EDEXCEL Advanced Level

Edexcel IAL Biology Unit

6 Summary©
List of all A-Level Biology experiments
Unit 1 experiments

1- Describe how the effect of caffeine on heart rate in Daphnia can be investigated
practically, and discuss whether there are ethical issues in the use of invertebrates. 2-
Describe how to investigate the vitamin C content of food and drink. 3- Describe how
membrane structure can be investigated practically, e.g. by the effect of alcohol
concentration or temperature on membrane permeability.
4- Describe how enzyme concentrations can affect the rates of reactions and how this
can be investigated practically by measuring the initial rate of reaction.
 Unit 2 experiments

1- Describe the stages of mitosis and how to prepare and stain a root tip squash in
order to observe them practically.
2- Describe how totipotency can be demonstrated practically using plant tissue culture
techniques.
3- Describe how to determine the tensile strength of plant fibres practically.
4- Describe how to investigate plant mineral deficiencies practically.
5- Describe how to investigate the antimicrobial properties of plants.

Unit 4 experiments
1- Describe how 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 abundance and
distribution of organisms and the measurement of abiotic factors, e.g. solar energy
input, climate, topography, oxygen availability and edaphic factors).
2- Describe how to investigate the effects of temperature on the development of
organisms (e.g. seedling growth rate, brine shrimp hatch rates).
3- Describe how DNA can be amplified using the polymerase chain reaction (PCR). 4-
Describe how gel electrophoresis can be used to separate DNA fragments of different
length.
5- Describe how to investigate the effect of different antibiotics on bacteria.

Unit 5 experiments

1- Describe how to investigate the effects of exercise on tidal volume and breathing
rate using data from spirometer traces.
2- Describe how to investigate rate of respiration practically.
3- Describe how to investigate habituation to a stimulus.
AS BIOLOGY UNIT 1 FOOD TESTS

Title: Semi-quantitative food tests (for reducing sugars).

Aim 1. To make the Benedict’s test semi-quantitative

2. To prepare different concentrations of glucose using serial dilution
3. To estimate the quantity of glucose (or % glucose) in a fruit juice solution.

By now you should be familiar with the use of the Benedict’s solution when testing for reducing and
non-reducing sugars. You should recall that the Benedict’s solution (blue) is an alkaline solution of
copper (II) sulphate (CuSO4). The aldehyde or ketone group of a monosaccharide sugar is able to
reduce Cu2+ ions to Cu+, itself being oxidised to a carboxyl (-COOH) group. A brick red precipitate of
copper (I) oxide is formed.

You should also remember that the reducing sugars include all monosaccharides, such as glucose and
fructose, and the disaccharide maltose.

The Benedict’s test can be made semi-quantitative, meaning that it is a rough estimation of the amount
of reducing sugar present in a solution. The final precipitate will appear green to yellow to orange to
red-brown with increasing amounts of reducing sugar. (The initial yellow colour blends with the blue
of the copper sulphate solution to give the green colouration.)

In this experiment, you are given a stock glucose solution of known concentration (2%). Using this
glucose solution and distilled water you should be able to create at least five different glucose
concentrations to obtain a range of colours with the Benedict’s test. Using these colour standards, the
percentage concentration of glucose in a fruit juice solution could be determined.

Requirements:
∙ 6 test tubes Method: water
∙ Test tube rack ∙ Fruit juice solution ∙
∙ Labels Benedict’s solution ∙ Stopclock
∙ Syringes – 1cm³ and 5cm³ ∙6 small beakers ∙ 1 large ∙ Measuring cylinder
beaker
∙ 2% glucose solution ∙ Distilled

1. Decide on the five concentrations of glucose that you will make for this experiment
(0.1%, 0.2%, 0.5%, 1% and 2%).
2. Label 5 beakers and 5 test tubes with the glucose concentrations.
3. Set up a water bath using the large beaker and water from the tap.
4. Make dilutions of the stock glucose solution (2%) using the syringes and distilled water; you will
require only 5cm³ of each dilution.

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AS BIOLOGY UNIT 1 FOOD TESTS

5. Use the following formula to guide you on making dilutions, and fill in the table:
C1V1 = C2V2
Where:C1 – initial concentration of the stock solution
V1 – volume of stock solution to be taken to perform the dilution
C2 – concentration of the diluted sample
V2 – final total volume of the diluted sample

Table 1 Concentrations and volume of 2% glucose to be used to make diluted solutions.

C1 (%) C2 (%) V1 (cm³) Volume of distilled water (cm³) V2 (cm³)

2 2 5 0 5

0.5

0.2
 0.1

Note: Volume of distilled water = V2-V1

6. Add 2cm³ Benedict’s solution to each of the test tubes.

7. Then add 0.5cm³ of each glucose solution and 0.5cm³ of the fruit juice to the appropriate test tubes
using a clean syringe each time.
8. Gently stir the contents to ensure mixing.
9. Place all 6 test tubes in the boiling water bath for exactly two minutes.
10. Very carefully remove all tubes from the boiling water bath and place them on the test tube rack
11. Observe the tubes carefully and record your observations in an appropriate format. 12. Remember
to describe what concentration of glucose your fruit juice contains.

Discussion Questions for Consideration

1. What is the Benedict’s test used for and how does it work?
2. What does semiquantitative mean, especially in the context of our aims?
3. What colours are you expecting to result from a semiquantiative Benedict’s test?
4. Describe your results and the trends or patterns you observed.
5. Explain your results – why did you get a range of colours, and what was the possible
concentration of your fruit juice?
6. Evaluate your procedure in terms of the variables manipulated and kept constant, precautions
you took when setting up and carrying out the experiment
7. State any assumptions you made about your results and the fruit juice.
8. What were the limitations of the experiment, as well as any errors experienced or anticipated?
9. How could the experiment be improved or be made more quantitative?

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AS BIOLOGY UNIT 1 FOOD TESTS 3

AS BIOLOGY UNIT 1 FOOD TESTS

Non-reducing sugar (Benedict's test)

Principles:

∙ Sucrose is a non-reducing sugar (not reduce CuSO4) ---> Benedict's test (-) . ∙ If it is
hydrolysed to form glucose and fructose ---> Benedict's test (+) .
∙ So sucrose is the only sugar that will give a (-) Benedict's test before hydrolysis and a (+) test
afterwards.

Steps:

∙ Test a sample for reducing sugars to be sure it does not contain reducing sugars. ∙ Boil the test
solution with dilute HCl for a few minutes to hydrolyse the glycosidic bond. ∙ Neutralise the
solution by gently adding small amounts of solid NaHCO3 until it stops fizzing. ∙ Test for
reducing sugar.

4
AS BIOLOGY UNIT 1 FOOD TESTS
∙ The higher the concentration of reducing sugar in a sample, the less time it would take for a colour
change to be observed

∙ To avoid issues with human interpretation of colour, a colourimeter could be used to measure the
absorbance or transmission of light through the sugar solutions of known concentration to establish a
range of values that an unknown sample can be compared against a calibration curve

The mass of precipitate also indicates the amount of reducing sugar present and makes the result more
quantitative.

1 – Caffeine and heart rate of Daphnia

Objective: Describe how the effect of caffeine on heart rate in Daphnia can be investigated
practically, and discuss whether there are ethical issues in the use of invertebrates.

Describe an experiment that the student could perform, using Daphnia, to confirm that
herbal tea has the lowest caffeine content.

Method

• idea of heart rate determined before treatment (in absence of caffeine) ; • idea that
daphnia need to be put into tea and allowed to acclimatise • practical detail e.g. use of
microscope ; -limit the movement of Daphnia (cotton wool ) • details of determining
heart rate described /eq ;
• ref to named controlled variable ; e.g. {source / size / age / type / eq} of Daphnia,
temperature, pH ;
• ref to {repeats /replicates} ;
• idea that heart rate of daphnia determined in {white tea (only) / known caffeine
concentration} ;
• reference to use of range of caffeine concentrations

Suggest how the student should position the Daphnia and the thermometer in order to
obtain valid results.

1. Daphnia positioned so heart visible;

2. idea of immobilising Daphnia
3. thermometer (bulb) submerged in pond water ; idea that thermometer positioned close
to Daphnia

Explain the effect of temperature on the heart rate of Daphnia.

1. increasing temperature increased heart rate / eq ;
2. idea that Daphnia will be at same temperature as the water e.g. they are cold-blooded ;
3. increased temperature increases kinetic energy / eq ;
4. comment on rate of enzyme controlled reactions / eq ;
5. idea of increased temperature increases { respiration / metabolism / eq ; } ;

Ethical issues:
For:
Daphnia are very simple organisms / Daphnia have basic nervous system / eq ;

Against:

use of (any) animal is wrong / how can we be sure what the Daphnia can feel / ref. to
possibility that the Daphnia could die / eq ;

2 – Vitamin C content
Objective: Describe how to investigate the vitamin C content of food and drink.

Describe how an investigation could be carried out to compare the effect of storage time
on the vitamin C content of the Paraná state camu-camu fruit with those from the Amazon
region.

Method
• reference to DCPIP
• reference to use of (camu-camu) juice ; -extract the juice
• idea of titrating juice with DCPIP ;
• correct reference to colour change e.g. from blue to {colourless / pink} ; • use of
calibration curve to determine vitamin C concentration , comparison of volumes of
DCPIP added to each / calculation of vitamin C concentration against known vitamin
C solution} ;
• reference to procedure being repeated at (regular) time intervals e.g. everyday ; •
reference to replication ;
• description of one controlled variable ; -volume / concentration} of {DCPIP / extract} •
reference to drawing graph of both sets of results ;
• REPEAT

Vitamin C acts as a reducing agent

3 – Temperature, alcohol & membranes

Objective: Describe how membrane structure can be investigated practically, e.g. by the
effect of alcohol concentration or temperature on membrane permeability.

Describe an experiment you have carried out to investigate the permeability of cell
membranes.

Method

• appropriate tissue named e.g. beetroot ;

• reference to {washing / soaking} {beetroot/ eq} (thoroughly) ;
• reference to waterbath (to maintain / change temperature) ;
• reference to {range / at least 5] {temperatures / alcohol concentrations} ; • appropriate
controlled variable named e.g. length of time, size of beetroot ; • indication of what is
being used to judge permeability colour of solution, absorbance, transmission ;
• description of how permeability can be assessed e.g. use of colorimeter, standard
solutions ;
• reference to repeats / replicates ;

Control variables in Beetroot

1. reference to pre-treatment e.g. rinsing method

2. {size / mass / surface area / volume / shape} of beetroot
3. beetroot storage conditions
4. source: {same / type / species } beetroot
5. {age of beetroot / storage time}
6. (incubation) time
7. {volume / concentration } of {water / solution}(added to beetroot)
8. pH , temperature
 1. same {wavelength / filter}

There was some red coloration in the tube containing only water. Suggest an explanation
for this.

1. {cells / membranes / eq} damaged (by cutting up of pieces)

2. (as a result pigment) could leak out of {vacuoles / cells}

Describe what the student should have done to reduce the red coloration in the tube
containing only water.

• rinse pieces (thoroughly) /dab pieces dry

Increased ethanol concentrations, increases intensity;

1. reference to {disruption } of membrane

2. ethanol is a (non–polar / organic) solvent
3. idea that {lipids } dissolve (in alcohol)
4. idea that increase in ethanol causes solution to be less polar
5. idea that orientation of phospholipids depends on water around it Explain

the effect of ethanol on the permeability of beetroot cell membranes.

1. idea that ethanol causes the membrane to be {disrupted / eq} ;

2. idea that this is due (phospho)lipids dissolve in ethanol
3. idea that (membrane) proteins denatured by ethanol
4. comment on the disruption of the vacuole membrane
5. idea that {betalain / pigment} can escape from the {cell / vacuole /eq } when the
membrane is disrupted

Increasing temperature increases the release of chloride ions from the cells of the carrot.

• reference to diffusion
• idea of rate (of diffusion) increases with temperature
• idea of increase temperature gives an increase in permeability of membrane •
idea of damage to membrane above 50 o C
• idea of damaged membrane described e.g. phospholipids disrupted, proteins
denature(d)
• above {60 / 61} o C equilibrium is reached / no longer a diffusion gradient / eq ;

4 – Enzyme concentrations and rate

Objective: Describe how enzyme concentrations can affect the rates of reactions and how
this can be investigated practically by measuring the initial rate of reaction.

The action of lipase can be investigated using a triglyceride as the substrate. Describe an
experiment, using lipase and a triglyceride, that could be carried out to collect data to plot
a graph

Method
1. reference to use of a range of substrate (triglyceride) concentrations ;
 2. idea of mixing (enzyme and substrate) ;
3. identification of a suitable dependent variable e.g. pH ;
4. description of how to measure the dependent variable e.g. use of pH indicator
; 5. reference to measuring time ;
6. description of how to calculate (initial) rate of reaction ;
7. idea of repeating experiment without the enzyme ;
8. idea of control of enzyme (lipase) concentration
9. reference to one other named controlled variable (e.g. temperature, type of triglyceride,
volume of solutions) ;
10. reference to {replicates / repeats} (using the same triglyceride concentration) ;

Describe how the catalase activity in two different types of mussel can be
compared. 1. reference to measuring volume of oxygen ;
2. description of how to measure the dependent variable -suitable reference to time e.g.
oxygen produced in unit time, time taken to produce same volume of oxygen ; 3. idea of
measuring the initial rate of reaction ;
4. reference to controlled variable in relation to the mussel e.g. age, part of mussel, mass,
surface area
5. reference to a controlled variable in relation to the experiment e.g. volume of hydrogen
peroxide, temperature, concentration, pH ; - description of how variable controlled ; 6.
suitable reference to repeats ;
7. idea of same volume of enzyme

Explain why it is necessary to measure the initial rate of reaction when investigating the
effect of enzyme concentration on the rate of reaction.

1. idea that there should be enough substrate molecules to saturate the enzyme
; 2. (to ensure that) substrate is not a limiting factor/ eq ;
3. {fastest / highest} rate / decreases after initial rate / eq ;
4. as reaction proceeds substrate concentration decreases / eq ;
5. as substrate gets used up {by enzyme / in reaction / eq } ;
6. substrate concentration should be constant (in each test) / eq ;
Explain the effect of changing enzyme concentration on the initial rate of
reaction. 1. idea that enzymes reduce activation energy ;
2. reference to active sites (of enzyme) ;
3. reference to effect on collisions between enzymes and substrates / enzyme substrate
complexes /
4. idea of number of active sites occupied ;
5. (levels off when) substrate becomes limiting factor ;

In this investigation, the substrate concentration was a factor that was kept constant.
Suggest two other factors that should be kept constant. For each factor, state how it can
be kept constant.

• pH ; buffer ;
• temperature ; water bath ; not room temperature
• time of reaction ; stopwatch ;
• volume of {enzyme / substrate} ; not amount measuring cylinder / pipette ; •
type of enzyme ; same batch of enzyme ;

5 – Investigating mitosis
Objective: Describe the stages of mitosis and how to prepare and stain a root tip squash in
order to observe them practically.

Method

• Treat with HCl to soften the tissue

• Rinse in distilled water and Name of the stain; toluidine blue
• Detail of removal of suitable region of root tip 1.5 mm (meristem)
• Break up tissue with rod/ maceration
• Allow time for staining
• Cover with cover slip
• Place in folded filter paper
• Apply firm pressure
• Avoid lateral movement of cover slip

In root tip squash sample is squashed between the slide and coverslip

Safety precautions:

• { safety goggles / safety glasses / gloves } when handling { acid / stain }

• care (with scalpel) when cutting root tip
• care with slide when squashing root tip

Describe how you would prepare an agar plate that would produce this result, using a
sterile Petri dish, sterile nutrient agar, a pure culture of a suitable bacterium in a bottle
and some garlic extract.

1. description of plate pouring

2. description of putting bacteria into molten agar and swirl / mix
3. description of how garlic extract added (well, filter paper)
4. incubate (at stated temperature,<35) ;{for appropriate time 1-3 days / upside down} 5.
credit description of aseptic precaution e.g.flaming neck of bacteria bottle / swabbing
bench at beginning / sterilising {loop /spreader} / using (lit) Bunsen (flame) at back/ 6.
any reference to reducing biohazard described e.g. pipette in disinfectant / autoclave
used eqpt./ swabbing bench at end / appropriate taping of lid (i.e. not all round)

Cell cycle – Revision

Cell cycle is the sequence of

events making up cell division.
One cell cycle of a eukaryotic cell
can be described as:
 • Interphase (G1, S, G3)
• Mitosis
• Cytokinesis

Interphase
Interphase is the period of non-division
in the cell cycle
and is further distinguished
into three stages:

Mitosis (PMAT)
Prophase:
The

chromosomes coil and condense

by a process of supercoiling. Towards the end of
prophase, it is possible to see that they consist of
two chromatids held together at the centromere.
The nucleolus gradually disappears and the
nuclear membrane breaks down. The centrioles move to opposite poles and
begin to form the spindle.

Metaphase: Spindles have now pulled the chromosomes

into the centre of the cell, where they line up on the
equator line.

Anaphase: The centromere of each chromosome

divides. The spindle fibres attached them shorten,

Hasan SAYGINEL HS 9
resulting in two chromatids being pulled by their centromeres to opposite poles of
the spindle. Once separated chromatids are referred to as
chromosomes back again.

Telophase: A nuclear membrane reforms around

both groups of chromosomes at the opposite ends
of the cell. The chromosomes decondense by
uncoiling, becoming chromatin again. One or more
nucleoli reform in each nucleus.

Cytokinesis
Cytokinesis is the division of the cytoplasm to form two daughter cells during cell
division. Vesicles from the Golgi apparatus are involved in cytokinesis of both
animal and plant cells.

In animal cells, a cleavage furrow (contractile fibres) develops in the middle of the
cell. Contraction of this cleavage pinches the cytoplasm in half. As this happens,
cell organelles become distributed between the two developing cells.

In

plant cells,
6 – Plant tissue cultures
Objective: Describe how totipotency can be demonstrated practically using plant tissue
culture techniques.

Describe how you could use a plant tissue culture technique to show totipotency in cotton
plant seedlings.

Method

• idea of using part of the seedling

• idea of using agar
• (agar contains) growth substances / hormones
• Idea of using aseptic technique
• Idea of covering the top of the container to prevent contamination by
bacteria🡪pathogenic bacteria
*OR loss of water;to maintain humid conditions🡪less water available for growth of
plant
• Idea of supplying light
• allow a suitable length of time for growth e.g. 1 to 6 weeks
• look for { roots / leaves / (complete) plant } forming

In this investigation, all the seedlings were grown from seeds from the same wheat plant.
Suggest why this would improve the validity of the results.{comparisons} that are valid ;

• {less/reduced} genetic variation/ reduced effect of genotype

• seeds are the {same age / produced under the same conditions}
7 – Tensile strength of plant fibres
Objective: Describe how to determine the tensile strength of plant fibres practically.

Suggest how you would use this apparatus to enable a valid comparison of the tensile
strength of fibres from two different plants.

Method

1. two different fibre variables taken into account e.g. length, width, age, mass,
hydration level, part of plant extracted from
2. environmental variable controlled, e.g. temperature, humidity
3. named procedural variable controlled e.g. size of masses used, retting method used
to extract fibres
4. idea of adding masses until fibre breaks /measure the mass [ that breaks the fibre}
5. repeat and find the { mean / average }
6. reference to action taken in case of { anomalous result / outlier } ;repeat or remove
7. description of how tensile strength calculated, e.g. conversion of mass to force or
reference to force divided by cross sectional area of fibre
8. reference to safety procedure

Give two variables to be controlled in this investigation. Describe how they could be
controlled.

• length of fibre ; 2. suitable measuring device so all the same

• cross-sectional area / diameter / radius / thickness of fibre ; 4. select after measuring
with micrometer
• humidity ; 6. chamber with suitable solution, which could just be water, to keep them
all the same
• temperature ; 8. {chamber / water bath} kept at {stated / fixed} temperature •
soaking time ; 10. use of stopclock for {stated / same} time
• 11. nature of fibre ;12. same {species / plant / location in plant/ age / eq

Suggest why all fibres were kept at the same temperature.

⮚ temperature is a variable e.g. results reliable, same effect on structure of fibre;

Suggest why increasing the mass by 50 grams each time, rather than 100 grams, could
increase the accuracy of the student’s results

idea that break mass would be to the nearest 50 grams (rather than 100 grams) / reference
to smaller percentage error
Suggest and explain a reason for the difference in the results obtained.

• second fibre had { less tensile strength / greater elasticity }

• different fibre { size /content / source } OR different environmental conditions e.g.
temperature or humidity
‘The length of the fibre has no significant effect on the tensile strength of the fibre.’
Explain how the information in the graph supports the statement.

• Only results for white coir fibres support the statement

• idea of no (significant) difference between { 5mm } and { 35mm} white fibres •
{ error bars / ranges / results } overlapping for white coir fibres
• use of data to illustrate overlap; eg for 5mm 180 is higher than 175 for 35mm

8 – Plant mineral deficiencies

Objective: Describe how to investigate plant mineral deficiencies practically.

The effect of varying nitrate ion concentration on the growth of one plant species can be
investigated in a laboratory. Describe how this investigation can be carried out to produce
reliable results.

Method

• (description of how to vary the independent variable) Idea of at least 5 different nitrate
(ion) concentrations
• Reference to repeats at each concentration
• (measuring of dependent variable) Increase in {length/mass/ height} • use plants that are
genetically {similar / same} / same age /same original {height/ size / mass} of plant; use
cuttings or clones
• time allowed for growth { weeks / months }
• Controlling abiotic factors:

▪ time (at least a week) allowed for growth

▪ other mineral ions constant
▪ temperature
▪ light (intensity)
▪ water provided
▪ pH of {solution / soil}
▪ CO2 concentration

• idea of control described, e.g. no nitrate/ soil with no extra nitrate

Suggest two factors, other than the time for growth and the source of the seeds, that
should have been kept constant in this investigation.

• volume of solution
• light
• temperature; initial status of seedlings e.g. height ; concentration of other mineral
ions; pH

Suggest why sweet potato plants need nitrate ions

• amino acids OR proteins

• nucleic acids / (organic) bases / DNA / ATP
 • (amino acids) for the synthesis of proteins, (proteins) as enzymes, (bases) for
synthesis of DNA, (nucleic acids) for cell division, (ATP) as an energy source
‘The optimum level of nitrate fertiliser for sweet potatoes is 30 kg per hectare.’ Suggest
reasons why this statement may not be valid.

• idea that { 3 / 4 / few } different levels of nitrate tested

• no indication of repeats { at (each level of nitrate) to calculate an average } •
intervals between values large / use narrower intervals
• optimum may have been between {0 and 30 / between 30 and 60 / between 0 and
60}
• other factors not controlled

Describe and suggest an explanation for the effects of a deficiency of magnesium and
nitrate on the growth of radish seedlings in this investigation. (6)

• {total / shoot / root / all eq} dry mass(es) less than all minerals (once only);

Magnesium:

• 2. credit comparative use of figures e.g. root growth down by 110 mg / shoot growth
down by 157 mg / total growth down by 167 mg ;
• 3. reference to needed to make chlorophyll ;
• 4. reference to effect on photosynthesis ;

Nitrate:

• 5. credit comparative use of figures e.g. root growth down by 64 mg / shoot growth
down by 94 mg / total growth down by 158 mg / eq ;
• 6. reference to needed for amino acids / protein / DNA / chlorophyll ; effect on
{metabolism / growth

State two variables, other than seed mass, that need to be controlled in this investigation.
For each variable, describe how it could be controlled.

• light intensity / wavelength 🡪 light bank / eq / use same bulb ;

• temperature 🡪 environment chamber / room /sensible use water bath ; •
carbon dioxide level 🡪 {cylinder/ enclosed chamber} / eq ;
• humidity 🡪 reference to enclosed chamber ;
• wind speed 🡪 reference to {enclosed chamber / fan} ;
• {source / variety / age / ref genes} of seed 🡪 from same packet / (parent) plant / eq ; •
pH (of mineral solution) 🡪 Use buffer
• Same concentration ((I) amount / volume) of all other minerals / minerals not
omitted 🡪 Use same concentration as in complete solution each time ;

9 – Antimicrobial properties of plants

Objective: Describe how to investigate the antimicrobial properties of plants. An
investigation was carried out to extract antimicrobial substances from black pepper.

Antimicrobial properties: ability to kill bacteria

Agar-can be the source of nutrient

Describe how the bacteria should be added to the Petri dish

• aseptic technique (used to prevent contamination of plate) e.g. use of sterile

equipment(pipette)
• idea of uniform spreading of bacteria e.g. lawn, spread (over agar), mixed in with
molten agar

Suggest one safety reason for covering the beaker with clear plastic film.

• idea of preventing { bacteria / fungi} FROM {contaminating / escaping / entering} •

reference to {harmful / pathogenic } {microorganisms } ;

Explain why the lid was secured in this way

• reduces contamination (of culture)

• allows { aerobic conditions / entry of air / entry of oxygen} / prevents anaerobic
conditions
• oxygen is required for respiration by bacteria
• reduces {growth} of {harmful / anaerobic} bacteria being {cultured } Suggest one

reason, other than for safety, for covering the beaker with clear plastic film.

• allowing light in (for photosynthesis) / reducing water loss / prevent entry of

organisms (that would affect plant growth)

Suggest why an incubation temperature of 37°C should not be used in a school or college
laboratory.

• encourages growth of bacteria that are {harmful / pathogenic } (to humans) •

optimum temperature for {growth of bacteria / enzyme activity }

Suggest which concentration of garlic extract has the strongest antimicrobial properties.

• 100% /full strength

• largest zone of inhibition; *no bacteria { growing / present } in clear area •
means most bacteria {killed / not reproducing / prevented from growing }
• faster diffusion at higher concentration

Control: disc {soaked (only) in water / 0% extract }

The discs were sterilised by being placed in alcohol and then left to dry before being soaked
in the extract. Suggest why the discs should be sterilised before being soaked in the extract.

• so no { bacteria/ fungi / microbes } (alive) on them / prevents contamination by microbes

• that could be {harmful / pathogenic } to be cultured
 • prevent competition between bacteria
• makes investigation valid

If not dried; 1.reference to increase in zone of inhibition ; alcohol would have killed {the
bacteria in the plate} / alcohol is antimicrobial OR reference to decrease in zone of inhibition
;extract can be{ diluted / effectiveness reduced by the alcohol }

An investigation was carried out to compare the antimicrobial properties of jambolan with
those of guava. This showed that jambolan was effective against more species of bacteria
than guava. Describe how this investigation would have been carried out to produce reliable
data.

1. extracts made using fruit of the same { mass / age } / extracts have same
concentration of fruit
2. extracts made using same solvent
3. same volume of extract tested on the bacteria / same diameter of wells in agar / same size
paper discs
4. idea of same species of bacteria used
5. incubated at the same temperature and for the same length of time / 25-30°C and 24h
6. zones of inhibition measured - (diameter of the clear zone)
7. replication qualified e.g. { repeats for each fruit extract / repeat the experiment / repeats to
calculate mean }

10 - Investigating ecosystems
Objective: Describe how 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 abundance and
distribution of organisms and the measurement of abiotic factors, e.g. solar energy input,
climate, topography, oxygen availability and edaphic factors).

Studying ecosystems is a crucial issue and a difficult task. Unlike a highly controlled
laboratory investigation, ecological investigations often involve many variables that cannot
all be adequately controlled. This means they need even more careful planning and cautious
interpretation.

Two important variables which ecologists investigate are:

Abundance of organisms: Number of individuals of one species in a particular area.

Distribution of organisms: Where a particular species is within the area

investigated.

When ecologists study habitats, they try to account for plant and animal abundance and
distribution, correlating them to the abiotic and biotic factors affecting the habitat. Abiotic
means non-living and examples of abiotic factors include light intensity, slope, humidity,
wind exposure, edaphic characteristics such as pH and soil moisture, and many more. Biotic
means living and examples of biotic factors include competition, grazing and predation.

Estimating population size and distribution in a habitat

It is important to obtain accurate data about what organisms are present in a habitat and
what is the pattern of their distribution. This will give you many clues about the complex
interrelationships within this habitat and the whole ecosystem. Ecologists use several
methods of actually recording abundance, which are selected according to the organism
being counted. The methods chosen must be both reliable and practical.

Quadrats

Quadrats are used for sampling plant communities and slow moving or stationary animals.
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 and may be subdivided into 25
smaller
squares, each 10 cm by 10 cm. The abundance of
organisms
within the quadrat is estimated. Quadrats may be placed
across the site to be sampled using random or systematic
sampling methods.
• It is important to sample enough quadrats to be representative of the site. 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 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:
 %"#$%& = ℎ)*+
ℎ)*+ + -)++%+× 100
Methods of assessing abundance

Individial counts: The simplest method to use provided numbers are reasonably low and
each individual is easily distinguished.

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-2)/

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 m2 quadrat frame, the percentage frequency
would be 40%.

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.

ACFOR scales: These are used for more approximate assessments where abundance is
measure on a five-point scale. ACFOR data are useful in displaying comparative overall
patterns but cannot be used statistically.

Sampling

It is normally impossible to measure everything in a whole habitat or ecosystem, therefore

samples of smaller areas are taken from which inferences can be drawn. This is useful
because it makes the investigation possible, but the final answer reached is just an estimate.
Sampling can also introduce bias.
• Random sampling

Having decided upon the method of actual counting, next step is to sort out how to take
samples. Habitats are so varied that you must select the most appropriate type of sampling
according to the area you are studying and the hypothesis you are investigating.

One of the most common approaches is to compare two fairly large areas to discover if there
are any significant differences between them. The steps are as follows:

⮚ A map of the habitat is marked out with gridlines along two edges of the area to be
analysed. Alternatively, tape measures put on the ground at right-angles to each other
can be used to mark out a sampling area.
⮚ Coordinates for placing the quadrats are obtained as sequences of random numbers,
using computer software, or a calculator, or published tables.
⮚ Within each quadrat, the individual species are identified, and then the density,
frequency, cover, that is the abundance of each species, is estimated.
⮚ Abundance estimates (density, frequency, and cover) are then quantified by measuring
 the total area of the habitat in square meters.

1#1234*)#5 +)7% = -%45 1%& 8249&4* × *#*43 4&%4

4&%4 #: %4"ℎ 8249&4*

• Systematic sampling

Random sampling may not always be appropriate. If conditions change across a habitat, for
example across a rocky shore, 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. Quadrats or point frames are placed at regular
intervals along the lone to make the required counts.

⮚ If the transect is short then you might use intervals where the quadrats touch each
other- this would form a continuous belt transect.
⮚ For much longer transects you would select intervals that give you a sensible number of
quadrats to assess but do not miss any obvious ecological changes along the line. This is
an interrupted transect.

Estimating animal populations

Quadrats cannot be used for mobile animals as these don’t stay in 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. Those in vegetation can be sampled using a pooter directly or
indirectly.
Mark-release-recapture

A known number of animals are caught, marked in some way, and then released back into
the community. Later, a number of individuals are collected randomly and the number of
marked individuals is recorded.

;#1234*)#5 +)7% = <*#*43 :#& *ℎ% :)&+* +4-13% × <*#*43 :#& *ℎ%
+%"#59 +4-13% < #: -4&=%9 )59)$)9243+ &%"41*2&%9

Measuring abiotic factors when sampling the environment

Assessment of the relationship between an abiotic factor and distribution of
the organism

• Plot graph of numbers of organisms against abiotic factors

• Investigate the correlation
• Use statistical analysis
• Use Spearman’s rank correlation test
Common questions

1-) Comparing the distribution of a plant on two different parts of land

- Measuring off two areas of the same size

- Use of a quadrat
- Use of random sampling
- Method of generating random coordinates e.g. computer program -
Estimation of abundance e.g. number of plants, percentage cover
- Use of several sample sites
- Method of recording quantitative data e.g. tally chart, table, graph

2-) Investigating the distribution of aquatic animals

- Taking sample of water / kick sampling / using a net

- Details of sampling method; same volume of water, kick sampling for same length of
time
- Systematic sampling / sampling at regular intervals along the river -
Counting the numbers of aquatic animals
- Measuring other abiotic factor; temperature / width / depth
 - Method of recording quantitative data; tallying, plotting a graph

3-) Measuring percentage cover 🡪 abundance

- Use a quadrat.
- Random / systematic sampling
- Count number of squares / determine area
- Calculate the percentage cover

4-) Measuring an abiotic factor

- Use of dedicated (light, temperature) sensor

- Taking several measurements

11 – Temperature and development

Objective: Describe how to investigate the effects of temperature on the development of
organisms (e.g. seedling growth rate, brine shrimp hatch rates).

Body temperature has huge impact on the rate of development of organisms. Metabolism of
an organism is largely regulated by enzymes. As temperature affects enzyme activity, it is not
unexpected that it also affects the metabolism of an organism, thus its development.

Increasing temperature and growth

• Increase in metabolic rate / enzyme activity as temperature rises

• Increase in kinetic energy of molecules as temperature rises
• Increase in enzyme-substrate complexes / energy of collisions as temperature rises •
Inactivation at lower temperatures / denaturation at higher temperatures of enzymes
• Temperature affects differentiation / growth / division

The effect of temperature on the development of various organisms can be investigated.

The effect of temperature on the hatching success of 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.

Method

Independent variable: temperature

Dependent variable: number of hatched shrimp

Controlled variables: light intensity, pH, salt content, presence of chlorine from tap water,
oxygen concentration

- Decide on a range of temperatures from 5°C to 35°C to be used.

- Place 2 g of sea salt into a 100 cm3 beaker.
 - Add 100 cm3 of de-chlorinated water and stir until the salt completely dissolves. -
Label the beaker with sea salt and the temperature at which it will be incubated. -
Place a tiny pinch of egg cysts onto a large sheet of white paper.
- Wet the piece of paper using a few drops of salt water. Dab the paper onto the white
sheet to pick up approximately 40 eggs.
- Use a magnifying glass to count the eggs.
- Cut the paper so that there are exactly 40 eggs.
- Put the paper with 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 with the water.
- Repeat steps for all the temperatures to be investigated.
- If possible replicate the treatments.
- Incubate the beakers at the appropriate temperatures, controlling exposure to light as
far as possible.
- The next day count the number of hatched larvae in each of the beakers. To do this,
place a bright light next to each 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. Repeat the counting daily for several days.
- Record the number of larvae that have successfully hatched at each temperature.

The effect of temperature on insect life cycles

Forensic entomologists use information on life cycles to estimate the time of death. The
hister beetle is an insect that may be found on a body.

The life cycle of this beetle consists of five stages:

• Egg
• First instar larva
• Second instar larva
• Pupa
• Adult beetle

The time taken for each of these stages depends on temperature.

Method

Independent variable: temperature

Dependent variable: length of life cycles

Controlled variables: humidity, mass of food, species

- Using a range of temperatures / 5 temperatures

- In a water bath / incubator.
- Timing starts when a new stage of life cycle appears.
- And ends when the next stage appears.
- Repeat / several organisms should be used at each temperature.
- Provide food for organism.
 - Appropriate controlled variable e.g. humidity, mass of food, species. -
Total length of life cycle can be measured directly.
The effect of temperature on the growth of duckweed

Duckweed is a small plant that floats on the surface of water. It could be a source of animal
feed as it grows very quickly. Duckweed absorbs dissolved mineral ions and this decreases
water pollution. Duckweed grows by producing more fronds, which then separate into new
plants.

Method

Independent variable: temperature

Dependent variable: growth of plants (increase in the length of roots / number of

fonds) Controlled variables: concentration of inorganic ions

- Idea of using solution of ions / complete medium:

The optimum growth solution for duckweed contains all mineral ions needed that duckweed
needs at the minimum concentration.

- Using a range of / minimum of 5 temperatures.

- Different temperatures will be achieved using waterbaths / incubators. -
Determining growth over a period of time.
- Assess growth; number / size / mass of fronds / plants, length of roots -
Same concentration of each inorganic ions
- Repeats to calculate a mean / average

11 – DNA profiling
Objectives:

6- Describe how DNA can be amplified using the polymerase chain reaction (PCR). 7-
Describe how gel electrophoresis can be used to separate DNA fragments of different
length.

DNA profiling relies on the fact that, apart from identical twins, every person’s DNA is
unique. A large amount of the DNA does not code for proteins. The non-coding blocks are
called introns and are inherited in the same way as genes within the coding regions (exons).
Within introns, short DNA sequences are repeated many times. The sequences of repeated
bases are known as short tandem repeats (STRs) or satellites. The same STRs occur at the
same locus on both chromosomes of a homologous pair. However, the number of times they
are repeated on each of the homologous chromosomes can be different. The number of
repeats at a locus also varies between individuals.

There is a large amount of variation in the number of repets at each locus. These facts
combined mean that two individuals are highly unlikely to have the same combination of
STRs. It is an important feature that enables scientists to create a virtually unique DNA
profile.

Stages in DNA profiling

A- Obtaining the DNA

Extracting DNA from plant material

- The tissue sample is broken down in a buffer solution that includes a salt and a
detergent to disrupt the cell membranes.
- The small suspended particles, including the DNA, are separated from the rest of the
cell debris by filtering or centrifuging.
- Protease enzymes are incubated with the suspension to remove proteins. -
Cold ethanol is added to precipitate out the DNA.

Creating the fragments

- The strands of DNA from a sample are chopped up using special enzymes known as
restriction endonucleases. These enzymes cut the DNA at particular points in the
intron sequences. There are many different restriction enzymes, each type cutting a
DNA molecule into fragments at different sites / specific base sequences known as
recognition sites.
B- Polymerase chain reaction (PCR) – Amplifying the fragments

The PCR is used to amplify target DNA sequences that are present within the DNA source. By
this process, a large number of copies of a fragment are produced. The technique involves
mixing DNA containing the target sequence with a mixture of reagents in a plastic PCR tube
in a machine called a thermal cycler.

Method

- The DNA sample is mixed with DNA primers, mononucleotides and DNA polymerase.
This mixture is placed into the PCR machine.
- The mixture is heated to 90-95 °C. The hydrogen bonds between the DNA strands
break and this separates the strands.
- The mixture is then cooled to 55-60 °C. Primers, small pieces of DNA with fluorescent
markers, attach at the start of the STR repeated sequence.
- The mixture is then heated to 75 °C. DNA polymerases attach. Nucleotides are added
extending the DNA from the primer. The STR repeated sequence and DNA adjacent is
 replicated.
- The cycle needs to be repeated several times to make several copies of the DNA.

Note: DNA polymerase from human resources is not suitable for use in a PCR machine
because human enzymes will not work at high temperature due to denaturation.

C- Gel electrophoresis – separating the fragments

- Fragments are loaded into the wells of an agarose gel in a tank.
- An electric current is applied.
- The negatively charged DNA moves towards the positive electrode. The fragments
separate into invisible fragments.
- DNA is transferred to a nylon or nitrocellulose membrane by solution drawn up
through the gel. DNA double strands split and stick to the membrane.
- Membrane is placed in bad with DNA probe. Single-stranded DNA probe binds to
fragments with a complementary sequence.
- If the DNA probe is radioactive, X-ray film is used to detect the fragments. If the DNA
probe is fluorescent it is viewed using UV light.

D- Analysis
- Looking at number of bands.
- Looking at size of bands.
- Looking at position of bands.
DNA profiling – Summary

• Proteomics
• Use of DNA profiling
• Obtaining tissue / cell sample
• Multiple copies of DNA made
• Using PCR
• Restriction enzymes to produce DNA fragments
• Gel electrophoresis
• Loading the DNA onto the agarose gel
• Electric current is applied
• Use of dye / fluorescent staining / UV light / Southern blotting / gene probes /
radioactive labelling

13 – Different antibiotics and bacteria

Objective: Describe how to investigate the effect of different antibiotics on bacteria.

When an antibiotic is taken, it may have one of two different effects. It may be
bacteriostatic, which means that the antibiotic or the dose completely inhibits the growth of
the micro-organism. This level of treatment is usually sufficient for the majority of everyday
infections because, combined with the actions of our immune system, it will ensure that the
pathogen will be completely destroyed. However, sometimes a particular drug, or dose of a
drug given, will be bactericidal.
The purpose of this experiment is to investigate the effect of different antibiotics on a
bacterium species using aseptic techniques. For this practical you will need to work in sterile
conditions (e.g. flaming the forceps in the Bunsen after every use).

Method

Independent variable: type of antibiotic

Dependent variable: area or diameter of the zone of inhibition

Controlled variables: concentration of antibiotic, amount of antibiotic, disc size, bacterial

species, temperature

- Wash hands.
- Prepare an agar seeded with bacteria.
- Label the Petri dish on the base at the edge the date and the type of bacterium it is
inoculated with.
- Flame the forceps and then use them to pick up an antibiotic disc. - Raise the lid of
the Petri dish and place the disc firmly in the centre of the agar; if individual discs
are used they will need to be spaced evenly around the dish. - Tape the dish
securely with two pieces of adhesive tape (but do not seal it completely), then keep
it upside down at 25-30 °C for 48 hours.
- 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.
The larger the inhibition zone, the more effective the antibiotic against that species.
Evalution

- Ensuring the discs are placed evenly on the Petri dish.

- Having good aseptic technique to prevent plate contamination. - Age of antibiotic, if
the antibiotic used is out of date it is likely to be less effective. - Repeats
- Accuracy of incubation temperature and time
- The lid is not completely sealed to prevent growth of anaerobic bacteria - The
incubation temperature must be lower than 37 °C to prevent the growth of human
pathogenic bacteria.

14 – Breathing and exercise

Objective: Describe how to investigate the effects of exercise on tidal volume and breathing
rate using data from spirometer traces.
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 a kymograph (pen writing on a rotating drum). 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
floating chamber. This absorbs the CO2 that the subject exhales.

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.

The recording apparatus is switched on and at the end of an exhaled breath the tap is turned
on so that the mouthpiece is connected to the spirometer chamber. The trace will move
down as the person breathes in.

🡺 Soda lime acts as a CO2 absorber. The trace on the kymograph will start eventually
going down.
🡺 As the student breathes out, the spirometer trace rises. As the student breathes in,
the spirometer trace falls.
🡺 Water ensures a more air-tight system so no air can enter or leave without being
registered on the kymograph.
🡺 Nose clip is used so that air is inhaled and exhaled through mouth only.
🡺 The counter balance removes the resistance for breathing.

Question: A spirometer can be used to measure tidal volumes and breathing rates. Explain
how you would use the traces from a spirometer to compare the tidal volumes and
breathing rates of male and female human subjects.

⎯ idea of calibration for volume

⎯ idea of calibration for time
⎯ description of how to calculate tidal volume (from trace) / eq
 ⎯ idea that one peak = one breath
⎯ reference to breathing rate is number of peaks per minute
⎯ idea of standardised group of males and females e.g. same age, non-smokers
⎯ idea that traces taken at rest
⎯ reference to replicates
⎯ description of how to calculate the mean from the trace

Question: Describe and explain two differences that you would expect to see in the
breathing patterns of this person following exercise as a result of the training programme.

⎯ increase in tidal volume after exercise not as great

⎯ increase in breathing rate after exercise not as great
⎯ faster return to normal breathing pattern
⎯ respiratory muscles stronger
⎯ alveolar capillary network increased
⎯ gaseous exchange more efficient

Question: Explain how a spirometer trace can be used to calculate the mean resting
breathing rate of a person.

⎯ one breath is peak to peak or trough to trough

⎯ count the number of peaks or troughs in a set time
⎯ number per minute
⎯ repetition to obtain a mean or improve reliability

Question: Suggest how breathing is controlled by the nervous system in response to

changing position from lying down to sitting on a chair

⎯ more {energy / respiration /oxygen } needed

⎯ {autonomic / sympathetic (increases) / parasympathetic (decreases) / somatic}
nervous system
⎯ ref. {ventilation / respiratory / inspiratory / expiratory } centre
⎯ (in) medulla
⎯ idea of chemoreceptors (carotid / aortic)
⎯ ref. changes in {carbon dioxide / pH / temperature} (in blood) detected
⎯ ref. (motor) cortex
⎯ idea that nerve impulses go to muscles involved in breathing
*The motor cortex is the region of the cerebral cortex involved in the planning, control,
and execution of voluntary movements.

15 – Rate of respiration
Objective: Describe how to investigate rate of respiration practically.

The rate of aerobic respiration can be determined using a respirometer by measuring the
rate of oxygen absorbed by small organisms. Any CO2 absorbed is absorbed by soda lime, so
that any oxygen absorbed by the organism results in the change in the reading.

Method
Independent variable: Time

Dependent variable: Position of dye in the measuring tube

Controlled variables: number of organism, temperature, time, amount of soda lime

- Place 5g of organisms (maggots) into the tube and replace the bung.
- Introduce a drop of dye into the glass tube.
- Open the connection to the gas 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). - Mark the
starting position of the fluid on the pipette tube with a permanent OHT pen. - Isolate the
respirometer by closing the connection to the syringe and the atmosphere and
immediately start the stop clock.
- Mark the position of the fluid on the pipette at 1 minute intervals for 5 minutes. - At the
end of 5 minutes open the connection to the outside air. 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. $#32-% = >&?ℎ.
- Record your results in a suitable table. Calculate the mean rate of oxygen uptake for 5
minutes.

Simple respirometer U-tube respirometer

Evaluation
Simple respirometer U-tube respirometer

Advantage Very simple to set up. Does not need to have an

Minimal number of additional control as the second
connections makes a good tube balances out the effects of
seal easier to obtain. changes in
temperature or atmospheric
pressure.
The syringe allows you to move the
liquid in the U to reset the
apparatus.

Disadvantage Does not allow you to reset Tendency for the connections to
it. It needs a control tube leak in elderly school models.
used alongside it. Expensive.
Conclusion and the biology behind it

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 = lower pressure). Atmospheric
pressure pushes the liquid along the tube, until the pressure in and outside the tube is equal.
Oxygen is the final electron acceptor, and 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.

Describe how to find the rate of respiration of woodlice using a simple respirometer.

1. reference to constant temperature

2. use of water bath / eq

3. reference to {suitable / stated / fixed time / eq}

4. Reference to measuring {volume / distance}

5. description of how to obtain volume

6. calculation of rate described / eq

7. reference to replicates

8. description of control e.g. no woodlice

9. idea of welfare of animals important

10. reference to {mass / eq} of woodlice ;

16 – Habituation to a stimulus
Objective: Describe how to investigate habituation to a stimulus.

Habituation is a very simple type of learning that involves the loss of a response to a
repeated stimulus which fails to provide any from of reinforcement (reward or punishment).
It allows animals to ignore unimportant stimuli so that they can concentrate on more
rewarding or threatening stimuli.
Lots of people, at some time in their childhood, will have touched a snail in the garden and
noticed that it withdraws into its shell when it is either inactive or threatened. When
touched, it withdraws to avoid danger. The purpose of this experiment is to investigate
whether the snails become habituated to the stimulus, ceasing to withdraw with repeated
stimulation.

Method

Independent variable: number of touches

Dependent variable: time taken for the snail to re-emerge

Controlled variables: replication using snails of approximately same size and age, equal
handling history, drying out

- 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.
- Dampen a cotton wool bud with water.
- 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.
- Repeat the procedure for a total of 10 touches, timing how long the snail takes to re
emerge each time.
- Record your results in a suitable table.
- Plot a scatter graph, with the number of stimuli on the x-axis, and time taken to re
emerge on the y-axis.
- Carry out a Spearman’s rank correlation test to investigate the relationship
statistically.
Evaluation

- 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 this experiment 🡪 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.

Conclusion and the biology behind it

As the number of stimuli increase, the time taken for the snail, to re-emerge decreases. This
is negative correlation. With repeated stimulation, Ca2+ channels in the presynaptic
membrane become less responsive. Less Ca2+ 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.
Therefore, the snail learns that the stimulus is not causing harm, and do 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.

Describe how you could investigate the habituation of a snail to a stimulus.

1. allow snail to adjust to environment before starting / eq

2. stimulus described e.g. tapping the snail / eq

3. idea of maintaining strength of stimulus e.g. same force

4. description of what will be recorded as a response / eq

5. idea of repeating the stimulus (after response)

6. idea of repetition of experiment after time delay OR with different snail ;