A2 Bio Practical Notes

23
Edexcel practical materials created by Salters-Nufeld Advanced Biology, University of York Science Education Group.
Student 1 of 8
Practical 5.1 Looking for patterns
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 nd 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 eld or grass paths; are there any patterns?
Once you have identied 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 eldwork 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 briey 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.
To carry out a study on the ecology of a habitat.
Purpose
24
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 eld
may be very different from the rest of the eld, or the species found upstream and downstream
of an outow 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 nd
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 feldwork.
A pilot study in advance of the main data collection will help you make these decisions.
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.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
peg marked 20
origin
peg marked
0
21 22 23 24 25 26 27 28 29 30
0.5 m 0.5 m
quadrat
no. 6
transect
continues
tape case
25
Student 3 of 8
Practical 5.1 (cont.) Looking for patterns
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 signicant differences, and explain the main
relationships between species and abiotic factors using scientic knowledge. Determine
if your original hypothesis was correct. If you have suitable data, you can calculate
correlation coefcients between your biotic and abiotic data. For example, you can see
if there is a signicant 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 modications that you could make to the
procedure.
26
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 eld may be very different to that in another
eld, or the species found under oak trees may be different to those under ash trees, or
the species upstream and downstream of an outow 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
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 rst random number gives the position on the rst
tape and the second random number gives the position on the second tape.
Figure 1 Using measuring tapes to dene a sample area.
1
2
3
4
5
1 2 3 4 5
position of 0.25 m
2
quadrat using random
numbers 2, 4
Tape measure 1
T
a
p
e

m
e
a
s
u
r
e

2
27
Student 5 of 8
Practical support sheet (cont.) Ecological sampling
If you are sampling xed 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 cm
3

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 2 m across a eld,
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 50 cm by 50 cm (0.25 m
2
)
and may be subdivided into 25 smaller squares, each 10 cm by 10 cm. 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 nd 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:
% cover 5
hits

____________

hits 1 misses
3 100
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6 of 8 Student
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 (m
22
). In many plant species (e.g. grasses) it is very
difcult 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 0.25 m
2
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.
multiple hit
metal spike
(such as a
tent peg)
inserted in
ground
holes
knitting needle
29
Student 7 of 8
Figure 3 Net and traps for sampling animals.
Pitfall trap for sampling arthropods
Tullgren funnel for collecting organisms
from the soil or leaf litter
Pooter for collecting insects
Sweep net
Alternatively a muslin bag of soil
surrounded by water can be used
to collect living organisms. This
is a Baermann funnel.
flat stone prevents
rainfall filling trap
stick support
soil sample
25 W bulb
60 W bulb
rod for
supporting bag
water
rubber
tubing clip
beaker
glass funnel
soil sample in
muslin bag
16 mesh flour sieve
ground slopes
away from trap
for drainage
bait of meat
or ripe fruit
glass collecting tube
clear plastic
tube
glass mouthpiece 80% alcohol
polythene
funnel
Organisms move
away from the heat
and light, falling
into the jar.
gauze covering
tube opening
air sucked
through
mouthpiece
air and arthropods
drawn into tube
cork or
rubber bung
specimen tube
where arthropods
collect
jam jar
sunk into
soil
This net is swept through
low-growing vegetation,
collecting any animals
in the mesh net.
30
8 of 8 Student
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 inuence 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 110 C until there is no further loss in mass. The % soil moisture
can be calculated using the equation:
% soil moisture 5
mass of fresh soil 2 mass of dry soil

________________________________

mass of fresh soil
3 100
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:
% organic matter in soil 5
mass of dry soil 2 mass of burnt soil

________________________________

mass of dry soil
3 100
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 eld, 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, 1 cm
3
of soil is shaken with distilled water before adding one spatula of barium sulphate
(low hazard). This helps to occulate (settle) the clay fraction, which is important as clay particles
are very small and will otherwise cloud the water for days. Then 1 cm
3
of pH indicator solution is
added and the pH recorded after the contents of the tubes have been allowed to settle.
31
Student 1 of 2
Practical 5.2 The effect of temperature on the hatching success of
brine shrimps
Brine shrimps
Brine shrimps are small, saltwater crustaceans; the adults are about 8 mm in length. They
are relatively easy to keep in the laboratory and will produce dormant egg cysts that hatch to
produce young shrimp larvae.
Drawings to show features of brine shrimps
Procedure
1 Decide on a range of temperatures from 5 C to 35 C to be tested.
2 Place 2 g of sea salt into a 100 cm
3
beaker.
3 Add 100 cm
3
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.
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.
Purpose
1 mm
eggs
second
antenna
female
(brownish red)
first
antenna
male
(blue/green)
Stirring rod
Magnifying glass
Pair of forceps
Fine glass pipette
Bright light
Access to refrigerator
Sheet of A4 white paper
Sheet of graph paper 3 cm 3 4 cm
You will need:
Brine shrimp egg cysts
2 g sea salt for each treatment
100 cm
3
de-chlorinated water for each
treatment
40 cm
3
beaker of salt water
100 cm
3
beakers (one for each temperature
to be tested)
Water baths or incubators (one for each
temperature to be investigated)
32
2 of 2 Student
Practical 5.2 (cont.) 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
ne 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 nd out more about brine shrimps, visit the British Ecological Society website.
33
Student 1 of 1
Practical 6.1 DNA gel electrophoresis
Procedure
You may have the opportunity to complete experimental work using restriction enzymes and
gel electrophoresis or you may use the simulation of this.
To use gel electrophoresis to separate DNA
fragments of different sizes.
Purpose
If you undertake gel electrophoresis make sure
you are aware of the hazards and follow the
instructions of your teacher very carefully.
Safety
34
1 of 1 Student
Practical 6.2 DNA amplification using PCR
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 amplied 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 amplied 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.
To use PCR to amplify DNA.
Purpose
If you undertake PCR make sure you are aware
of the hazards and follow the instructions of
your teacher very carefully.
Safety
35
Practical 6.3 Which antibiotic is most effective?
To investigate the effect of different
antibiotics on bacteria.
Purpose
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.
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 nd 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
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 rmly 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.
Marker pen
Autoclaved forceps
Mast ring or antibiotic-impregnated paper
discs
Adhesive tape
Eye protection
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
Student 1 of 2
36
Practical 6.3 (cont.) 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?
2 of 2 Student
37
Practical 7.1 Measuring the rate of oxygen uptake
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
1 Assemble the apparatus as shown in the diagram below.
A simple respirometer
2 Place 5 g of maggots or peas into the test tube and replace the bung.
To demonstrate the uptake of oxygen in
respiration.
To measure the rate at which an organism
respires.
Purpose
Wear eye protection when handling soda lime.
Soda lime is corrosive. Do not handle directly:
use a spatula.
Safety
scale
1 cm
3
pipette or glass tube
syringe
glass tubing
small organisms
gauze
soda lime
three-way tap
coloured liquid
Soda lime
Coloured liquid
Dropping pipette
Permanent OHT marker pen
Solvent (to remove the marker)
Cotton wool
Stopclock
Eye protection
You will need:
Respirometer (see diagram
below)
5 g of an actively respiring
organism
Student 1 of 2
38
Practical 7.1 (cont.) Measuring the rate of oxygen uptake
3 Introduce a drop of marker uid into the pipette or glass tube using a dropping pipette.
Open the connection (three-way tap) to the syringe and move the uid 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 uid 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 uid 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 pr
2
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?
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.
soda
lime
wire
mesh
organism to
be studied
capillary
tube
drop of
liquid
2 of 2 Student
39
Practical 7.2 Investigating breathing
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-lled 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.
To investigate lung volumes
and rate of breathing.
Purpose
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 ll
the spirometer.
Safety
Student 1 of 4
40
Practical 7.2 (cont.) 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 oating
chamber. This absorbs the CO
2
that the subject exhales. In which direction will the pen move
when the subject inhales?
Procedure
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 oating-
chamber 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. 500 cm
3
) and make a second mark
on the kymograph trace.
2 Repeat this procedure until the chamber has been completely lled 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 ts 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 1 dm
3
.
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 1 mm 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 lled 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.
Disinfectant solution
Eye protection
You will need:
Spirometer
Kymograph, chart recorder or computer
Soda lime (for the spirometer canister)
2 of 4 Student
41
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.
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 0.5 dm
3
.
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 1 dm
3
) 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
vital
capacity
tidal
volume
V
o
l
u
m
e

0
2
/
d
m
3
Time/minutes
Student 3 of 4
42
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, nd 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.
4 of 4 Student
43
Student 1 of 2
Practical 8.1 Can snails become habituated 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 nd out if
habituation to a touch stimulus does occur in these organisms.
Procedure
1 Collect one giant African land snail, and place it on a clean, rm 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.
To investigate habituation of snails to a stimulus.
Purpose
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.
Safety
You will need:
One giant African land snail (or a garden snail if not available)
One dampened cotton wool bud
Suitable clean, frm surface for the snails (e.g. a plastic chopping board)
Stopwatch
44
2 of 2 Student
Practical 8.1 (cont.) 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 ndings 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 r
s
correlation test to determine if there is a statistically
signicant 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 D
2
9 Use a table of critical values to accept or reject your null hypothesis. If your calculated
Spearman rank value (r
s
) is greater than the critical value, then the null hypothesis is
rejected. If your calculated r
s
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 r
s
the number of pairs of data
the signifcance level
the critical value
whether the null hypothesis is being accepted or rejected.

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