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Practical one
Sampling techniques
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)

It is virtually impossible to identify and count every organism in a habitat. So small
sections or patches of the habitat are usually studied in detail. These areas are
called sampling areas. If, a large number of samples are studied and these are
representative of an areas as a whole, any conclusions drawn from the findings
will be valid

Quadrat sampling

Random sampling:
SystemaEc sampling used for es=ma=on of
used for es=ma=ng species diversity,
changes as we move popula=on density or
along a habitat abundance of organisms,
within a habitat

Belt transect: Line transect:

for study of distribu=on for study of distribu=on
and abundance of of organisms (no
organisms indica=on of abundance)



• A quadrat is the basic tool used for sampling of organisms with an
ecological site
• It is a sturdily wooden frame, often designed so that it can be folded to
make it more compact for storage and transport
• It is placed on the ground, the species present within the frame are
identified, and their abundance recorded.
• Where the species are small and/or densely packed, one or more of the
smaller squares within the frame may be used rather than the quadrat as a
• Sampling with a quadrat may be random or systematic.
• Quadrats should be placed randomly so that a representative sample is
taken. This increases the statistical significance of the data
• You should look at the results from several quadrats in an area to reduce
the effect of an unusual distribution
• The results are more reliable when you look at the results from many

• Quadrat size is highly variable, depending on the type of organism and size
of the habitat being studied
• Shape of the quadrat may be square, circular or triangular. However, the
square shape is the most commonly used quadrat because of the ease of
calculation and uniform size of smaller squares.


A. Random sampling
• It can be as simple as throwing the quadrat over one’s shoulder and
counting the species within it whenever it falls.
• Even with the best intentions, it is difficult not to introduce an element
of personal bias using this method.
• A better form of random sampling is to lay out two long tape measures
at right angles to each other, along two sides of the study area.
• Using random numbers generated on a computer or certain calculators,
a series of coordinates can be obtained. The quadrat is placed at the
intersection of each pair of coordinates and the species within it

Random sampling (quadrats placed at randomly generated positions):
• Used where the habitat is uniform
• Removes observer bias
• Used in a large area
• Randomness makes it statistically acceptable
• Used if time is limited

Place the quadrat where
the coordinates for the

random numbers

B. Systematic sampling
• It involves placing the quadrat at regular intervals along a transect

Systematic sampling may be
• Used to show zonation
• Used where there is continuous variation
• Used to sample linear habitats

Belt transect:
• A rope or tape measure is laid along a sampling area, which is a strip of land
known as a transect.
• An environmental gradient usually exists along the transect. For example,
the concentration of sulfur dioxide generally decreases as we move away
from the city center, or the abundance of shade tolerant plants increases as
we move from the forest edge towards the center.
• Quadrats are placed at regular intervals, 2m in this case, along the transect
• The species within the quadrats are carefully recorded. This gives a record
of species in a continuous belt along the line. This is called as a belt or
ladder transect

Belt transect:
• Produces more data, gives detail about species abundance down the line as
well as range
Measuring tape
• Shows species dominance down the line


Line transect
• A line transect is used so that systematic sampling of an area can be carried
• A string or tape is stretched out along the ground in a straight line. A record
is made of the organisms touching or covering the line all along its length,
or at regular intervals.
• This technique is particularly useful where there is a transition (change) of
plantation or vegetation and animals across the area, down a seashore for

Line transect
• Used where time is limited
• Used to visually illustrate how species change along a line

For both belt and line transects, the interval at which samples are taken will
depend on the individual habitat, as well as on the time and the effort which
can be allocated to the survey:
• too great an interval may mean many species actually present are not
• too small an interval can make the sampling time consuming, as well as
yielding more data than needed.


Methods of measuring abundance

a. Population density (the number of individuals of a species per unit area of
the ground)

Area = 1 m2
Buttercup density is 13/m

b. Percentage frequency (number of squares in which the species appears)

Percentage frequency of buttercup= (11/25) x 100 = 44%


c. Percentage cover (the percentage of the ground covered by a species)
Count the number of squares within the quadrat that the plant completely
covers, then count those that are only partly covered (at least 50%) and
estimate the total number of full squares that would be completely covered
by that species

• The squares that are marked with a blue circle represent components on
the grid which have a cover of at least 50%

% cover = number of squares covered with the vegetation/ total number in the quadrat
= 11/25 = 44%


Sampling animals

It is impossible to find and count all the animals in an area. You can get an idea of
the variety and number by taking a sample

a. Pitfall traps
• They are often used to sample the small invertebrates living on the ground.
You are likely to trap beetles and other insects, as well as spiders and slugs.
• Pitfall traps must be properly set up. The top of the carton should be level
with the soil surface. Cover the trap with a stone or piece of wood to keep
out the rain, to make it dark and to stop birds eating your catch
• The traps must be checked often to avoid the animals escaping or being
eaten before they are counted
• As with most methods, a large number of traps makes results reliable and
minimizes the effects of unusual results.

b. capture-recapture techniques
• This method of estimating the size of a population is useful for mobile
animals, which can be tagged, or in some other way marked.
• A known number of animals are caught, clearly marked, and then released
into the population again
• Sometime later, a given number of individuals are collected randomly and
the number of marked animals recorded.
• The size of the population is calculated on the assumption the proportion
of marked to unmarked individuals in the second sample is the same as the
proportion of marked to unmarked animals in the population as a whole


Estimated size of population =
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!"#$%& !" !"#$%& !"#!$!#%&'( !"#$%&'!"(

• The estimate calculated is called Lincoln index


Measurement of environmental factors

• Light: use a light meter; light readings can vary with the time of the day and
cloud cover
• Oxygen concentration: in aquatic ecosystems, oxygen probes can be used
to measure oxygen concentration
• Humidity: relative humidity can be measured using a whirling hygrometer
• Soil water: a sample of soil is dried at 100oC until there is no further loss in
mass; the % soil moisture can be calculated using the equation:
!"## !" !"#$! !"#$!!"## !" !"# !"#$
% soil moisture = x 100
!"## !" !"#$! !"#$

• soil organic matter: a dry soil sample of known mass is strongly heated in a
crucible for 15 minutes to burn off all the organic matter; the mass is re-
measured after the soil sample has cooled.
!"## !" !"# !"#$!!"## !" !"#$% !"#$
% organic matter in the soil= x 100
!"## !" !"# !"#$

• pH: pH indicator or a pH meter can be used to test pH after mixing a soil
sample with water.

Risk assessment
• Hand washing and sanitization: use of alcohol gels or other hand sanitizers
with paper towels
• Avoid sunburn: wear cap or sunscreen lotions
• Avoid injury when using and carrying tools or heavy loads of unfamiliar
equipment; wear boots or gloves to avoid injury
• Ethical issues: it is useful to consider how the act of surveying the area and
collecting plants might damage or change the environment surveyed


Core practical 2
Effect of temperature on development of organisms

A. Effect of temperature on brine shrimp hatching rate

• Independent variable: environmental temperature
• Dependent variable: number of hatched shrimp or hatch rate of
• Confounding variables:
ü pH (above 7 or alkaline)
ü salinity
ü oxygen concentration
ü light intensity
ü presence of chlorine from tap water

• Equipment:
ü Brine shrimp egg cysts
ü 2 g sea salt for each treatment
ü de-chlorinated water for each treatment
ü beakers
ü Water baths or incubators
ü Forceps
ü Bright light
ü pipette

• Procedure
ü Decide on a range of temperatures from 5 °C to 35 °C to be
ü Place 2 g of sea salt into a 100 cm3 beaker.
ü Add 100 cm3 of de-chlorinated water and stir until the salt
completely dissolves.
ü Label the beaker with sea salt and the temperature at which it
will be incubated.
ü Place a tiny pinch of egg cysts onto a large sheet of white
paper. Wet the piece of graph paper using a few drops of salt

water. Dab the paper onto the white sheet to pick up
approximately 40 eggs.
ü Use a magnifying glass to count the eggs.
ü Put the paper with the 40 eggs into the beaker (eggs-side
ü After 3 minutes, use a pair of forceps to gently remove the
paper, making sure that all the egg cysts have washed off into
the water.
ü If possible replicate the treatments.
ü Incubate the beakers at the appropriate temperatures,
controlling exposure to light as far as possible.
ü The next day count the number of hatched larvae in each of
the beakers. To do this, place a bright light next to the beaker.
Any larvae will swim towards the light.
ü Using a fine glass pipette catch the brine shrimps and place
them in a small beaker of salt water. Brine shrimps are very
delicate and care must be taken when handling them.
ü Record the number of larvae that have successfully hatched at
each temperature.

• Outcome
ü The majority of the shrimp should hatch at the optimum
temperature between 25 and 30 ̊C. (optimum at 28 ̊C).
ü Stats tests could be used to show evidence for data. Difference
(student t-test or mann whitney U) Correlation (spearman’s
rank )

• Ethical issues
ü ethics of hatching shrimp under different conditions
ü use of animals in experiments effect of light intensity

• Limitations
ü may be a difference in light in each sample fluctuating
ü not accurate salt measurements
ü may not have counted exactly 40 eggs
ü may miss seeing some of the baby shrimp


ü some eggs may not be viable anymore and wont hatch

B. Effect of temperature on seedling growth rate

• Independent variable: temperature; the temperature could be varied
by keeping the experimental setup in an incubator at different
• Dependent variable: any parameter that can measure growth;
increase in length, mass or number of leaves could be used
• Confounding variables that need to be controlled
ü Type and concentration of nutrients
ü Size of the seedlings at the start of the experiment (soak seeds of
the same plant in a beaker containing water at 25oC; then transfer
the soaked seeds to a nutrient medium to germinate under the
same temperature and water availability)
ü Oxygen availability for roots (bubble air into the nutrient
ü Competition (prevent algal growth by wrapping the glass tube
with black paper to prevent light penetration into the medium)
ü Light intensity (could be controlled by adjusting the distance
between the light source and the apparatus)
ü Duration of exposure to light intensity

Recording of raw data

Temp. oC Initial mass of seedlings Final mass of seedlings / Mean

/g g percentage
increase in
mass (%)

Trial Trial Trial Mean Trial Trial Trial Mean

1 2 3 1 2 3








(!"#$ !"#$% !"##!!"#$ !"!#!$% !"##)
Percentage increase = × 100
!"#$ !"!#!$% !"##

Presentation of the results

• Results can be presented on a line graph

Methods of data analysis
• Suitable statistical test can be used to analyze and interpret the data and
draw a valid and unbiased conclusion such as correlation coefficient

• The light bulb heats up very quickly; so avoid direct contact with the skin


Core practical 3
Polymerase chain reaction (PCR)

• Thermocycler
• DNA sample
• Taq polymerase
• Nucleotides primers
• Buffer solution

Method and outcome
• DNA sample is placed into tube in thermocycler with nucleotides, primers
and polymerase:
1. Step 1: denaturation = DNA heated for 1min at 94°C to denature it. This
breaks the H bonds between nucleotides and makes the double stranded
DNA, single stranded.
2. Step 2: annealing = temperature reduced to 54°C. Bonds form between
primers and the template strands. This will allow the polymerase enzyme to
start to copy the template.
3. Step 3: extension = carried out at 72°C. This is the optimum for taq
polymerase enzyme. The bases are placed in their correct position,
extending the strand from the primer.


Evaluation issues

• The amplification factor = 2n, where n is the number of replication cycles;
for example, if the cycle is run for 20 times then the DNA quantity increases
220 times.
The amount of DNA doubles each cycle (steps 1-3) therefore a considerably
amount of copied DNA can be made for use in DNA fingerprinting etc;
• 35 cycles (a few hrs) = 34 billion copies

Safety and precautions

• Aseptic techniques: extraction of DNA from human body cells might cause
contamination or infection; students should handle their own samples, all
the sample must be disposed off appropriately after use and any spill is
immediately disinfected

• Restriction enzymes could cause allergic reactions; avoid contact with the
skin, wear rubber gloves


Core practical 4
Gel electrophoresis

• Selected restriction enzymes
• agar gel (agarose gel)
• gel tank
• electrical supply
• micropipettes
• DNA sample
• Loading dye
• UV light
• Camera
• Buffer solution
• DNA restriction ladder

• Mix DNA with desired restriction enzyme and loading dye.
• Prepare agar and pour into electrophoresis mould.
• Once set, fill electrophoresis tank with buffer solution.
• Use micropipette to load restriction ladder into first well then DNA samples
cut with restriction enzyme into the other wells.
• Connect to electrical supply, turn on and leave until the dye has moved to
the opposite end of the gel tank.
• Switch off and disconnect electrical supply.
• Carefully remove the gel from the tank and view under UV light.
• Take picture if desired.

• DNA will be separated out through the agar gel, with the heaviest (biggest)
DNA strands near the wells and the lightest (smallest) will be at the
opposite end.
• The DNA restriction ladder can be used as a ‘ruler’ to measure the size of
the different fragments.


Core practical 5
Effect of Antibiotics on bacteria

• Independent variable: antibiotic
• Dependent variable: diameter of inhibition zone
• Confounding variables:
ü Concentration of antibiotic
ü Amount of antibiotic
ü Disc size
ü Bacterial species
ü Temperature
ü Ruler

• Equipment
ü Samples of different antibiotics on mast ring or filter paper discs
ü Petri dishes
ü Agar gel
ü Disinfectant
ü Bunsen burner
ü Forceps
ü Marker pen
ü Adhesive tape
ü incubator

1. Step one: preparation of a nutrient medium; a selective medium can be
chosen as it will allow only a specific type of bacteria to grow and will
inhibit the growth of other types
2. Step two: sterilization of the nutrient medium; the nutrient medium is
autoclaved at 121oC for 15 min at a pressure of 103 kPa


3. Step three: plate pouring
• Remove the liquefied hot agar medium and allow it to cool in water bath to
about 50oC
• Collect one bottle of sterile molten agar from the water bath
• Hold the bottle in the left hand; remove the lid with the little finger of the
right hand
• Flame the neck of the bottle; flaming the mouth of the bottle for a few
seconds is done to produce an upward flow of air from the bottle so that
any organisms in the area will not fall into the bottle; it is not done to kill
• Lift the lid of the petri dish slightly with the right hand and pour the sterile
molten agar into the petri dish and replace the lid
• Flame the bottle again and replace the lid
• Gently rotate the dish to ensure that the medium covers the plate evenly
• Allow the plate to solidify
• Seal and incubate the plate in an inverted position

4. Step four: inoculation of the solidified agar plates
• Introducing the bacteria into the nutrient medium is called inoculation
• Use a sterile pipette to transfer a sample of the bacteria (from a sample
bottle) to the surface of the nutrient agar in the petri dish

5. Step five: streaking or spreading of bacteria
• Using a sterile spreader or an inoculating wire loop to streak the surface of
the agar surface

Glass spreader Inoculating wire loop


Streaking the plate with Spreading bacteria with
an inoculating wire a glass spreader

Step six: preparing a solution of the antibiotic with an optimum concentration;

transfer a 0.1 cm3 of the antibiotic solution to an antibiotic disc; place the disc
onto the surface of the inoculated agar medium
The same procedure is repeated with few more antibiotics and place the discs
into the same petri dish
A control can be soaked in distilled water
Agar plates should only taped closed with a couple of strips of cellotape; this will
promote the growth of less harmful aerobic bacteria

Step seven: incubation of the culture medium at 25oC in an incubator; the dishes
are incubated upside down to prevent the condensation of water vapor on the
agar surface


Step eight: interpretation of the results

Measure the maximum diameter of the clear zone for each disc; use Vernier
calipers to increase accuracy of the experiment; take three measurements in
different directions for each disc and find the mean diameter


Core practical 6
Spirometer traces

Changes in lung volume as shown

by a spirometer trace

Lung volumes can be measured by using a sprirometer


• Spirometer
• Kymograph
• Disinifectant
• Eye protection
• Soda lime

• 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
the floating chamber. This absorbs the CO2 that the subject exhales.
• In which direction will the pen move when the subject inhales.
• 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.
• Switch on the recording apparatus and at the end of an exhaled breath turn
the tap so that the mouthpiece is connected to the spirometer chamber.
• The trace will move down as the person breathes in. After breathing
normally the subject should take as deep a breath as possible and then
exhale as much air as possible before returning to normal breathing.
• See trace example below.


• The tidal volume is the volume of air breathed in and out in one breath at
• The tidal volume for most adults is only about 0.5 dm3.
• Vital capacity is the maximum volume of air that can be breathed in or out
of the lungs in one forced breath.
• Breathing rate is the number of breaths taken per minute.
• Minute ventilation is the volume of air breathed into (and out of) the lungs
in one minute. Minute ventilation = tidal volume × rate of breathing
(measured in number of breaths per minute).
• Some air (about 1 dm3) always remains in the lungs as residual air and
cannot be breathed out. Residual air prevents the walls of the bronchioles
and alveoli from sticking together. Any air breathed in mixes with this
residual air.

Calibration of the y-axis-breathing volume
• Fill water into the tank and pump oxygen into the space above the water by
using the facemask tube; the volume readings can be calibrated by making
marks on the chart with the pen before and after a known volume of
oxygen is added to the air tank

We can calibrate the scale as 10 small squares (10 mm) representing 1 dm3
volume of oxygen


Calibration of the x-axis-time scale
• The speed of kymograph can be set at a specific value; it may be convenient
to set the time scale to 1 mms-1

Calculation of breathing rate
• Breathing rate is calculated by counting the number of breaths in a given
time; in the trace below, AB represents exhalation and BC represents
inhalation; so ABC represents one complete breath

Calculation of tidal volume
• The volume of air breathed in and out is calculated from the vertical
movements of the trace; the spirometer has been calibrated as 1 dm3
equals 10 mm

The tidal volume of the trace between A and B is 3-1.6= 1.4 dm3


Measuring oxygen consumption
• Each time we take a breath, some oxygen is absorbed from the air in the
lungs into the blood. An equal volume of carbon dioxide is released back
into the lungs from the blood
• When we use the spirometer, each returning breath passes through soda
lime, which BSORBS carbon dioxide, so less gas is breathed back into the
spirometer chamber than was breathed in. if we breathe into and out of
the spirometer for about 1 minute, a steady fall in the spirometer trace can
be seen.
• The gradient of the fall is a measure of the rate of oxygen absorption by the
blood, and so is a measure of the rate of respiration by the body

x= 1.65-1.05= 0.6 dm3
y= 29.5-7.5= 22 s
rate of oxygen consumption = x in dm3 / y in s
= 0.6/22
= 0.03 dm3 s-1
• If a person had been exercising, the rate of oxygen consumption would be
higher, so the slope would be steeper.

• Soda lime may be corrosive; wear eye protection
• Disinfect the spirometer mouthpieces with ethanol and dry by allowing
ethanol to evaporate


Core practical seven

Confounding variables
• No of organisms
• Temperature (maintained by a water bath)
• Time
• Amount of soda lime

• Respirometer
• Soda lime
• Coloured liquid
• 5g Organisms e.g. maggots, germinating peas, woodlice
• Cotton wool
• Stop clock
• Marker pen

• Place 5g of organism (maggots) into the tube and replace the bung.
• Introduce a drop of dye into the glass tube.
• Open the connection (three-way tap) to the syringe and move the fluid to a
convenient place on the pipette (i.e. towards the end of the scale that is
furthest from the test tube).
• 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. 6. 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. (Remember the volume
used = πr2 × distance moved, where r = the radius of the hole in the
• Record your results in a suitable table.

• Calculate the mean rate of oxygen uptake during the 5 minutes.

• 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.


Evaluation issues
• Simple respirometer
Disadvantages: does not allow you to reset; it needs a control tube used
alongside it; no scale so measurements likely to be less accurate.
Advantages: very simple to set up; minimal number of connections makes a
good seal easier to obtain.
• U-tube respirometer
Disadvantages: tendency for the connections to leak in elderly
school/college models (making the equipment useless); expense.
Advantages: does not need to have an additional control as the second
tube balances out the effects of changes in temperature or atmospheric
pressure; the syringe allows you to move the liquid in the U to reset the

• KOH is corrosive, so wear eye protection and wash any splashes off the skin
• Precaution should be taken while assembling the apparatus

Ethical issues
• No ethical issues if using plant material for this investigation
• If using invertebrates, it is important to handle the organisms with care and
respect, ensuring that they don’t come in contact with KOH solution
• Return the animals promptly to their holding tank or natural environment
after the investigation


Core practical 8

• Independent variable: number of pokes

• Dependent variable: retraction time

• Confounding variables:
ü Replication using snails of approx same size and age
ü Equal handling history
ü Drying out

• Apparatus
ü 1 giant African land snail
ü 1 dampened cotton wool bud
ü Clean firm surface
ü Stop watch

1. Allow the animal to acclimatize: Collect one giant African land snail, and
place it on a clean, firm surface. Allow the snail to get used to its new
surroundings for a few minutes until it has fully emerged from its shell.
2. Apply the stimulus: 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.
3. Measure the response: Measure the length of time between the touch and
the snail being fully emerged from its shell once again, with its eye stalks
fully extended.
4. Replicate the results: Repeat the procedure in step 3 for a total of 10
touches, timing how long the snail takes to re-emerge each time.
5. Record your results in a suitable table.
6. Present your results in an appropriate graph.


• Evaluation issues
ü Snails already handled before the experiment may not react in the
same way
ü Determining when a snail has fully emerged
ü Lack of moisture may encourage snail to stay more in its shell
ü Measuring eye stalk length instead

• Ethics and safety
ü Take sensible hygiene while handling snails
ü Treat snails with respect
ü Return them to their habitat after the experiment


Statistical analysis of data

• Data from any investigation will require statistical analysis to draw a valid and
unbiased conclusion
• The chart below may help you decide which statistical test could be applied
to your data to draw a valid conclusion
Is your analysis
concerned with
differences or

Differences Associa=ons

Is your data normally Do you have Have you taken

Is your data paired? distributed? Is your data skewed? categories? measurement?

Spearmean's rank
Wilcoxon matched Mann-whitney χ2 order
paits test (W test) t-test
U test Chi-squared analysis
correla=on coefficient


1. Correlation coefficient

• The correlation coefficient is used to determine whether there is a significant
association between two measured variables
• Some examples of data that can be analyzed by the correlation coefficient are
the relationship between:
ü Temperature and rate of reaction
ü Time of the day and body temperature of lizards
ü Speed of water current and number of nymphs

• The general format of the table
Independent variable/unit Dependent variable/ unit

• Null hypothesis: there is no significant relationship between the independent
and the dependent variable

• It is not required to know how to calculate the correlation coefficient; the
value will be given in the question

• Critical value:
ü it is given in the correlation probability table

• Significance level:
ü we always use the 5% significance level or p=0.05 level; therefore, we
are at least 95% confident in our conclusion

• Degree of freedom:
ü this takes into account the sample size
ü Degree of freedom = n-1 (where n= number of paired


• Drawing a valid and unbiased conclusion:
ü Compare the calculated value with the critical value at 5%
significance level
ü If, the calculated correlation coefficient is greater than the critical
value, then reject the null hypothesis

2. Chi-squared test

• This test is used to test whether the difference between a set of observed
values are significantly different from the expected values

• For example:
If 100 maggots are placed into the center of a long tube containing rotten
meat at both ends; then the maggots have an equal chance of moving
either direction; so the expected ration is 1:1 (50:50); however, when the
experiment is performed the observed results differ (45:55); the chi-
squared test can tell us whether the difference is significant or not

• Null hypothesis:
ü there is no significant difference between the observed and the
expected values; any difference between is due to random chance

• Critical value:
ü is given in chi-squared probability table at 0.05 significance level

• Significance level:
ü 5% significance level or p=0.05 level

• Degree of freedom:
ü degree of freedom = n-1 (where n is the number of pairs of observed
and expected values)

• Draw a valid and unbiased conclusion:


ü If the calculated χ2 value is greater than the critical value at 5%
significance, then reject the null hypothesis
3. t-test

• To determine whether the differences between the mean values of 2 sets of
data measuring the same variable is significant or not; it can be used for
data that shows a normal distribution

• null hypothesis: there is no significant difference between the mean values
of data set one and data set two

• It is not required to calculate the t-test; the calculated value will be given in
the question

• Critical value: it is given in t-test probability table at 5% significance level

• Significance level: 5% significance or p=0.05 level

• Degree of freedom:
ü degrees of freedom= (n1-1) + (n2-1)
ü where n1 is the number of samples in data set 1
n2 is the number of samples in data set 2

• Draw a valid and unbiased conclusion
ü if the calculated t value is greater than the critical value at 5%
significance, then reject the null hypothesis


4. Mann whitney U test

• This test is used to determine whether the differences between the median
values for the two sets of data is significant or not; the two sets of data
don’t show a normal distribution

• Null hypothesis
ü There is no significant difference between the median values of
sample 1 and sample 2

• It is not required to calculate the Mann whitney u test; it is given in the

• Critical value: it is given in U test probability table at 5% significance level

• Significance level: 5% significance or p=0.05 level

• Drawing a valid and unbiased conclusion:
ü If the calculated U test value is greater than the critical value at 5%
significance, then accept the null hypothesis
ü There will be two calculated U values: U1 and U2; the smaller U value
is the calculated value


5. Wilcoxon matched pairs test

• The test is used to determine if the difference between two sets of data
measuring the same variable is significant or not;
• The data must be in matched pairs
• Usually bar graphs, with bars in pairs can be used to represent the
paired data

• For example:
ü The pulse rate of students before and after exercise; the before exercise
rate of one student can only be matched with the after exercise rate for
the same student

• Null hypothesis:
ü There is no significant difference between the values in data set one and
data set two

• It is not required to calculate the W value; the calculated value is given
in the question

• Critical value: it is given in the W test probability table at 5% significance

• Significance level: 5% significance or p=0.05 level

• Drawing a valid and unbiased conclusion
ü If the calculated W value is greater than the critical value at 5%
confidence level, then accept the null hypothesis
ü There will be 2 calculated W values: W1 and W2; the smaller value is the
calculated value


Planning an investigation

In planning your method, you need to make sure you include all the points listed

Identify a problem or question that needs probing

Frame a Hypothesis

Design an experiment to test the hypothesis
Clearly identify the independent variable (IV) and the
dependent variable (DP)
Identify the variables to be controlled (confounding
Consider if a control is needed

Perform the experiment as planned to get a fair test

Collect data- either experimental or observational
Make accurate measurements

Analyse and interpret the data to draw a valid conclusion

Use statistical tests (A2 only) or simple calculations of
percentage or means for analysis and comparison of data
Consider standard deviation (error bars), range bars,
systematic or random errors, outliers, correlation or
causation and conflicting evidence, while interpreting the


Limitations: these should be genuine sources of error that would be encountered
Safety and ethical issues to be considered
• This will vary from question to question; However, look for any factor that
could cause harm or damage to the experimenter; do not hesitate to
include any thought that might pass through your mind, however silly it
may seem, as there is no negative marking and only the right answers will
be marked
• There may not be an ethical issue always. So, if you cannot think of any
ethical issue then move on and ponder over it as you write
• Do not get stuck at a question for a long time as time management is a
major factor in this paper

Suggestions for preliminary work
• Carry out the method in advance to ensure it will work and to check if this
proposed method will provide meaningful data
• Relate marking scheme information to the context of the question and
make a clear indication of what exactly you are going to do as a preliminary
• State the variables and any other relevant information
• Identify the independent and the dependent variables and state them

Detailed method explaining exactly how you would carry out the investigation
and how important variables are to be controlled or monitored

• Even though there is no specified format to write this section, it is useful to
write under subheadings, as it encourages elaboration of points and also
helps to acquire the marks organization and sequence of writing
• State the IV and DV and all the other variables that can influence the
dependent variable
• Give a detailed explanation about how each variable is to be controlled or
monitored and a brief consequence of variations in these variables; quote
approximate values whenever possible, instead of making statements like a
known amount, or fixed distance, or specified volume etc
• Describe at least five variables to be controlled; it is always better to state
more variables than the marking scheme usually has; remember when you


are writing the experiment you are not sure which variables are awarded so
it is better to play it safe and describe as many variables, however take time
management into account
• Give full practical details of how each variable may be controlled or

Measuring the dependent variable
• The dependent variable is to be measured to give you data for final
analysis; choose a method that can be measured accurately; it should also
provide reliable data
• Repeating helps us to check the reliability by giving us an idea about the
variation in repeated experiments

A clear explanation of how the data is to be analyzed
• Data are to be recorded in a table; the table should have columns with
appropriate headings and units; it should match the method used in the
• A hypothetical graph is drawn; you should label the axes appropriately and
state the units; it may also show the trend of the expected results
• State the statistical test used to analyze the data; the statistical test will test
the validity of the null hypothesis
• State the rule of acceptance or rejection of the null hypothesis
• State the significant level used

They are genuine sources of errors that cannot be controlled