P5 PLANNING,ANALYSIS AND

EVALUATION NOTES
ALEVEL BIOLOGY

DR. NIDA MALIK

 P5 BIOLOGY NOTES

SOME IMPORTANT NOTES YOU DEFINETLY NEED TO READ BEFORE PLANNING

THE INVESTIGATION!

CO2:

● Usually, most of the CO2 absorbants are with -OH eg: NaOH or KOH
● Sources of CO2 are usually those with the -CO3 eg: CaCO3, H2CO3
● a non-chemical source: gas cylinder
● the gas is supplied through a bubbler.
● to measure CO2 concentration: use probe with meter

Temperature:

● method of controlling: use an electronic water-bath (a beaker of boiling water)-depends

on the experiment
● use an electronic thermostat
● heat screen*/ heat filter

*for uniform distribution of heat.

● incubator
● digital/mercury thermometer
● for food tests, such as Benedict's test, the temp must be above 80'C.

Time:
Always mention:
- the method of timing (stopwatch/ wall clock)
-precise time
-mention the time according to the need
(Teacher's advice: Don't be vague when mentioning the time period; be sensible, realistic
and precise! )

Sample Size:

● the larger the sample size, the more reliable the results.
● When making concentrations, the minimum number you should make is 3. Ideally, the
number of conc you're going to make must be 5.
● mass must be the same when comparing two samples.

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 P5 BIOLOGY NOTES

Measuring:

● use mm calipers
● a ruler- calibrated to cm or mm
● measuring cylinders
● syringe, pipette
● weighing scale/ electronic balance
● digital/mercury thermometer

MEASURING PLANT LENGTH-

1. use ruler
2. use a thread (remind me to explain what this means if i didn't by the end of this post
please)

● MOVEMENT AND TIME

1. measure distance moved at a certain time (or)

2. measure time taken for certain distance moved

● VOLUME OF CAPILLARY TUBING:

1. mention the diameter

2. and the surface area

● RATE OF TRANSPIRATION=

DIAMETER (of capillary tubing)/ DISTANCE MOVED(by the droplet) orLENGTH

Reliability:

● never use the same sample when you are going to repeat the experiment
● always reset- whenever resetting the exp. always refresh the specimen with the same
mass and volume you were using in the first exp.
● repeat and take average/ plot a graph

Accuracy:

● use the right apparatus

● gas syringe for measuring volume of gas
● look at "Measuring" for more information ^^

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 P5 BIOLOGY NOTES

Plants:

● always use same species

● same developmental age
● keep in mind the size of leaves, size of roots, and the number of roots (and leaves).
● ENVIRONMENTAL CONDITIONS:

1. light intensity
2. temperature
3. humidity
4. CO2 and O2 concentrations
5. wind
6. water
7. mineral content

● same mass of germinating seeds

● shoots of same size/length
● when the plant is placed in water, the stem must be cut slanted under water to prevent
any air-locking
● use of bung or air-tight screw to prevent evaporation of water
● in some questions, you'll see a screw: it's there for resetting the apparatus
● accuracy factor: measure properly and cut using a thread
● to control the surrounding temperature, plant experiments must be done in a
thermostatically controlled room
● When using a suspension, always use either same mass or same volume
● in a question on dry mass (and you're dealing with seeds): the water is removed
(evaporated) by placing seeds in an incubator or oven but never use bunsen flames-it
damages the seed!
● when cutting the leaf in discs for comparing the rate of photosynthesis (or cutting
anything, such as a potato):

1. the size and cross-section must always be the same

2. always cut the curved edges out and cut out a flatter surface from the center

DNA:

● if the question is on electrophoresis, the distance is always taken

● for accuracy: always cut out the same size of DNA fragments
● restriction enzymes are used to cut at any specific place

Light:

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 P5 BIOLOGY NOTES

● Keep three things in mind:

1. keeping light constant

2. varying it
3. excluding it

● in any experiment, you can only test for one factor at a time.

Varying Light:

● same wattage of bulb & varying distances

● same distance and varying bulb wattage
● different number of lamps at same distance
● a dark room with light of fixed illumination
● lamp at same distance and use light filter with different thicknesses.

Measuring Light Intensity:

● light meter
● light-dependent resistor
● photometer
● camera meter
● photodiode

Methods of Eliminating Light:

● card board box, black paper, dark room, black bag

● place in a cupboard

Enzymes:

● temperature, pH, and substrate concentration

● When varying the concentration of the enzyme, then the substrate concentration must be
kept constant
● temp control: use water bath
● pH control: use buffer solution

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 P5 BIOLOGY NOTES

Exp on Effect of Chemicals on Enzyme Action:

● must think whether the chemical is an inhibitor

● when dealing with beads of enzyme: always use the same number/ same mass of beads

KEY: Remember the 4 factors affecting enzyme activity plus the effect of inhibitor
Indicator:

● used to show the presence of a substrate

● it always shows a change in it's original color to mark the end point of a reaction
● the color change of the indicator will always be mentioned in the question
● For any exp: note color change at a fixed time (or)
● note the time taken for the color change to take place
● when repeating the experiment, always replace/refresh the indicator with the same
volume and concentration that was used in the previous exp

● the specimen is always wrapped to exclude a certain environmental factor when an

absorbent, such as CO2, is added
● wrapping is also done to prevent evaporation

Humans:

● measurement of height, sex, heart rate, disease

● Reliability factors:

1. age group
2. gender
3. body mass/size
4. genetics/race
5. state of health
6. time of the day the test is being conducted
7. any tolerance or addiction
8. use of any stimulant or depressant
9. metabolism

Metabolic activity decreases with age!!!

Population:

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 P5 BIOLOGY NOTES

● sigmoid curve: drawing, labelling, and the reasons behind every phase
● What decreases population?

1. destruction of habitat
2. disease
3. food availability
4. migration and emigration
5. increase in predators
6. increase in parasite
7. lesser nesting places
8. hibernation
9. accumalation of toxic waste
10. for plants: -> increased grazing -> environmental factors: natural disasters, soil erosion ->
deforestation: causes soil erosion

● IMPORTANT LIMITING FACTORS!!!!

● Food Availability and Disease!!!
●

Wind:

● use a fan
● for varying wind: vary the fan speed or the distance from the specimen

MICROrganism Growth:

● source: corn syrup, glucose, protein, low grade NH3

● never write nutrient broth
● mention 2 examples at least
● same amount or conc of nutrient broth to the two sets of specimen
● the nutrient supply must be kept constant
● mention flow rate through fermenter
● For batch culture: note the amount of time the organism is left in the fermenter
● keep in mind the O2 supply, temperature, pH
● sterility of the fermenter is very important [so that no other organism grows and acts as a
competitor]
● sterility is important in both batch and continous culture- in fact, every time you set up a
batch culture, sterility must be mainatained

Reliability:
> give time for calibration
(Calibration time is adjusting time)
>Repeat 3 times to be certain that the results are consistent-do not change the parameter
>large sample size

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 P5 BIOLOGY NOTES

Why repeat?

● increases the certainty that the results are consistent

● so that anomalous results can be removed
● permit variance from mean
● to take an average

Accuracy:

● means measuring in a reliable manner. Eg

1. weighing scale
2. thermometer
3. Vernier caliper
4. measuring cylinder
5. gas syringe

● use of a buffer solution to maintain pH

● using sol of known conc (by serial dilution)
● comparing colors of sol by a colorimeter
● larger number of known conc
● washing syringes and pipettes
● mixing and stirring for uniformity to prevent settling of suspension
● in microscopy: eye-piece graticule
● to measure the surface area, the specimen is placed on a grid, where the full squares, half
or more than half are taken into consideration
● FILTERING AND CENTRIFUGING: the suspension spins and the more dense sinks at
the bottom

Control:

● in an exp with living organisms, the control must be a dead organism

● whatever factor is being used in the question is emitted from the control

COUNTING CHROMOSOMES:

For counting chromosomes and making them visible, the growing regions of the plant
are cut

● cut surface of the specimen

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 P5 BIOLOGY NOTES

● chromosomes are counted by placing cut surface under a high power light
microscope(with high magnification)
● How to make chromosomes visible?
● > add dye/stain them
● > Examples of dyes:

1. methylene blue
2. aceto-carmine
3. aceto-orcein

DR. NIDA MALIK

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 P5 BIOLOGY NOTES

Planning investigations
One of the questions involves writing a plan for an investigation. You will be given some
information about the investigation and this will be enough material for you to write your plan.
The skills that you are being tested on are:

1. Identifying key variables.

2. Describing a workable practical procedure.

3. Selecting appropriate methods for measuring the independent variable.

4. Selecting appropriate methods for varying and measuring the dependent variable.

5. Selecting appropriate methods for controlling other variables.

6. Suggesting a suitable control experiment.

7. Suggesting a quantitative, testable, prediction.

8. Selecting equipment of a level of precision appropriate to ensure accuracy.

9. Planning to collect sufficient results to ensure reliability.

10. Describing how results will be recorded.

11. Suggesting how results will be analysed.

12. Risk assessing the practical procedure.

• When you read through the information provided on the paper, try to work out three main
things:

1. what should be changed – this is the independent variable

2. what is going to be measured – this is the dependent variable

3. what should be kept the same – these are the control variables

• You should organise your plan under several headings and then write as concisely as possible.
Suitable headings are:

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 P5 BIOLOGY NOTES

○ hypothesis and/or prediction

○ variables

○ risk assessment

○ method

○ collecting results

○ analysis of results.

• Some investigations need to have two parts.

○ The experimental – which measures the process being studied and contains the living
organism, part of an organism (e.g. a leaf) or enzyme being tested.

○ The control – which will be exactly the same as the experimental except that the living
organism will be missing or replaced by something non-living. The control shows that the results
are due to the activity of the living organism and is not due to the apparatus or an
environmental factor.

• Make sure you explain carefully how to standardise the control variables; for example, ‘put
test-tubes in to a thermostatically-controlled water bath’ is better than ‘keep the test-tubes at
the same temperature’.

• All investigations should be repeated to increase the reliability of the results. If the same
results are achieved (or the results are very similar) then they are reliable. You can also include
the calculation of means and standard deviation in your plan under the heading of analysis of
results.

• Always give quantities in appropriate terms – avoid the use of the word ‘amount’ as this does
not convey precise meaning to any specific quantity. ‘Amount’ could mean volume, mass or
concentration. For example, you can give the volume in cm3 , mass in grams and concentration
in an appropriate unit, such as grams 100 cm–3.

• Suggest appropriate volumes and concentrations in your plan. Include instructions on making
up dilutions either by serial dilution or proportional dilution. You should have learnt how to do
this when preparing for Paper 3.

• Choose apparatus that will give precise results. For example, if you are measuring using a
syringe or measuring cylinder it may be difficult to measure to the nearest cm3 . You should
think about ways in which the precision can be improved before writing your answer.

• Write out your method as a list of numbered steps as if you are writing a set of instructions
for someone else to follow. Think of your method as a recipe.

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• Carry out a risk assessment on your plan and include a section headed risk assessment or
safety precautions. Analysing data, making conclusions and evaluation In preparation for Paper
3, you will have learnt how to analyse data, draw graphs, evaluate data and experimental
methods, and make conclusions. You will be tested on these skills in Paper 5.

In addition, you should know about some statistical methods and apply them to the data
provided. There is always a question that asks you to analyse the data from an investigation. You
should know about the following aspects of statistics:

• calculating standard deviation and standard error (formulae will be provided)

• using two statistical tests – the chi-squared test and the t-test

• making a null hypothesis. You should know when and how to use these methods. There are
several different styles of questions that test your understanding of these statistical methods.
The best preparation is to look at the way data is presented in past paper questions and see
what sort of questions are asked.

SUGGESTED TOPICS FOR P5 PLANNING QUESTIONS:

● Respirometer
● Potometer
● Gel electrophoresis
● Reaction time
● Enzyme activity
● Photosynthesis
● Sampling (random and systematic)
● Osmosis
● Microscopy
● Chromatography

SAMPLE QUESTIONS
OSMOSIS IN PLANT

1. EFFECT OF SUCROSE CONCENTRATION ON POTATO CELLS:

● prepare different concentrations of sucrose solution by simple dilution/serial dilution; by using
1% sucrose solution and distilled water; prepare 1%,0.8%,0.6%,0.4%,0.2% and 0%
● same vol of sucrose sol
● plant (same age, type, same species, pieces of same size & dimension e.g. 5cm
● method of cutting (using scalpel and ruler, cut)
● maintain temp (thermostatically controlled room)
● put the plant pieces in diff sucrose dilutions, they should be completely submerged, start the
time, eg 15min
● remove after 15min, dry the pieces using paper towel
● measure the length/mass before and after

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 P5 BIOLOGY NOTES

● repeat 3 times and take a mean

● graph
● precaution, hazard🡺scalpel; risk🡺 cutting ; precaution🡺cut away from fingers, on a tile
hazard🡺plant ; risk🡺allergen🡺 ; precaution🡺wear gloves
(if any chemical used)hazard🡺chemical name ; risk🡺irritant/harmful/toxic/corrosive ;
precaution🡺 wear gloves/goggles/tweezers

2. ESTIMATE THE WATER POTENTIAL OF A PLANT TISSUE

● prepare different concentrations of sucrose solution by simple dilution/serial dilution; by using
1% sucrose solution and distilled water; prepare 1%,0.8%,0.6%,0.4%,0.2% and 0%
● same vol of sucrose sol
● plant (same age, type, same species, pieces of same size & dimension e.g. 5cm
● method of cutting (using scalpel and ruler, cut)
● maintain temp (thermostatically controlled room)
● put the plant pieces in diff sucrose dilutions, they should be completely submerged, start the
time, e.g. 15min
● remove after 15min, dry the pieces using paper towel
● measure the length/mass/angle of curve of the plant tissue (before and after)
● repeat 3 times and take a mean
● repeat the exp with new conc of sucrose to get closer to the equivalent sucrose conc
● graph
● precaution, hazard🡺scalpel; risk🡺 cutting ; precaution🡺cut away from fingers, on a tile
hazard🡺plant ; risk🡺allergen🡺 ; precaution🡺wear gloves
(if any chemical used)hazard🡺chemical name ; risk🡺irritant/harmful/toxic/corrosive ;
precaution🡺 wear gloves/goggles/tweezers

MEASURING ENYMES ACTION:

E.g. EFFECT OF DIFF SUBSTRATE CONC ON ENZYME INHIBITION (USING COLORIMETER)
● prepare 5 diff dilutions of substrate
● suggest the range (1%,0.8%,0.6%,0.4%,0.2%,0.0%)
● maintain temp (thermostatically controlled water bath)
● equilibrate enzyme and substrate before mixing (Wait till the temp gets constant)
● same/known/constant conc of inhibitor
● same/known/constant vol of inhibitor and same vol of d substrate
● measure absorbance/intensity of color using colorimeter after set time
● repeat 3 times and take a mean
● repeat without inhibitor as well
● graph
● hazard🡺enzyme ; risk🡺 irritation ; precaution🡺 wear gloves

EG. MEASURE THE Km AT DIFF TEMP(at different substrate conc) IN AN ENZYME CONTROLLED
REACTION

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 P5 BIOLOGY NOTES

● carry out the exp at 5 diff temp e.g. 10oC,20OC,30OC,40OC,50OC

● Use a thermostatically controlled water bath/incubator
● Same vol and conc of enzyme e.g. 20cm3 of 10% enzyme sol
● Same vol of substrate e.g. 20cm3
● Use same vol of buffer sol to keep pH constant
● Incubate enzyme and substrate separately at 10oC
● Mix enzyme and substrate at 10oC
● Note the reading at 30s
● Find the initial rate of reaction of each conc of substrate at 10oC,
Rate=amount of substrate disappeared/time; rate =amount of product appeared/time
● Now repeat the exp at other temperatures and test each conc at each temp and note the
reading after 30s each
● Plot a graph of initial reaction at diff substrate conc at 10oC, find Vmax & read off Km value at
1/2Vmax
● Repeat 3 times and take a mean
● Precautions
NOTE: THE SAME EXPERIMENT COULD BE ASKED WITH VARYING pH
JUST REPEAT THE SAME METHOD BUT WITH VARYING PH INSTEAD OF TEMP e.g. pH
2,4,6,8,10,12

HUMAN REACTION TIME :

1. USING AN ONLINE COMPUTER CLICK TIMER TEST:
● Same number of people,50people in each group
● Same ages, same number of male female, or same gender
● Same time of the day, same level of activity
● Same type of online app, computer
● No noise in the room.no other people in the room
● Consent form (volunteer)
● Avoid people with any condition or disease e.g. poor eye sight, neurological disorders
● No neurostimulant drugs, caffeine or alcohol
● Same hand to be used
● Repeat 3 times on each individual and take a mean
● Low risk exp

2. USING THE RULER CAPTURE METHOD:

● Same number of people,50people in each group
● Same ages, same number of male female, or same gender
● Same time of the day, same level of activity
● No warning when ruler to be dropped
● Held vertically
● Use same hand
● Avoid people with any condition or disease e.g. poor eye sight, neurological disorders
● No neurostimulant drugs, caffeine or alcohol

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 P5 BIOLOGY NOTES

● Same hand to be used

● Repeat 3 times on each individual and take a mean
● Low risk exp

3. REACTION TIME/HEART RATE AFFECTED BY CAFFEINE(presence or absence)

● Same number of people,50people in each group
● Same ages, same number of male female, or same gender
● Same time of the day, same level of activity
● Avoid people with any condition or disease e.g. poor eye sight, neurological disorders,
pregnancy, asthma etc.
● Subjects shouldn’t be consuming neurostimulant drugs, or alcohol
● Consent form (volunteer)
● Subject should be away from others during test, and should stay quiet
● They shouldn’t be taking any caffeine drink at least 5 hours before the test
● Measure reaction time and heart rate before giving the drinks as well
● Give same vol of drinks to all subjects e.g. 100cm3
● Prepare caffeine dinks of same conc
● Give one group caffeine drinks, and other non-caffeine of same vol
● Control🡺 give same volume of water as that of caffeine drink to a few subjects
● Take the measurements only after 45min of giving the drink
● Method of measuring heart rate🡺 count the number of heart beats e.g. clicker counter/tally
counter/video
● Repeat and mean
● Precaution🡺 fill up a health questionnaire and allow the subjects to stop the test if they feel
unwell
NOTE: THE SAME EXPERIMENT COULD BE ASKED WITH VARYING CONC OF CAFFEINE
INSTEAD OF ABSENCE/PRESENCE
JUST MENTION PREPARE DIFF CONC OF CAFFEINE BY SERIAL/SIMPLE DILUTION, E.G.
100mg/dm3,80mg/dm3,60mg/dm3 ,40mg/dm3,20mg/dm3

RATE OF TRANSPIRATION USING POTOMETER:

AT DIFF LIGHT INTENSITIES:

● Cut stem using scissors under water

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● Use petroleum jelly to make air tight

● Removing tube from water to introduce air bubble
● 5 diff light intensities by keeping the lamp of same wattage(75W) at different distances
e.g. 10cm,20cm,30cm,40cm,50cm/ OR lamps of diff wattage
(20W,40W,60W,80W,100W) at same distance e.g. 20cm
● Temp constant by keeping the apparatus in a thermostatically controlled room
● Same wavelength by using the same lamp with a same color filter
● Remove the background light—dark room/closed blinds
● Allow plant to equilibrate to the surrounding e.g. wait for 10min before taking
measurements
● Reset the start position of air bubble between measurements
● Measure the distance travelled by the air bubble in a set time e.g. 15min / OR measure
the time taken to travel a set distance
● Repeat 3 times and take a mean
● hazard🡺scalpel; risk🡺 cutting ; precaution🡺cut away from fingers, on a tile
hazard🡺plant ; risk🡺allergen🡺 ; precaution🡺wear gloves
(if any chemical used)hazard🡺chemical name ; risk🡺irritant/harmful/toxic/corrosive ;
precaution🡺 wear gloves/goggles/tweezers

MEASURE LOSS OF WATER VAPOUR IN UPPER & LOWER EPIDERMIS:

● Cut stem under water

● Use same leaf/type pf leaf/species of plant/same plant
● Use upper and lower leaf surface
● Air tight seal on the cup so water vapor doesn’t escape
● Suck water into the graduated tube and then close the clip
● temp constant by keeping the apparatus in a thermostatically controlled room
● Same humidity e.g. 80% by using a humidifier
● Same air flow/wind speed by using fan at a constant low speed at same distance/closed
fans/windows closed
● Same CO2 level 0.4%(1000-1300ppm) by using NaHCO3
● Temp constant by keeping the apparatus in a thermostatically controlled room
● Allow plant to equilibrate to the surrounding e.g. wait for 10min before taking measurements

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● Same light intensities by keeping the lamp of same wattage(75W) at same distances(10cm)
● Reset the water level between measurements by opening then clip and sucking up through the
mouth piece, then close the clip
● repeat 3 times and take a mean
● hazard🡺scalpel; risk🡺 cutting ; precaution🡺cut away from fingers, on a tile
hazard🡺plant ; risk🡺allergen🡺 ; precaution🡺wear gloves
(if any chemical used)hazard🡺chemical name ; risk🡺irritant/harmful/toxic/corrosive ;
precaution🡺 wear gloves/goggles/tweezers

OBSERVING EFFECTS OF CHEMICAL ON OPENING/CLOSING OF STOMATA

● Use same leaf/type pf leaf/species of plant/same plant

● cut the epidermal strips using scalpel/scissors
● cut the strips to same size using ruler/calipers
● put a few drops of chemical solution on the slide
● now place the strip on the microscopic slide
● cover with a cover slip to prevent evaporation
● carry out the exp in a dark room with fixed light of constant light intensity
● temp constant using thermostatically controlled room/incubator
● observe the slide using the light microscope at x40
● count same fixed number of stomata in each field of view e.g. observe 5 stomata in each field of
view
● count the number of stomata open/closed out of the fixed 5 stomata in one field of view
● then change the field of view and count again
● repeat 3 times and take a mean to identify anomalies
● hazard🡺scalpel; risk🡺 cutting ; precaution🡺cut away from fingers, on a tile
hazard🡺plant ; risk🡺allergen🡺 ; precaution🡺wear gloves
(if any chemical used)hazard🡺chemical name ; risk🡺irritant/harmful/toxic/corrosive ;
precaution🡺 wear gloves/goggles/tweezers
FOR EXAMPLE:
Describe a method that the student could use to investigate the effect of different
concentrations of KCl on the opening of stomata. The description of your method should be
detailed enough for another person to follow.
1. Put epidermal strips into solutions in Petri dishes.
2. Use distilled water for one of the Petri dish to act as a control.
3. Cut them to equal length by using a 15 cm ruler and a scalpel
4. Keep them in the dark when in solution and use a water bath to prevent evaporation.
5. Cover Petri dish with a lid to prevent evaporation
6. Mount them on a slide and use a light microscope with the same magnification to count
the number of stomata.
7. Record the number of stomata that are open.
8. Make 3 counts on each leaf strip and take a mean to identify anomalies.

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9. It is a low risk experiment but wear gloves to avoid contact with leaves in case of allergy
to plants.

OBSERVING EFFECTS OF GIBERELLIN (GA) ON GERMINATION OF SEEDS:

1. Carry out a serial dilution to give 5 dilutions of GA
2. Soak the same number of grains in respective GA solutions of the same volume for 1 day in a
petri dish.
3. Keep the concentration of starch in agar and the depth of agar the same.
4. Use distilled water instead of GA solution to act as a control
5. Keep the temperature the same by putting the Petri dish in a water bath of 30oC
6. Cover the petri dishes to prevent evaporation
7. Replicate the experiment 2 more times and take a mean to identify anomalies.
8. Safety: Wear gloves to prevent contact with plants in case of allergies.

CHROMATOGRAPHY:
Describe a method that the student could use to prepare and use chromatograms to compare
the changes in the products of hydrolysis of the protein by the two different proteases over
time. Your method should be detailed enough for another person to follow.
1. Use a pencil to draw the base line
2. Use a capillary tube to give a spot of hydrolysed extract from the protease around 5 cm apart
on the chromatography paper.
3.Allow the spot to dry and repeat to build up a small, concentrated spot of sample.
4. Repeat 1 and 2 for the hydrolysed extract by the other protease.
5. Concentrate the extract by drying between adding spots.
6Keep the number of spots added for each extract.
7Place the filter paper in a beaker and pour in the solvent such that the solvent line is just below
the base line.
8 Cover the beaker to prevent evaporation and maintain a saturated environment
9 Run all chromatograms for 10min.
10 . Find the Rf value of each spot by measuring the distance travelled by the spot / solvent
front.
11 Compare the Rf values of both chromatograms of different proteins.
12 Run at least 3 chromatograms for both enzyme and take a mean of Rf values for each
spot to identify anomalies
13 Safety: Wear gloves to avoid contact with dye in case of allergies to dyes.

Plant chromatogram
1. Cut the leaves into small pieces and place them in a mortar.
2. Add some propanone .
3. Grind the leaves with the pestle until you have a dark green solution of
chlorophyll.

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4. Filter the mixture and collect the solution in a beaker.

5. Draw a pencil line 2.5cm from one end of the paper.
6. Secure each end of the paper with a pin
7. Use a capillary tube to give a spot of extract on the chromatography paper.
8. Allow the spot to dry and repeat to build up a small, concentrated spot of
sample.
9. Place the filter paper in a beaker and pour in the solvent such that the solvent
line is just below the base line.
10. Cover the beaker to prevent evaporation and maintain a saturated
environment
11. Run all chromatograms for 10min.
12. Find the Rf value of each spot by measuring the distance travelled by the spot /
solvent front.
13. Compare the Rf values of both chromatograms of different proteases.
14. Run at least 3 chromatograms for different leaves and take a mean of Rf values
for each spot to identify anomalies
15. Safety: Wear gloves to avoid contact with dye in case of allergies to dyes.

Immobilised enzymes
Outline how the student could immobilise the enzyme ethanol dehydrogenase.

1. Mix enzyme with sodium alginate solution in a beaker.

2. Stir well to prevent setting
3. Use a syringe to transfer droplets of this solution into a beaker containing calcium chloride
solution
4. The jelly beads formed contains the immobilised enzyme
5. Separate the beads from CaCl2 by filtration
6. wash the beads with distilled water
FOR EXAMPLE: Describe a method the student could use to find the activity of the
immobilised and free ethanol dehydrogenase using methylene blue as an indicator of enzyme
activity(methylene blue becomes colorless when oxidized). Assume that the immobilisation
traps all of the available enzyme from the solution..
1 Use same volume of enzyme for making beads and testing free enzymes and put them in
respective beakers.
2 Transfer the same volume of ethanol and NAD for the enzymes into another beaker.
3 Add 3 drops of methylene blue into each beaker.
4 Keep pH constant by adding a buffer to this beaker.
5 Keep temperature constant by placing beakers in water bath.
6 Wait for 20min to reach temperature equilibrium before mixing enzyme and substrate.
7 Mix enzymes and substrates and start stop watch immediately.
8 Use a stopwatch to measure time taken for methylene blue to decolourise
9 Repeat at least 3 times and find mean time to avoid anomalies
10 Safety: Alcohol is flammable hence avoid any open flames. Wear gloves to avoid contact
with enzymes in case of allergies to enzymes.

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RESPIROMETER:

DR NIDA

1. Use a funnel to pour 5 cm3 of potassium hydroxide solution into each

respirometer vessel.
2. Make sure none of the potassium hydroxide touches the sides of the vessels.
3. Add small rolls of filter paper to act as wicks.
4. Fill the cage with respiring material and put it into vessel B.
5. Make sure that the seeds or invertebrates are not touching the potassium
hydroxide or the wick.
6. Add water to vessel A to match the volume of respiring material in vessel B
7. Fit vessel A with a bung holding two connecting tubes – one with a screwclip on
a flexible hose.
8. Fit vessel B with a bung holding a 1 cm3 syringe and a connecting tube
9. Draw some coloured fluid into the manometer U-tube using a syringe.
10. The fluid must be free of bubbles and come to about the middle of the scale on
each side.
11. Open the screw clip and remove the syringe, then connect the manometer U-
tube.
12. To check that the apparatus is airtight, move the marker fluid in the manometer
to one end with the syringe and leave for a few minutes. The fluid should not
move.
13. Record new positions of the manometer fluid at regular intervals for 30
minutes.
14. Find the amount of oxygen absorbed by germinating seeds in a period of 30
minutes at 20 °C. This is Vol1.

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15. Remove the potassium hydroxide solution from both vessels and wash them
out with water.
16. Replace the basket containing seeds or invertebrates in vessel B, an equivalent
volume of water in the other vessel and the bungs in both.
17. Set up the respirometer a 20 °C again and record any increase or decrease in gas
volume over the next 30 minutes. This is Vol2.
18. Calculate the volume of carbon dioxide produced. 16. Calculate the respiratory
quotient.
19. The volume of oxygen absorbed is Vol1. This is recorded in the first part of the
investigation.
20. Vol2 is the volume of carbon dioxide produced minus the volume of oxygen
absorbed. This is recorded in the second part of the investigation. Therefore,
Vol1 + Vol2 = the total volume of carbon dioxide produced.

Using simple respirometers to measure the effect of temperature on

the respiration rate of germinating seeds or small invertebrates
1) respirometer is placed in water baths at different temperatures
2) organisms to be investigated are placed in one tube and non-living material (glass
beads) of the same mass in the other tube
soda lime is placed in both tubes to absorb carbon dioxide
3) coloured fluid is poured into the manometer reservoir and allowed to flow into the
capillary tube (ensure that there are no air bubbles and volume of liquid is the same in
both tubes)
4) rubber bungs are fitted on both tubes; spring clips are closed, and the manometer is
then attached to the bent glass tubing (ensure that it's airtight)
5) open spring clips (to allow pressure throughout the apparatus to equilibrate with
atmospheric pressure)
6) as organisms respire, oxygen is taken from air in the tube, reducing the volume and
pressure, causing the manometer fluid to flow towards the organisms
7) carbon dioxide is removed by the soda lime which ensures that distance moved by the
fluid is only affected by oxygen uptake
8) distance moved by the manometer fluid can be calculated using πr 2h
9) volume of oxygen taken up can be calculated if the diameter of the tube is known
10) Precaution

Effect of factors such as temperature and substrate concentration on the rate of

respiration of yeast using a redox indicator (e.g., DCPIP or methylene blue)

Mechanism of redox indicators when determining respiration rates

1) dehydrogenation happens regularly throughout the different stages of aerobic respiration

2) the hydrogens that are removed from substrate molecules are used in oxidative phosphorylation

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and are transferred by NAD and FAD

3) when DCPIP and methylene blue are present, they can also take up hydrogens and get reduced
(blue → colourless)

4) faster the rate of respiration, the faster the rate of hydrogen release and faster the dyes get
reduced and turn colourless

5) therefore the rate of colour change can correspond to the rate of respiration in yeast

6) rate of respiration (sec-1) = 1 / time (sec)

Effect of temperature on the rate of respiration of yeast using a redox indicator

1) add a redox dye such as DCPIP or methylene blue to a suspension of yeast cells

2) add the test tubes to a temperature-controlled water bath

3) record the time taken for a colour change to occur once the dye is added (& repeat across a range of
temperatures)

4) when reduced, the blue dyes become colourless (rate of change from blue to colourless is a measure
of the rate of respiration of the yeast)

Effect of substrate concentration on the rate of respiration of yeast using a redox indicator

1) add different concentrations of a substrate to the suspension of yeast cells (e.g. 0.1%, 0.5% glucose)
2) record the time taken for a colour change to occur once the dye is added (& repeat across a range of
temperatures)

3) when reduced, the blue dyes become colourless (rate of change from blue to colourless is a measure
of the rate of respiration of the yeast)

Variables to be controlled in redox indicator experiment to investigate respiration rate in yeast

1) volume of dye added

2) volume of yeast suspension

3) type of substrate

4) concentration of substrate

5) temperature

GEL Electrophoresis

DNA ELECTROPHORESIS:

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1. Put 0.32g powdered agarose into conical flask

2. Add 50ml of TBE (0.5mol) into conical flask.(TBE:Buffer)

3 Place a sponge above conical flask to prevent loss of solution

4 Heat it using a microwave to make it dissolve for around 30s to prevent boiling.

5 Stir and put it in for another 15s.

6 Cool it down.

7 Put 2 carbon electrodes at each end of the plastic tank

8 Slot combs into plastic tank which will make wells
9 Pour agarose gel solution into tank
10 Wait for solution to set to form gel
11 Add TBE until the gel is covered which will act as buffer
12 remove comb
13 Stain each sample with a dye.
14 . Use a micropipette set to 20mml to transfer sample into each well.
15 Use a separate micropipette to transfer difference samples
16 Connect the end at which samples are to the negative electrode and apply a potential
difference across both ends of the tank
17 Keep the current running for about 1h or till the dyes are 1cm from the end
18 Observe bands using UV light
19 Safety: wear gloves to avoid contact with stains in case of allergy, do not touch
electrical conductors with wet hands to prevent electric shock

Electrophoresis of proteins

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• gel electrophoresis of proteins is used to separate polypeptides produced by different

alleles of the same gene e.g., the haemoglobin variants (⍺-globin, β-globin, and the
sickle cell anaemia variant of βglobin)
• they can be separated as the different polypeptides have different net charges
• the charge on proteins is dependent on the ionisation of R groups of amino acids
• the charge of the R groups depends on the pH
• therefore, buffer solutions are used during the separation of proteins to keep the pH
constant
• once electrophoresis has been performed, the results can be compared against known
industry standards (e.g., from a bioinformatics database)

Sampling
There are two types of sampling – random sampling and systematic sampling.
a) Random sampling This is a sample that fairly represents a population
because each member has an equal chance of inclusion.
When should random sampling be used?
1) when an area looks reasonably uniform
2) no clear pattern to the way the species is distributed
3)samples must be taken randomly to avoid bias
b) Systematic sampling
Systematic sampling is used investigate species distribution where physical conditions
change e.g., altitude, soil moisture content, soil pH, light exposure/ intensity.
Methods of systematic sampling
1) line transects 2) belt transects

Methods to assess the distribution and abundance of organisms in a local area

METHOD TYPE

1) frame quadrats random

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2) mark-release-recapture random

3) line transects systematic

4) belt transects systematic

When assessing the distribution and abundance of organisms, the following can be used
when recording species for accuracy
1) dichotomous key
2) drawings or photographs with identification
3) timed research throughout the area being studied
4) if the species is unidentified, photograph and label them as ‘species A’, ‘species B’, etc.

A) RANDOM SAMPLING: done by:

a) Frame quadrats &
b) mark-release recapture

a) FRAME QUADRAT:
A frame quadrat is a square frame divided into a grid of 100 smaller squares. Each small
square is 1x1m
How to use a quadrat
DR NIDA

1) measure area and form a grid

2) take 2 random numbers and use these as coordinates on your grid

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3) lay your quadrat down

4) count the number of species and record results
5) repeat the process again in different parts of the area
6) ensure that – - there is random assignment of grids - the bigger the sample, the better
(the validity)
7) limitations –
- it will be hard to count fast moving animals, this method is limited to vegetation and
slow-moving or stationary animals
- the quadrat must also be placed on a fairly flat piece of land
The results of this can be used to calculate –
a) species frequency – measure of a chance of a particular species being found within any
one quadrat

b) species density – measure of how many individuals there are per unit area e.g., per
square metre

c) percentage cover – the percentage area inside the quadrat that is occupied by each
species

• if you have 100 little squares in 1 quadrat, then you count the squares in which the plant
species is present – you count a square only when it is half or more covered by the plant

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• so, if the plant is in about 25 squares within the quadrat you can say the plant covers 25%
of the area
• not always equal to 100%; can be less or more

2) Mark-release-recapture
This is a method of estimating the population size of mobile organisms.
1) as many individuals as possible are caught
2) each individual is marked, in a way that will not affect its future chance of survival
(e.g., cutting its fur)
3) marked individuals are counted
4) marked individuals are returned to their habitats to mix randomly with their
population
5) after enough time has elapsed a large sample is recaptured
6) number of marked and unmarked individuals are counted
7) the population size can then be estimated using

– Precautions that should be taken to ensure that the results obtained from the mark-
release-recapture method are valid.
1) sample from a large area
2) ensure there is a long enough time interval, for marked individuals to mix into the
population / between capture and recapture
3) the marking technique must not be toxic
4) the marking technique must not increase/decrease chances of survival
5) marking technique must not fall off / be rubbed off the animal
6) time is not so long that migration / life cycle changes have occurred

B) Transect sampling
a) line transect (continuous) – all individuals touching the line are recorded
b) belt transect (can be continuous or interrupted) – all individuals within the quadrats
placed in the locations are recorded

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c) Line transect

1) randomly select a starting point in the habitat being examined

2) unroll the transect line (a measuring tape can be used) along the gradient identified

3) the species touching the line may be sampled along the whole length of the line, which is
called a transect (continuous sampling)

4) to sample, record the identity of the organisms that touch the line

5) a line transect will give qualitative data that is represented as a drawing

b) Belt transect
two types:

a) Continuous belt transect

b) Interrupted belt transet

1) extend a measuring tape from one side of the habitat to another

2) place a quadrat at 0m on the tape

3) count the numbers/estimate percentage cover of each species

4) use a key to identify each species

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5) record results in a table

6) move the quadrat along the measuring tape

7) repeat step 3-5, this can be done via two different methods - interrupted sampling –
repeat steps 3-5 in regular intervals e.g., recording the organisms present at every 2m -
continuous – repeat steps 3-5 throughout the whole length of the measuring tape, recording
all organisms in this distance

8) continue until the full length of the measuring tape has been sampled

9) calculate the average of each species

10) a bar chart, kite diagram or a histogram can be drawn to show the data obtained

Random sampling of Biodiversity

State the data that the students would have collected from the grazed and ungrazed
areas to calculate Simpson’s Index of Diversity.
Answer:
Number of individuals of each type of species present and total number of individuals of
all species
Sample question:

Quadrat A group of students investigated the effect of grazing by domestic herbivores

on the plant biodiversity of a grassland as measured by Simpson’s Index of Diversity.
They investigated two areas. One area was grazed by herbivores and the other area
was not grazed for many years because it was surrounded by a fence to keep out the
herbivores.
Describe a random (unbiased) method which the students could have used to collect
the data needed to calculate the biodiversity of the plant species in the two areas
1. Mark out an area of grazed area to be sampled with 2 measuring tapes forming an x
and y axis.
2. Use a random number generator app to mark out coordinates of the sampling points
in relation to the 2 measuring tapes
3. Place frame quadrant with area 1m2 at coordinates
4. Identity species using a nature guide and count number of individuals of each species
present
5. Repeat step 2 to 4 3 times with different plots in a given area and take a mean to
identify anomalies

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6. Mark out the same size plot in ungrazed area and use the same quadrat to take the
same number of samples in the plot.
7. Sample at different seasons
8. Safety: Wear gloves to avoid contact with plants in case of allergies

Mark-release-recapture This is a method of estimating the population size of mobile

organisms.
1) as many individuals as possible are caught
2) each individual is marked, in a way that will not affect its future chance of survival
(e.g., cutting its fur)
3) marked individuals are counted
4) marked individuals are returned to their habitats to mix randomly with their
population
5) after enough time has elapsed a large sample is recaptured
6) number of marked and unmarked individuals are counted
7) the population size can then be estimated using

– Precautions that should be taken to ensure that the results obtained from the mark-
release-recapture method are valid.
1) sample from a large area
2) ensure there is a long enough time interval, for marked individuals to mix into the
population / between capture and recapture
3) the marking technique must not be toxic
4) the marking technique must not increase/decrease chances of survival
5) marking technique must not fall off / be rubbed off the animal
6) time is not so long that migration / life cycle changes have occurred

PHOTOSYNTHESIS:
Q) Describe how you would carry out an investigation into the effect of wavelength of
light (/light intensity) on the rate of photosynthesis of a plant, using a redox indicator
such as DCPIP.
1) grind spinach leaves with ice-cold buffer solution using a pestle and mortar.
2)centrifuge the resulting suspension to remove unwanted debris
3) A buffer solution is added to maintain pH at 7.0
4) add 5 cm3 of the buffered chloroplast suspension with 10 cm3 of DCPIP into test
tubes
5) place the test tubes in different light intensities or in different wavelengths of light
6) the rate can be measured using one of two methods: method 1 - measure time for
blue colour of DCPIP to go colourless (getting reduced) - calculate rate using 1/t method

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2 - leave for a fixed amount of time and measure with a colorimeter - calculate rate as
change in colour value divided by time
7) repeat and take the average for accuracy or calculate the mean
8) plot a graph (method 1 – wavelength on x-axis, and calculated rate on y-axis //
method 2 – time on x-axis, and colorimeter reading/calculated rate on y-axis) 8) this is
known as the Hill reaction

investigating the effect of light intensity or light wavelength on the rate of

photosynthesis of a plant, using a redox indicator such as DCPIP The Hill reaction

(This Hill reaction investigates the light dependent reactions of photosynthesis which
take place in the thylakoid membranes of chloroplasts. The reaction can only occur if the
thylakoid membranes are illuminated as the light-dependent stage stops in the dark. •
This reaction involves isolating chloroplasts from living cells and suspending them in a
coloured electron acceptor such as DCPIP. • The reaction then depends on the electrons
released during the light-dependent stage of photosynthesis being picked up by DCPIP.
When oxidised, DCPIP is blue, and when reduced, it’s colourless. Therefore, it is possible
to monitor the loss of blue colour as an indication that DCPIP has accepted electrons. In
this experiment, DCPIP takes the place of NADP (which is usually reduced in the light-
dependent reaction), allowing photolysis to continue even when the supply of NADP has
been exhausted because the DCPIP can continue to accept the electrons from the
electron transport chain)
1 • independent variable – light intensity/wavelength reaching the chloroplast samples
2• dependent variable – amount of DCPIP reduced (can be judged based on the colour
of the mixture.
3 • control variables – use chloroplasts from the same species of plant
4) amount of chloroplast/buffer solution added to each test tube should be same
5) duration – all test tubes must be left for the same amount of time before comparing
the colour of the mixture
5) pH should be the same – add buffer solution to maintain the pH at 7.0

investigating the effects of light intensity, carbon dioxide and temperature on the
rate of photosynthesis using whole plants (e.g., aquatic plants such as Elodea and
Cabomba)
These experiments can be carried out using a variety of different apparatus such as –
11) micro burette

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12) photosynthometer

13) EFFECT OF TEMPERATURE USING PHOTOSYNTHOMETER:

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Q) Describe how you would carry out an investigation into the effect of temperature
on the rate of photosynthesis of an aquatic plant.
1) the experiment can be carried out with the use of a photosynthometer (or micro
burette or gas syringe)
2) the shoot of the plant should be cut and placed in hydrogen carbonate solution, to
provide carbon dioxide
3) ensure that the plant is well illuminated before use
4) use a water bath for maintaining the temperature (use at least five different
temperatures e.g., 10°, 20°, 30°, 40°, 50°)
5) allow sufficient time for the shoots to acclimatise to the conditions
6) keep the lamp at a fixed distance from the shoots
7) count the number of bubbles produced during a fixed amount of time
8) to prevent the gases in the bubbles given off from dissolving in water, aerate the
water well (by bubbling air through it) before use
8) repeat the experiment twice more and calculate the mean values
9) use these values to calculate the rate of photosynthesis
10) plot a graph to show the results

To investigate the other factors that affect photosynthesis, the same experiment setup
can be used, with the following changes made –
• light intensity – by altering distance, d, of a small light source from the plants (light
intensity is proportional to 1/d2) - the light needs to be a white light and should not get
hot (LED lights are the best for this) - set up the experiment as shown:

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ALTERNATIVES:
• wavelength of light – by using different colour filters, making sure that they each
transmit the same light intensity
• concentration of carbon dioxide – by adding different quantities of sodium
hydrogencarbonate to the water surrounding the plant

Spearman’s rank correlation and Pearson’s linear

correlation
These are both used to analyse the relationships between the distribution and
abundance of species and abiotic or biotic factors.
The formula for these correlations will be provided in the exam.
Correlation
A measure of the extent to which two factors vary together, and thus of how well either
factor predicts the other.
• plot a scatter graph or calculate correlation coefficient (Pearson’s correlation
coefficient, denoted by r) - the correlation coefficient is measured on a scale that varies
from +1 (positive correlation) through 0 to -1 (negative correlation

• strength of correlation = how close the points are to the straight line

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1) Spearman’s rank correlation

Spearman's rank correlation determines whether there is correlation between variables that
don't show a normal distribution.

When used –

1) data points are independent of each other

2) data is correlated, but not linear (draw scatter diagram first)

There are three steps to take when using the Spearman’s rank correlation test

– 1) state the null hypothesis “there is no correlation between ___ and ___”

2) calculate the Spearman’s rank correlation coefficient

3) test the significance of the result:

- if the value calculated for is greater than the critical value at p=0.05 (confidence level 95%), then
the null hypothesis can be rejected, meaning there is a correlation between two variables

- if is less than the critical value, accept the null hypothesis – there is no correlation between the two
variables

- the degree of freedom used is (n − 2) where n is the number of samples

- a positive sign for indicates a significant positive relationship and a negative sign indicates a
significant negative relationship

2) Pearson’s linear correlation

● Pearson's linear correlation is a statistical test that determines whether there is linear
correlation between two variables
● Data must be quantitative and show normal distribution.

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Steps when using Pearson’s linear correlation test

1) state the null hypothesis “there is no correlation between ___ and ___”
2) calculate the Pearson’s linear correlation coefficient
3) test the significance of the result - if r is close to 1 or -1, then it can be stated that there is a
strong linear correlation between the two variables and the null hypothesis can be rejected
4) to see if the relationship found in a sample is significant, see if the value of r is greater than
the critical value at p=0.05 - the degree of freedom used is (n − 2) where n is the number of
readings

Simpson’s Index of Diversity (D)

A measure of biodiversity between 0 and 1 that takes into account both species richness and
species evenness.
• used to quantify the biodiversity of an area
• as species richness and evenness increase, biodiversity increases
• values near 1 indicate high levels of biodiversity
• values near 0 indicate low levels of biodiversity

Where:
• n is the total number of organisms of one species
• N is the total number of organisms of all species

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t-TEST
● A statistical test called the t-test can be used to compare the means of two sets of
data and determine whether they are significantly different or not
o The formula for the t-test will be provided in the exam, but formulae for how to
calculate the number of degrees of freedom is not provided in the exam and must
be learnt
● The sets of data must follow a rough normal distribution, be continuous and the standard
deviations should be approximately equal
● The standard deviation (s) must be calculated for each data set before the t-test can be carried
out
● A null hypothesis should also be given
o This is a statement of what we would expect if there is no significant
difference between two means, and that any differences seen are due to chance
● If there is a statistically significant difference between the means of two sets of data, then the
observation is not down to chance and the null hypothesis can be rejected

Using the t-test to compare two means

● The steps below outline the general steps in a t test; for a worked example see the next page
● Null hypothesis: there is no statistically significant difference between the means of sample 1
and sample 2
● Step 1: Calculate the mean for each data set:

● Step 2: Calculate the standard deviation for each set of data, s1 = standard deviation of
sample 1 and s2 = standard deviation of sample 2

Step 3: Square the standard deviation and divide by n (the number of observations) in each
sample, for both samples:

● Step 4: Add the values from step 3 together and take the square root:

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● Step 5: Divide the difference between the two means (see step 1) with the value calculated in
step 4 to get the t value:

●
●

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DR NIDA MALIK
●

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MALIK
DR NIDA

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DR NIDA MALIK

T TEST:

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MALIK
DR NIDA

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