A2 Bio Practical Notes

8/10/2019 A2 Bio Practical Notes

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Edexcel practical materials created by Salters-Nuffield Advanced Biology, University of York Science Education Group.

Student 1 of 8

Practical 5.1 Looking for patterns

Observing patternsHave 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 patternsLook 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 thestudents 161section 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


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2 of 8 Student

Practical 5.1 (cont.) Looking for patterns

Completing a transect studyOne 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.

Procedure1 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 eldwork.

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 marked0

21 22 23 24 25 26 27 28 29 30

0.5m0.5m

quadratno. 6

transectcontinues

tape case


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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 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 notprovecause 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.


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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 attwo 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

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 randomnumbers 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.

Figure 1 Using measuring tapes to define a sample area.

1

2

3

4

5

1 2 3 4 5

position of 0.25m2

quadrat using randomnumbers 2, 4

Tape measure 1

Tapemeasu

re2


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Student 5 of 8

Practical support sheet (cont.) Ecological sampling

If you are sampling fixed objects within an area, for example the area ofPleurococcus

(analga) 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 samplingRandom 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 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 50 cm by 50 cm (0.25 m2)

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 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:

% cover 5hits

____________

hits1misses3100


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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 (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 0.25 m2quadrat 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 atent peg)inserted inground

holesknitting needle


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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 preventsrainfall filling trap

stick supportsoil sample

25 W bulb

60 W bulb

rod forsupporting bag

water

rubbertubing clip

beaker

glass funnel

soil sample inmuslin bag

16 mesh flour sieveground slopesaway from trapfor drainage

bait of meator ripe fruit

glass collecting tube

clear plastictube

glass mouthpiece 80% alcohol

polythenefunnel

Organisms moveaway from the heatand light, fallinginto the jar.

gauze coveringtube opening

air suckedthroughmouthpiece

air and arthropodsdrawn into tube

cork orrubber bung

specimen tubewhere arthropodscollect

jam jarsunk intosoil

This net is swept throughlow-growing vegetation,collecting any animalsin the mesh net.


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8 of 8 Student

Measuring abiotic factors when sampling the environmentAngle 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.

LightUse 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 5mass of fresh soil 2mass of dry soil

________________________________

mass of fresh soil 3100

Soil organic matterA 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 5mass of dry soil 2mass of burnt soil

________________________________

mass of dry soil 3100

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, 1 cm

3

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 1 cm3of pH indicator solution is

added and the pH recorded after the contents of the tubes have been allowed to settle.



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Student 1 of 2

Practical 5.2 The effect of temperature on the hatching success ofbrine shrimps

Brine shrimpsBrine 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 cm3beaker.

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

secondantenna

female(brownish red)

firstantenna

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 34 cm

You will need:

Brine shrimp egg cysts

2 g sea salt for each treatment

100 cm3de-chlorinated water for each

treatment 40 cm3beaker of salt water

100 cm3beakers (one for each temperature

to be tested)

Water baths or incubators (one for each

temperature to be investigated)


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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 faras 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.


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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


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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 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). Comparingthe 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


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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

IntroductionWhen 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

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.

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


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Practical 6.3 (cont.) Which antibiotic is most effective?

5 Wash your hands with soap or handwash and clean the bench again using the Virkonspray.

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


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Practical 7.1 Measuring the rate of oxygen uptake

Respirometers

Respirometers range from relatively simple pieces of equipment used in school sciencelabs 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

cm3pipette 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


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Practical 7.1 (cont.) 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 5pr23distance

moved, where r5the 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 ofA2 Biologyshow two other types of

respirometer. What advantages and disadvantages do these have compared to the oneyou 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.

sodalime

wiremesh

organism tobe studied

capillarytube

drop ofliquid

2 of 2 Student


17/2239


Practical 7.2 Investigating breathing

Using a spirometerThe 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), achart 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 goodhealth.)

A trained member of staff should use an oxygen cylinder to fill

the spirometer.

Safety

Student 1 of 4


18/2240


Practical 7.2 (cont.) Investigating breathing

Tubes run from the chamber to a mouthpiece and back again. Breathing in and out throughthe 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 CO2that 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 floating-

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 cm3) 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 1 dm3.

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 breathing5 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.

Disinfectant solution

Eye protection

You will need: Spirometer

Kymograph, chart recorder or computer

Soda lime (for the spirometer canister)

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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 inand 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 dm3.

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 5tidal volume 3rate of breathing (measured in number of breaths

per minute).

Some air (about 1 dm3) 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 respirationEach 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

vitalcapacity

tidalvolume

Volume02/dm3

Time/minutes

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chamber than was breathed in. If we breathe into and out of the spirometer for (say) 1minute, 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.

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Student 1 of 2

Practical 8.1 Can snails become habituated to a stimulus?

Touching snailsLots 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.

Procedure

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.

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, rm surface for the snails (e.g. a plastic chopping board)

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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 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 rscorrelation 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 signicance level

the critical value

whether the null hypothesis is being accepted or rejected.

A2 Bio Practical Notes

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