Biology Core Practicals Edexcel AS Level

EFFECT OF CAFFEINE ON DAPHNIA HEART RATE

Aim: To investigate the effect of caffeine on heart rate in Daphnia.
Independent Variable: Caffeine concentration (M)
Dependent Variable: Heart rate of Daphnia (beats per minute)
Control Variables:
 Temperature – measure temperature with thermometer. Carry out in same place
 Volume of solutions – use same number of drops on Daphnia each time
 Stress of Daphnia – try to minimise stress of Daphnia (explained in Ethical
Considerations section)
 Size of Daphnia – try to pick Daphnia of around the same size for repeats
 Time to acclimatise – leave Daphnia in caffeine solution for same amount of time to
acclimatise (e.g. 5 minutes)
Ethical Considerations: Daphnia are most likely not complex enough to suffer physical and
mental stress. Nevertheless, there is still debate over whether or not animals should possess
rights as humans do. Due to a lack of consent from the Daphnia, we can instead try to
minimise the amount of suffering by considering animal welfare. The purpose of the
investigation can be justified if the Daphnia doesn’t suffer as much. We can minimise the
suffering by:
 Returning the Daphnia to their natural habitat after use
 Storing the Daphnia in conditions that replicate their natural environment
 Working at a good pace to minimise time Daphnia are under any possible stress
 Turning off the microscope lamp when not in use, as Daphnia are poikilotherms
(cold-blooded)
 Not using an excessive amount/concentration of caffeine
Why Use Daphnia? Daphnia (otherwise known as water fleas) are very common and so
there is no real threat to the species’ existence or its dependent species (via food webs).
There is also no threat to Daphnia reproduction because they reproduce asexually as
genetic clones – hence no loss of genetic variation. Daphnia possess a less developed
nervous system compared to humans, so they have a reduced awareness of pain. Finally,
Daphnia are transparent and so the heart is visible, which avoids the need for dissection.
Equipment:
 Microscope  5 different caffeine concentration
 Counter solutions
 Cavity Slide  Distilled water
 Dropping pipettes  Beaker containing Daphnia in pond
 Stopwatch water
 Tissue

Control: Count heart rate of the Daphnia when caffeine concentration is 0M (in distilled
water).
Method:
1. Remove 1 Daphnia with a pipette and place it in a cavity slide
under a microscope.
2. Dab around the Daphnia with a tissue to remove the pond
water and replace with drops of caffeine solution (e.g. 0.1M).

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Biology Core Practicals Edexcel AS Level

3. Leave the Daphnia for 5 minutes to acclimatise and then observe & count (using a
counter) the heart rate under the microscope for 30 seconds (multiply number by 2 to
calculate beats per minute).
4. Repeat this for measurements across 5 different caffeine concentrations (e.g. 0.2M,
0.3M, 0.4M and 0.5M). Repeats can then be carried out with two other Daphnia.
Results & Calculations: Make sure to present your data in an appropriate table and graph.
Work out any mean values from your repeats. The standard deviation can be displayed on
the graph using error bars.
Conclusion: An increase in caffeine concentration results in a faster heart rate (more beats
per minute, bpm). This shouldn’t come as a surprise as we know that caffeine is a cardiac
stimulant (from our GCSE knowledge). Beyond the specification and for your own interest,
in humans, caffeine binds to receptors within the heart. This blocks an enzyme called
phosphodiesterase from working with another enzyme cyclic AMP to keep the heart at its
normal rate. Therefore, heart rate increases.
Evaluation Points:
 Left for too long under microscope, temp increased (due to lamp) = increased heart rate
(random error) – turn off microscope lamp when not in use next time
 Different Daphnia showed different results due to genetic variation (random error) –
carry out more repeats with different Daphnia to get an ‘average genes’ mean value,
which is more accurate
 Too high concentration of caffeine kills Daphnia (systematic error) – use lower caffeine
concentrations
 Daphnia moving around can make it hard to count heart beats (random error) – place
Daphnia in cotton wool next time to limit movement
 Water from Daphnia extraction may have diluted the caffeine concentration (random
error) – use a micro-sieve to filter out all pond water
 Heart rate counted only for 30 seconds so may have missed a few heart beats when
calculating beats per minute (systematic error) – record heart rate for full 60 seconds
____________________________________________________________________________
MEASURING THE CONTENT OF VITAMIN C IN FRUIT JUICE
Aim: To investigate the vitamin C content in different fruit juices.
Independent Variable: Type of fruit juice
Dependent Variable: Volume of juice (cm³) required to decolourise 1cm³ of DCPIP
Control Variables:
 Temperature – measure temperature with thermometer. Carry out in same place
 Concentration of DCPIP – 1% solution used each time
 Volume of DCPIP – 1cm³ of DCPIP solution used each time
 Shake each tube same number of times (e.g. 3 times)
 Same end point colour – until the blue colour of DCPIP just about disappears
Equipment:
 1% DCPIP solution
 1% vitamin C solution
 Range of fruit juices
 Test tubes (Version 2)
 Conical flasks (Version 1)
 Pipette accurate to 1cm³

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Biology Core Practicals Edexcel AS Level

 Burette (Version 1)
Control: Use a 1% vitamin C solution to decolourise 1cm³ of 1% DCPIP solution
Method (Version 1):
1. Pipette 1cm³ of 1% blue DCPIP into a conical flask.
2. Fill up a burette with the first type of fruit juice to be used and take a
note of the start value.
3. Use the burette to slowly add the fruit juice to the DCPIP drop by drop.
Swirl the contents of the conical flask with one hand whilst controlling
the tap with the other.
4. Close the tap as soon as the DCPIP loses its blue colour and note the end value.
5. Work out how much volume of the fruit juice was needed to decolourise the DCPIP and
note this down in an appropriate table.
6. Repeat this procedure for the other fruit juices available. Repeats can be carried out 2
times to obtain mean results.
Method (Version 2):
1. Pipette 1cm³ of 1% blue DCPIP into a test tube.
2. Use an accurate pipette to add 1-3 drops of the first fruit juice to the same test tube and
then shake the mixture 3 times. Continue to add drops and shake the contents of the test
tube until the blue colour of the DCPIP disappears. Note down the volume of fruit juice
that was used up.
3. Repeat this procedure for the other fruit juices available. Repeats can be carried out 2
times to get mean results.
Results & Calculations: Results can be recorded in a table as well as a bar chart. Mean
values should be calculated from the repeats to produce a more accurate result. We can
also work out the mass of vitamin C required to decolourise 1cm³ of DCPIP along with the
mass of vitamin C present in the fruit juice samples. We know that 1cm³ of 1% vitamin C
solution should contain 10mg of vitamin C:

Mass of vitamin C to decolourise 1cm³ of DCPIP =

10mg × volume of vitamin C used

Mass of vitamin C in fruit juice sample =

mass of vitamin C to decolourise 1cm³ of DCPIP × volume of sample required to decolourise
1cm³ of DCPIP

Conclusion: We would expect to see that different volumes of the different fruit juices are
required to decolourise the DCPIP. This is due to the varying levels of vitamin C in the
different fruit juices. You should see that as the vitamin C content increases, the volume
required to decolourise the DCPIP decreases; a negative correlation.
Evaluation Points:
 Difficulty in controlling temperature (random error) – use a thermometer
beforehand to make sure it is constant and not affected if the fruit juice was
refrigerated and is cold, for example
 Too much shaking adds oxygen which will slightly restore the DCPIP to blue
(systematic error) – shake contents to mix reactants but keep it to a minimum
 Misjudgement of end point (random error) – stop adding fruit juice once blue just
about disappears. Carry out repeats to minimise the effect of misjudgement errors

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 Accuracy of measuring equipment (systematic error) – use equipment with more

precise measurements
___________________________________________________________________________
THE EFFECT OF TEMPERATURE ON CELL MEMBRANES
Aim: To investigate membrane structure, including the effect of temperature on membrane
permeability.
Independent Variable: The temperature of the water in the water bath (Degrees Celcius)
Dependent Variable: The percentage transmission of light through the resulting solution
Control Variables:
 Volume of distilled water – 10cm³ of distilled water should be used to fill the boiling
tubes each time
 Time left in water – leave each boiling tube containing beetroot for 30 minutes
 Size of beetroot piece – use a ruler and knife to cut cylindrical beetroot pieces of 1cm in
length
 Colorimeter used – same colorimeter should be used on the same blue/green setting
each time, measuring percentage absorbance. Calibration with distilled water should be
carried out each time
 Volume of beetroot solution – 2cm³ of beetroot solution should be added to a cuvette
each time
Why Use Beetroot? Beetroot cells contain pigment called betalains
in their vacuoles. We can observe the effect of temperature on cell
membranes in beetroot by observing the leakage of this pigment,
indicating the weakening of the cell membrane. Betalains display as
a dark purple colour in this case.
Equipment:
 Raw beetroot  Water baths
 Size 4 cork borer  Boiling tubes
 White tile  Thermometers
 Knife  Colorimeter with cuvettes
 Ruler  Stopwatch
 Beaker  Distilled water
 Forceps  Filter paper/tissue
 Pipette

Control: Keeping a beetroot piece in 10cm³ of distilled water at room temperature can
provide control results.
Method:
1. Use a cork borer and knife to cut 8 x 1cm lengthed cylinders of beetroot over a white
tile.
2. Place all the cut pieces in a beaker of distilled water and leave overnight to remove
any dye (betalains) released when the beetroot was cut.
3. Wash and blot dry (with filter paper or a tissue) the 8 pieces of beetroot.
4. Fill 8 boiling tubes each with 10cm³ of distilled water and place them into 8 separate
water baths of different temperatures (e.g. 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C,
70°C).

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Biology Core Practicals Edexcel AS Level

5. Once at the desired temperature, add a piece of beetroot to each boiling tube and
leave for 30 minutes.
6. Remove the beetroot pieces gently with a pair of forceps and then shake the tubes to
disperse the dye.
7. Set a colorimeter to percentage absorbance on the blue/green filter. Calibrate by
filling a cuvette with distilled water first then add 2cm³ of beetroot solution from the
first temperature to a new cuvette.
8. Place this cuvette into the colorimeter to read the percentage absorbance. Repeat
this for all other pieces.
Results & Calculations: In order to obtain the percentage transmission for each beetroot
solution in the colorimeter, we can use the following equation:
Percentage transmission = 100 – percentage absorbance

Recordings can be noted down in an appropriate table as well as a graph.

Conclusion: As temperature increased, the percentage transmission slightly increased to a

point at which it greatly increased. The betalains pigment was contained in the vacuole of
the beetroot cells. The cell membrane contains the contents of the cell like a barrier. The
cell membrane is made up of the phospholipid bilayer and protein molecules. These
proteins are made up of linked amino acids. The hydrogen bonds within the protein
determines its 3D shape. However, as heat increased, the hydrogen bonds got weaker due
to the increased kinetic energy of individual atoms. Greater kinetic energy created more
gaps in the phospholipid bilayer for the betalains to leak through. At a certain point, broken
hydrogen bonds caused proteins to change shape and denature – leaving larger holes in the
cell membrane. This is when even greater amounts of betalains leaked through the
membrane and so coloured a solution more strongly. In summary, an increase in
temperature caused further destruction of the cell membrane, which allowed more pigment
to leak out via diffusion.

Evaluation Points:

 Some beetroot may have skin on affecting surface area (random error) – use a bigger
beetroot and use cork borer to obtain pieces free from skin
 Difficulty in maintaining temperature (random error) – set water baths for higher
temperatures and set refrigerators for lower temperatures
 Accurate reading of the colorimeter (systematic error) – use more precise
colorimeter and close cap to ensure outside light does not interfere with reading.
Make sure that distilled water is used for calibration
 Accurate size of beetroot (random error) – cut many different pieces from different
beetroot and use the most similar sized pieces for the experiment
 From the different parts of the root (random error) – use a large beetroot and take
all samples from the round part of the root
 Ensuring same amount of time at the different temperatures (random error) – have
7 other helpers to make sure all 8 boiling tubes are extracted at the same time after
30 minutes
___________________________________________________________________________

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Biology Core Practicals Edexcel AS Level

THE EFFECT OF CHANGING ENZYME CONCENTRATION ON THE

RATE OF REACTION
Aim: To investigate the effect of enzyme concentration on the initial rate of reaction.
Independent Variable: Protease concentration (measured in percentage)
Dependent Variable: Time taken for enzyme to breakdown substrate (in seconds)
Control Variables:
 Temperature – measure temperature of water bath with thermometer and make
sure it is constant for repeats
 Volume of enzyme solution – 2cm³ of the protease enzyme solution will be used
each time
 Volume of substrate – 5cm³ of casein solution will be used each time
 Concentration of substrate – same concentration of casein should be used for all
repeats
Equipment:
 Range of protease (trypsin)  Water bath
concentrations (e.g. 0.2%, 0.4%,  Stopwatch
0.6%, 0.8% and 1%)  Test tubes
 Casein solution  Test tube rack
 Thermometer  Distilled water
 Marker pen

Control: Enzyme concentration of 0.0% (distilled water) can be used and added to the
casein solution. Results can be compared to this control result.
Method:
1. Set up a water bath so that the temperature can be kept constant.
2. Mark an ‘X’ on one side of a test tube using a marker pen. Fill this test tube with 5cm³
of casein solution and place it into the water bath alongside a second tube containing
2cm³ of the lowest concentration trypsin (e.g. 0.2%).
3. Allow both substances to acclimatise for 3 minutes so that at they are both at the
same temperature.
4. Add the test tube of trypsin to the casein and start the stopwatch. Time how long it
takes for the casein solution to turn transparent. This is when you can clearly see the
shape of the ‘X’ mark.
5. Repeat this a further 2 times and then repeat for the other concentrations of trypsin
(e.g. 0.4%, 0.6%, 0.8% and 1%).
Results & Calculations: Any recordings should be noted down in an appropriate table and a
graph can also be drawn. Mean values should be calculated using repeat data. The initial
rate of reaction can be calculated using the following equation:
Rate = 1 ÷ Time (seconds)

Conclusion: The initial rate of reaction is directly proportional to the enzyme concentration
because the more enzymes that are present, the greater the number of active sites that are
available to form enzyme-substrate complexes. The increase in rate will continue in this

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Biology Core Practicals Edexcel AS Level

linear fashion assuming that there is an excess of substrate. This trend is shown on the
image below.
Only a specifically shaped substrate will induce the correct
change in shape of an enzyme’s active site. The slight change in
shape of the active site enables the substrates to react. This is
known as the induced fit theory and is currently more widely
accepted than the lock-and-key theory.

Evaluation Points:
 Failure to maintain a constant temperature (random
error) – carry out reactions at room temperature.
 Identifying end point inconsistently when ‘X’ mark was hard to see (random error) –
use colorimeter method instead to measure transparency/transmittance of light
through the casein/trypsin mixture at regular intervals.
___________________________________________________________________________
OBSERVING MITOSIS
Aim: To be able to prepare and stain a root tip to observe the stages of mitosis under a
microscope.
Equipment:
 Garlic roots  Test tube rack
 Sharp knife  2 pipettes
 1moldm¯³ of hydrochloric acid  Microscope slides
 Acetic alcohol  Forceps
 Orcein ethanoic stain  Mounted needle
 Ice-cold distilled water  Filter paper
 Water bath (60 ̊C)  Microscope with x100 & x400
 2 watch glasses magnification
 Test tube
Method:

1. Place a test tube of 2cm³ HCl (1moldm¯³) into a test tube rack in a 60 ̊C water bath.
2. Cut off 1-2cm of a root tip from garlic root. Put the tip in a watch glass containing
2cm³ of acetic alcohol for at least 12 minutes.
3. Remove the tip and then place into another watch glass containing 5cm³ of ice cold
distilled water. Leave for 4-5 mins, and then remove and dry.
4. Place the tip into the heated HCl for 5 minutes then repeat the process again by
placing tips back into acetic alcohol etc. Tip: The tips will be very fragile at this point.
5. Transfer 1 tip onto a microscope slide and cut 4-5mm from the growing tip (site of
mitosis) and keep it.
6. Gently break up (macerate) the root tip with a mounted needle.
7. Add 1 small drop of orcein ethanoic stain and leave for 2 minutes.
8. Add a coverslip and blot with filter paper.
9. View under a microscope and identify the stages of mitosis.

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Biology Core Practicals Edexcel AS Level

Results & Calculations: The percentage of cells in each stage of mitosis can be observed and
noted down. A Mitotic index can also be formed:
Number of cells containing visible chromosomes ÷ Total number of cells in the field of view

Conclusion: Mitosis consists of four different sections: prophase, metaphase, anaphase and
telophase.

 Prophase:
o Chromosomes condense (chromatids joined by centromere)
o Spindle fibres join to both centrioles
o Nuclear envelope breaks down
 Metaphase:
o Centromeres attach to spindle fibres at the equator
o Chromosomes line up
 Anaphase:
o Centromeres split
o One chromatid from each chromosome is pulled to either end of the cell
 Telophase:
o Chromosomes unravel
o Two nuclear envelopes reform

Cytoplasmic division then occurs where the cell surface membrane divides to form two
daughter cells. Interphase then takes place (G1, S and G2 sub-phases). The cell cycle then
repeats.

Evaluation Points:

 Low resolution of microscope (systematic error) – use a more detailed microscope

with a greater magnification.
 Human error in counting numbers of cells (random error) – take a picture of the
image in the lens as this should make counting easier.
 Not enough time in the solutions to enable successful maceration or staining
(systematic error) – leave for 5 minutes longer in future trials.
___________________________________________________________________________
INVESTIGATING TOTIPOTENCY USING PLANT TISSUE CULTURE
Aim: To observe totipotency and tissue culture.
Independent Variable: N/A
Dependent Variable: N/A
Control Variables:
 Mass of agar used.
 Volume of distilled water added to agar.
 Size of explants.
 Source of explants.
 Time of incubation.
 Temperature – 20 to 28°C.

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Biology Core Practicals Edexcel AS Level

Equipment:
 Seeds of white mustard
 Agar
 Distilled water
 Damp sponge
 Cling film
 Short-necked test tubes or
McCartney bottles
 Weighing scales
 Plastic tray
 250ml beaker
 Glass rod
 Scissors
 Sunny window sill
Method:
1. Sprinkle seeds on damp sponge
and allow to germinate. Use when
just starting to unfold their cotyledons (seed leaves).
2. Make up Agar gel and pour 2cm height of gel into McCartney bottles and allow to
set.
3. With sharp scissors, cut the tops off just below the shoot apex (including the
cotyledons). This is called an explant.
4. Push the stem of the explant into the gel (making sure cotyledons don’t touch agar)
5. cover with cling film and place on sunny windowsill.
6. Observe over 10 days.
Results:
Explant grows roots and leaves continue to grow. You need to be able to
explain why they are covered in cling film and why they continue to grow even when
covered. Also, why they shouldn’t be opened again.
Conclusion:
It is important to seal with cling film to prevent:
 Pathogens (bacteria, fungi, etc.) from entering.
 Pathogens (bacteria, fungi, etc.) from competing with the plants for nutrients and
water.
 Entrance of microorganisms from entering and causing disease to the plants.
 Evaporation of water.
Advantages of tissue culture:
o Produces large quantities of genetically identical plants.
o New plants are disease free.
o New plants can be produced at any time of the year.
o Conserve plant species which are at risk of extinction.
Disadvantages of tissue culture:
o Reduced gene pool increases vulnerability to new diseases.
o Reduces the chances of producing new antibiotics.
Evaluation Points:
 Unwanted pathogens growing in the gel as it is a good source of water and nutrients.
 Wrong part of the plant cut and inserted into gel.
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Biology Core Practicals Edexcel AS Level

___________________________________________________________________________

THE STRENGTH OF PLANT FIBRES

Aim: To determine the tensile strength of plant fibres.
Independent Variable: The type of fibre used (from different plants)
Dependent Variable: The amount of mass that can be added before the fibre snaps
Control Variables:

 Length of fibre – each fibre should be roughly the same length for a fair comparison
 Size of each individual mass – a set of the same weights can be used
Equipment:

 Stems of stinging nettles or celery

 Bucket
 Gloves
 Paper towels
 2 clamp stands
 A set of the same type of weights (that can be added in increments)
 White tile
 Sharp knife
Method:
1. The plant material should be left to soak in a bucket of water for about a week in
order for the fibres to be easily extracted (retting).
2. Once the fibres have been removed, connect them between 2 clamp stands and
gradually add mass in the middle until the fibre snaps. Note the mass required to
snap the fibre.
3. Try this again but with individual fibres from different plants and different ways of
combining fibres (e.g. twists). You can also compare the tensile strength of the stem
to the individual fibres.
Results: You should observe that different species of plants have different tensile strengths
of their fibres. This is related to the plants ecological niche and how the plant has adapted
to it. You may also observe that more fibres bundled together results in greater strength,
requiring a greater mass to snap.
Conclusion: The strength of the fibres is thanks to the chemical nature and structure of the
cells that make up the fibre. Cellulose is a key component of cell walls and has cross-linking
thanks to strong, horizontal glycosidic bonds between glucose molecules and vertical
hydrogen bonds between neighbouring chains (forming microfibrils). A mesh of microfibrils
is then glued together with pectin and hemicellulose which allows for greater strength and
flexibility.

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Biology Core Practicals Edexcel AS Level

Lignin is a chemical found in cell walls as well which gives cells support and waterproof
capabilities. Middle lamella joins adjacent cell walls together with calcium pectate – adding
to the strength of the plant fibres. The fibres may also contain sclerenchyma fibres – these
form long tubes for strength and support, featuring lignified walls as well. All of these
features give the plant fibres great tensile strength.
Evaluation Points:

 Variation within fibres (random error) – use a large sample of fibres with repeats for
reliability. Also, try to pick plants of roughly the same age and in the same location
for validity.
___________________________________________________________________________

INVESTIGATING PLANT MINERAL DEFICIENCIES

Aim: To investigate the effect of plant mineral deficiencies on plant growth.
Independent Variable: Minerals present
Dependent Variable: Physical characteristics of the plant
Control Variables:
 Volume of mineral solution used – half a test tube will be filled with each
 Same species of the plant – Bryophyllum daigremontianum (Mexican Hat plant) used
 Size of plantlets used – roughly same length plantlet cuttings should be used for each
solution
 Amount of light received – all test tubes placed in test tube rack which is positioned
on a sunny window sill
 Same amount of time growing – all test tubes should be left for approximately one
week before examining plantlets

Equipment:
 5 Mexican Hat plantlets (roughly equal length)
 Ruler
 5 test tubes
 Test tube rack
 Solution containing all minerals
 Solution containing all minerals except magnesium ions
 Solution containing all minerals except nitrate ions
 Solution containing all minerals except calcium ions
 Solution containing no minerals (distilled water)
 Aluminium foil
 Control: Solution containing all minerals acts as positive control and solution
containing no minerals (just distilled water) acts as negative control.

Method:
1. Half fill a test tube with the solution containing all nutrients.
2. Cover the top of the tube with aluminium foil and push down on covering so that
there is a well in the centre.

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Biology Core Practicals Edexcel AS Level

3. Gently push the roots of Mexican Hat plantlet through the hole so it is in the solution
below.
4. Repeat steps 1 to 3 with the other 4 solutions.
5. Wrap all tubes in aluminium foil and place them in the test tube rack on a sunny
window sill.
6. Leave plantlets for approximately one week.
7. Observe characteristics and growth of the plantlets, comparing each solutions’
effects.

Results:
The following results should be observed:
 Solution containing all minerals – full growth and no abnormalities in characteristics.
 Solution containing all minerals except magnesium ions – leaves should appear
yellow between veins, with reddish-brown tints. Growth should be stunted.
 Solution containing all minerals except nitrate ions – yellow leaves and stunted
growth.
 Solution containing all minerals except calcium ions – growth stunted and may even
be shorter than before. Plantlet should be soft and lacking support.
 Solution containing no minerals (distilled water) – no growth should be observed,
plant should have died.

Conclusion: The characteristics of each plantlet relates to the function of each mineral ion.
Magnesium ions are used to synthesise chlorophyll molecules for photosynthesis.
Therefore, plant lacks green colour and growth is stunted as energy source becomes less
efficient. Plants use nitrate ions to build amino acids and proteins/polypeptide chains.
Proteins are largely responsible for growth and repair and so growth is reduced. The yellow
colour is a result of a lack of chlorophyll molecules due to a lack of proteins/enzymes being
made. Finally, calcium ions are used to strengthen cell walls and for membrane
permeability. Reduced support from weaker cell walls causes a ‘floppy’ stem. Reduced
metabolic activity due to reduced membrane permeability results in stunted growth.

Evaluation Points:
 Different volumes of each solution added (random error) – use a measuring cylinder
first and measure 100cm³ of each solution.
 Microorganisms possibly grew in mineral solution (random error) – heat each
mineral solution first and allow to cool before submerging plantlet root into solution.
 Insufficient time to see an effect (systematic error) – take pictures of each plantlet
every few days for 2 weeks.
___________________________________________________________________________
INVESTIGATING THE ANTIMICROBIAL EFFECT OF PLANT
EXTRACTS
Aim: To investigate and compare the antimicrobial properties of garlic and mint.
Independent Variable: Substance whose antimicrobial properties are being tested (garlic or
mint)

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Biology Core Practicals Edexcel AS Level

Dependent Variable: Zone of inhibition made by substance (area measured in cm²)

Control Variables:

 Same concentration of plant material used

 Type and amount of bacteria used – E.coli will be used as the bacteria and it will be
evenly spread across an agar medium
 Volume of plant material used for each disc – 0.1cm³ can be used per sterile paper
disc each time
 Contamination of culture – aseptic techniques and sterile equipment used to avoid
contamination of bacteria culture
 Temperature of cultures – all Petri dishes should be incubated overnight at the same
temperature
 pH of medium – an agar jelly will be used in each case with a consistent, neutral pH
Why compare the effects of garlic and mint?
The inspiration behind this practical is the fact that most kinds of toothpaste contain a mint
extract. Since toothpaste is used to remove bacteria in and around the mouth, this practical
will test the effectiveness of mint as an antibacterial by comparing it to the antibacterial
effect of garlic.
Equipment:

 3 Petri dishes where the agar is seeded with E.coli bacteria

 Garlic and mint plant material
 Pestle & Mortar
 20cm³ industrial denatured alcohol
 Measuring cylinder
 3 sterile pipettes
 12 sterile paper discs
 Sterile forceps
 Hazard tape
 Marker pen
 Ruler
Control: Four sterile paper discs soaked in distilled water can be placed on a Petri dish
seeded with E. coli bacteria. This will act as a negative control to see if the bacteria die
regardless of plant material being used.
Method:
1. Crush 3g of garlic with a pestle & mortar and use a measuring cylinder to add 10cm³
of denatured alcohol to the mixture. Shake the mixture occasionally for 10 minutes.
2. Repeat step 1 but this time using 3g of the mint plant material.
3. Pipette 0.1cm³ of the garlic extract solution onto 4 of the sterile paper discs. Allow
each disc to dry. Repeat this process for the mint extract solution.

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Biology Core Practicals Edexcel AS Level

4. Label the other Petri dishes for garlic, mint and control solutions – include the date.
5. Use the sterile forceps to place all 4 discs of each type of extract onto their
corresponding Petri dish. Close each dish and seal with hazard tape. Make sure that
a small gap is left so that oxygen can enter and there is no build-up of anaerobic
bacteria.
6. Leave the cultures to incubate overnight.
7. Open each Petri dish and use a ruler to work out the zone of inhibition for each
paper disc.
Results & Calculations:

 Use a ruler to calculate the radius for the circular clear zones around each paper disc
– this is the zone of inhibition.
 Use the equation A=πr² where r, cm, is the radius to work out the area of the zone of
inhibition in cm².
 You should observe that the mean zone of inhibition for the garlic extract is greater
than that of the mint’s.
Conclusion: The reason that the zones of inhibition for mint are greater is because it has
stronger antimicrobial properties. There is no current evidence that mint possesses
antimicrobial properties despite its component, menthol, being a mild anaesthetic. On the
other hand, garlic is a fairly strong natural antibacterial. The active ingredient in garlic is
allicin, which interferes with lipid synthesis and RNA production in bacteria. This inhibits
growth and leads to the death of bacteria.
Evaluation Points:

 Contamination of microbes (random error) – use improved aseptic techniques. Clear

and wash the area of work before and after with alcohol gel and wash hands before.
Wear sterile gloves and set up Petri dishes under a naked flame.
 Not shaking extract enough to ensure enough active ingredient (random error) – use
a centrifuge to separate and mix the extract.
 Uneven bacteria growth (random error) – ensure same lighting conditions used by
keeping cultures under a lamp.
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