experience any unusual
breathing problems or feel 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.
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.
39
2 of 4 Student
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.
Finding the vertical scale

The vertical scale measures the volume of air in the chamber. The spirometers floatingchamber lid has markings on it showing how much gas it contains.
1 First, empty the chamber completely and, if using a kymograph, make a mark on the
paper while it is stationary, to show where the pen lies when there is no gas in the tank.
Then force a known volume of air into the tank (e.g. 500cm3) and make a second mark
on the kymograph trace.
2 Repeat this procedure until the chamber has been completely filled with air. If using a
kymograph, if the trace is too large or too small, the length of the arm supporting the pen
can be adjusted so that the trace fits onto the paper.
3 Write the values next to your calibrating marks they will help with interpretation of the
trace later. Once the marks have been made on the paper it should be possible to count
how many squares on the trace represent 1dm3.
Finding the horizontal scale

4 On most kymographs there is a switch allowing you to set the speed at which the drum turns.
Choose a speed close to 1mm per second. This is your horizontal scale. Make a note of the
speed on your trace, so that the trace can be interpreted once the experiment is complete.
Collecting data on breathing

5 After calibration, the spirometer is filled with oxygen. A disinfected mouthpiece
is attached to the tube, with the tap positioned so that the mouthpiece is connected to
the outside air. The subject to be tested puts a nose clip on, places the mouthpiece in
their mouth and breathes the outside air until they are comfortable with breathing
through the tube.
40
Student 3 of 4
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.
Collecting data on rate of respiration

Each time we take a breath, some oxygen is absorbed from the air in the lungs into our
blood. An equal volume of carbon dioxide is released back into the lungs from the blood.
When we use the spirometer, each returning breath passes through soda lime, which absorbs
the carbon dioxide so, with the canister in place, less gas is breathed back into the spirometer
41
4 of 4 Student
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.
42
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.

Effects of exercise on breathing rate and volumes can be investigated using the spirometer.
Although this is not detailed on the activity sheet, students do need to be able to describe
how to investigate the effects of exercise on tidal volume and breathing rate.
18
Using a spirometer with a datalogger

One advantage of collecting the data electronically is that the data can be moved from the
datalogging software to a spreadsheet or a word processing program. The data collected can
then be made available to students involved in the investigation.
Sensors such as motion sensors can be used to record the up and down motion of the airfilled spirometer lid.
A motion sensor can be mounted above the large top surface of the moving lid (see below).
As the lid moves up and down the change in distance can be recorded. Make sure the limits
of the motion sensor are known and the minimum recordable distance is available when the
lid is at its closest to the sensor.
If known series of gas volumes is introduced into the spirometer, and the distance between
the motion sensor and the surface of the lid is recorded, it is possible to produce a
calibration curve of distance versus volume. Most datalogging software can convert distance
measurements recorded by the sensor to volume data.
In a similar way, a linear motion sensor (e.g. rotary motion or pendulum sensor) can be
attached to the spirometer as shown below. With a pendulum sensor, a string weighted with a
small counterweight can be run from the end of the lid over the sensors pulley wheel. When
the lid moves up and down, the string moves over the pulley. The sensor records a changing
distance or angle. Calibration relating the volume of gas in the spirometer tank to the sensors
reading will be required.
rotary motion/position sensor
A
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).
19
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
20
1 cm
Spirometer trace for spirometer used with

soda lime and filled to 9 dm3 with oxygen
Volume 02(dm3)
5
Kymograph speed:
64 mm min1
Sample reference spirometer trace
21
Technician 1 of 1
Practical 8.1 Can snails become habituated to a stimulus?
Purpose
To investigate habituation of snails to a stimulus.
Notes
Giant African land snail (or large garden snail)
Giant African land snails can be purchased in some

pet shops or from Biological suppliers such as Blades
Biological.
If purchasing giant African land snails they should
be bred in this country, to reduce chance of
them having parasites. They are easy to keep
in a glass or plastic aquarium tank containing
several centimetres of compost, organic compost
is recommended. There are several publications
available about the care of land snails. If giant land
snails are not available, a large garden snail can be
used.
Snails should be kept in moist conditions before
the lesson, to encourage activity. If snails are still
inactive, they can be paced for a short time in warm
water.
Safety
Wash your hands thoroughly after handling the snail.
Equipment and surfaces should be disinfected with
1% Virkon.
Cotton wool bud

Small beaker
Any small container for water to allow cotton wool

bud to be dampened.
Board
Clean, firm surface for the snails, for example a

plastic chopping board.
Stop watch
157
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.
43
2 of 2 Student
Can snails become habituated to a stimulus?
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.
44
Purpose
Safety
To investigate habituation of
snails to a stimulus.
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.

In this investigation students find out if habituation to a touch stimulus occurs in snails.
A giant African land snail is best, but large garden snails can be used. The student sheet
that accompanies this activity contains a procedure for this experiment but students could
be asked to plan the investigation themselves. The experiment is more reliable if snails are
handled little prior to the experiment, so avoiding them becoming habituated to stimuli.
The completion of Spearmans rank correlation provides an extension to this activity. The
null hypothesis to be tested would be: there will be no correlation between the number of
stimuli the snail receives and the time taken for the snail to re-emerge.
Further investigations might include how long the habituation lasts, or how handling prior to
the experiments affects the results.
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.
22

Edexcel A2 Biology Practicals (Complete)

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Technician 1 of 1

Practical 5.1 Looking for patterns and correlations

To carry out a study on the ecology of

Teachers/lecturers must follow their LEA/school/college

A 50m tape measure. If this is not available a

A piece of rope with 0.5m intervals marked on it will also work.

Small, numbered pegs

To protect students notes if it rains.

The geography department may have these already.

The geography department may have one already.

Small plastic vials or dishes

For holding invertebrates while identifying them on site.

A key to organisms likely to be found in the area

The Field Studies Council produces a range of excellent guides.

To describe the aspect.

Access to an OS map of the area

To accurately pinpoint the site and for background knowledge.

For pitfall traps.

Practical 5.1 Looking for patterns

Practical 5.1 (cont.)

Looking for patterns

Completing a transect study

Comparing two sites

Practical 5.1 (cont.)

Looking for patterns

2 Collect the data.

Practical support sheet Ecological sampling

Why sample in ecology?

Practical support sheet (cont.)

Practical support sheet (cont.)

Methods of measuring abundance

Estimating animal populations

Practical support sheet (cont.)

Pitfall trap for sampling arthropods

Tullgren funnel for collecting organisms

air and arthropods

Alternatively a muslin bag of soil

Figure 3 Net and traps for sampling animals.

Practical support sheet (cont.)

Measuring abiotic factors when sampling the environment

mass of fresh soil

Soil organic matter

mass of dry soil

Practical 5.1 Looking for patterns

To carry out a study on the ecology of

Teachers/lecturers must follow their LEA/school/college

Notes on the procedure

Select a site carefully

Practical 5.1 (cont.)

Looking for patterns

Identification dont panic!

Practical 5.1 (cont.)

Looking for patterns

FieldWorks: an invaluable IT resource

Practical 5.2 The effect of temperature on the hatching success of

Brine shrimp egg cysts

100cm3 beakers (one for each temperature to be tested)

Tap water can be de-chlorinated by leaving it to stand for 48 hours.

40cm beaker of salt water

Students could be supplied with the salt water already prepared.

A range of temperatures between 5 and 35C is recommended. The

White A4 sheet of paper

Fine glass pipette

For counting larvae on the second day.