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Core Practical one Sampling techniques

Objective:

Describe how to carry out a study on the ecology of a habitat to produce valid and reliable data (including the use of quadrats and transects to assess abundance and distribution of organisms and the measurement of abiotic factors, e.g. solar energy input, climate, topography, oxygen availability and edaphic factors)

Sampling 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
Quadrat sampling
SystemaEc sampling used for es=ma=ng changes as we move along a habitat
SystemaEc sampling
used for es=ma=ng
changes as we move
along a habitat
Random sampling: used for es=ma=on of species diversity, popula=on density or abundance of organisms, within
Random sampling:
used for es=ma=on of
species diversity,
popula=on density or
abundance of organisms,
within a habitat
Belt transect: for study of distribu=on and abundance of organisms
Belt transect:
for study of distribu=on
and abundance of
organisms
Line transect: for study of distribu=on of organisms (no indica=on of abundance) 1
Line transect:
for study of distribu=on
of organisms (no
indica=on of abundance)
1

Quadrats

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 identi fied, 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 whole.

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 reliab le when you look at the results from many quadrats.

reliab le when you look at the results from many quadrats. • Quadrat size is highly

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.

2

A. Random sampling

can be as simple as throwing the quadrat over one’s shoulder and counting the species within it whene ver it falls.

It

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,

series of coordinates can be obtained. The quadrat is placed at the intersection of each pair of coordinates and the species within it recorded.

a

Random sampling (quadrats placed at randomly generated positions) :

Used where the habitat is uniform

Removes observer bias

Used in a large area

Randomness mak es it statistically acceptable

Used if time is limited

Place the quadrat where the coordinates for the random numbers intersect 3
Place the quadrat where
the coordinates for the
random numbers
intersect
3
acceptable • Used if time is limited Place the quadrat where the coordinates for the random

B. Systematic sampling

It i nvolves 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 environmenta l 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 t ransect:

Produces more data, gives detail about species abundance down the line as well as range

Shows species dominance down the line

Measuring tape
Measuring tape
detail about species abundance down the line as well as range • Shows species dominance down
detail about species abundance down the line as well as range • Shows species dominance down

4

Line transect

A line transect is used so that systematic sampling of an area can be carried out.

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 i ntervals.

This technique is particularly useful where there is a transition (change) of plantation or vegetation and animals across the area, down a seashore for example.

Line transect

Used where time is limited

Used to visually illustrate how species c hange along a line

Used to visually illustrate how species c hange along a line For both belt and 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 noted

too small an interval can make the sampling time consuming, as well as yielding more data than needed.

5

Methods of measuring abundance

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

of individuals of a species per unit area of the ground) Buttercup density is 13/m 2

Buttercup density is 13/m 2

Area = 1 m 2

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

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

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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 covere d by that species

squares that would be completely covere d by that species • The squares that are marked

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%

7

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 s tone 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.

reliable and minimizes the effects of unusual results. b. capture - recapture techniques • This method

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 kn own 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 as sumption 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

8

Estimated

size

of

population

=

!"!#$ !"#$%& !" !"#!$!#%&'( !" ! ! ! !"#$% !"#$%& ! !"!#$ !"#$%& !" !"#!$!#%&'( !" ! ! ! !"#$%& !"#$%&

!"#$%& !" !"#$%& !"#!$!#%&'( !"#$%&'!"(

The estimate calculated is called Lincoln index

!"#!$!#%&'( !"#$%&'!"( • The estimate calculated is called Lincoln index 9

9

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 humid ity can be measured using a whirling hygrometer

Soil water : a sample of soil is dried at 100 o C 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

10

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 shrimp

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 tested.

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

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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 in to the beaker (eggs- side down).

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 plac e 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 temperatures

ü not accurate salt measurements

ü may not have counted exactly 40 eggs

ü may miss seeing some of the baby shrimp

12

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 incuba tor at different temperatues

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 25 o C; 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 medium)

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. o C

Initial mass of seedlings /g

Final mass of seedlings /

Mean

g

percentage

 

increase in

mass (%)

Trial

Trial

Trial

Mean

Trial

Trial

Trial

Mean

 

1

2

3

1

2

3

10

                 

20

                 

30

                 

13

40 50 60
40
50
60

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

Presentation of the results

× 100

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

Safety

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

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Core practical 3 Polymerase chain reaction (PCR)

Equipment

Thermocycler

DNA sample

Taq polymerase

Nucleotides primers

Buffer solution

Method and outcome

DNA sample is placed into tube in thermocycler with nucle otides, 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 t he template stran ds . 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.

for taq polymerase enzyme. The bases are placed in their correct position, extending the strand from

15

Evaluation issues Thermocycler • T he amplification factor = 2 n , where n is

Evaluation issues

Thermocycler

T he amplification factor = 2 n , where n is the number of replication cycles; for example, if the cycle is run for 20 times then the DNA quantity increases 2 20 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 ; avo id contact with the skin, wear rubber gloves

16

Core practical 4 Gel electrophoresis

Equipment:

S elected restriction enzymes

agar gel (agarose gel)

gel tank

electrical supply

micropipettes

DNA sample

Loading dye

UV light

Camera

Buffer solution

DNA restriction ladder

Method

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 fr om the tank and view under UV light.

Take picture if desired.

Outcome

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.

17

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

Method

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 121 o C for 15 min at a pressure of 103 kPa

of the nutrient medium; the nutrient medium is autoclaved at 121 o C for 15 min

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3.

Step three: plate pouring

Remove the liquefied hot agar medium and allow it to cool in water bath to about 50 o C

Collect one bott le 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 microorganisms

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

Fla me 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

Introduci ng 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 st erile spreader or an inoculating wire loop to streak the surface of the agar surface

wire loop to streak the surface of the agar surface G l a s s s

Glass spreader

wire loop to streak the surface of the agar surface G l a s s s

Inoculating wire loop

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

Streaking the plate with an inoculating wire

Streaking the plate with an inoculating wire Spreading bacteria with a glass spreader Step six: preparing

Spreading bacteria with a glass spreader

Step six: preparing a solution of the antibiotic with an optimum concentration; transfer a 0.1 cm 3 of the antibiotic solution to an antibiotic disc; place the disc onto the surface of the inoculated agar medium The same procedure is r epeated 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 bacter ia

will promote the growth of less harmful aerobic bacter ia Step seven: incubation of the culture

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

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Step eight: interpretation of the results

Step eight: interpretation of the results Measure the maximum diameter of the clear zone for each

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

21

Core practical 6 Spirometer traces

Core practical 6 Spirometer traces Changes in lung volume as shown by a spirometer trace Lung

Changes in lung volume as shown by a spirometer trace

traces Changes in lung volume as shown by a spirometer trace Lung volumes can be measured

Lung volumes can be measured by using a sprirometer

traces Changes in lung volume as shown by a spirometer trace Lung volumes can be measured

22

Equipment

Spirometer

Kymograph

Disinifectant

Eye protection

Soda lime

Method

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 containi ng 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 disinfecte d 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 comfortabl e 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 norma lly 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.

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Outcome

The tidal volume is the volume of air breathed in and out in one breath at rest.

The tidal volume for most adults is only about 0.5 dm3.

Vital capacity is the maximum volume of air that can be breathed in or out

of the lungs in one forced breath.

Breathing rate is the number of breaths taken per minute.

Minute ventilation is the volume of air breathed into (and out of) the lungs

in one minute. Minute ventilation = 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 o ut. 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 wate r 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

and after a known volume of oxygen is added to the air tank W e can

W e can calibrate the scale as 10 small squares (10 mm) representing 1 dm 3

volume of oxygen

is added to the air tank W e can calibrate the scale as 10 small squares

24

C alibration 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

it may be convenient to set the time scale to 1 mms - 1 Calculation of

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

represents inhalation; so ABC represents one complete breath Calculation of tidal volume • The volume of

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 dm 3 equals 10 mm

spirometer has been calibrated as 1 dm 3 equals 10 mm The tidal volume of the

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

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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

and so is a measure of the rate of respiration by the body x= 1.65 -

x= 1.65 - 1.05= 0.6 dm 3 y= 29.5 - 7.5= 22 s

rate of oxygen consumption = x in dm 3 / y in s

= 0.6/22

= 0.03 dm 3 s - 1

If a person had been exercising, the rate of oxygen consumption would be higher, so the slope would be steeper.

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

26

Core practical seven Respirometry

Confounding variables

No of organisms

Temperature (maintained by a water bath)

Time

Amount of soda lime

Equipment

Respirometer

Soda lime

Coloured liquid

5g Organisms e.g. maggots, germinating peas, woodlice

Cotton wool

Stop clock

Marker pen

Method

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 wil l 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 pipette.)

Record your results in a suitable table.

27

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

the mean rate of oxygen uptake during the 5 minutes. Outcome • Oxygen molecules are absorbed

Outcome

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 th e 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.

28

Evaluation issues

Simple resp irometer Disadvantages: does not allow you to reset; it needs a control tube used alongside it; no scale so measurements likely to be less accurate. Adv antages: 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 apparatus.

Safety

KOH is corrosive, so wear eye protection and wash any splashes off the skin immediately

Precaution should be taken while assemblin g 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

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Core practical 8 Habituation

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

Method

1. Allow the animal to acclimatize : Collect one giant African l and 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 ha s 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 .

30

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 handl ing snails

ü Treat snails with respect

ü Return them to their habitat after the experiment

31

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 associa=ons?
Is your analysis
concerned with
differences or
associa=ons?
Differences Is your data paired? Is your data normally distributed? Is your data skewed? Mann-whitney
Differences
Is your data paired?
Is your data normally
distributed?
Is your data skewed?
Mann-whitney
Wilcoxon matched
paits test (W test)
t-test
U test

32

Associa=ons
Associa=ons
Do you have categories? χ 2 Chi-squared analysis
Do you have
categories?
χ 2
Chi-squared analysis
Have you taken measurement? Spearmean's rank order correla=on coefficient
Have you taken
measurement?
Spearmean's rank
order
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 gen eral format of the table

Independent variable/unit

Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit

Dependent variable/ unit

Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
Independent variable/unit Dependent variable/ unit
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

measurements)

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Drawing a val id 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 events

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 :

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ü If the calculated χ 2 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

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

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

S ignificance level : 5% significance or p=0.05 level

Degree of freedom :

ü degrees of freedom= (n 1 - 1) + (n 2 - 1)

ü where n 1 is the number of samples in data set 1 n 2 is the number of samples in data set 2

D raw 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

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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 question

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

S ignificance level : 5% significance or p=0.05 level

Drawing a valid and unbiased conclu sion:

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: U 1 and U 2 ; the smaller U value is the calculated value

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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 an d 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 level

S ignificance 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: W 1 and W 2 ; the smaller value is the calculated value

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Planning an investigation

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

Identify a problem or question that needs probing

Identify a problem or question that needs probing Frame a Hypothesis Design an experiment to test

Frame a Hypothesis

a problem or question that needs probing Frame a Hypothesis Design an experiment to test the

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 variables) Consider if a control is needed

(confounding variables) Consider if a control is needed Perform the experiment as planned to get a
(confounding variables) Consider if a control is needed Perform the experiment as planned to get a
(confounding variables) Consider if a control is needed Perform the experiment as planned to get a
Perform the experiment as planned to get a fair test
Perform the experiment as planned to get a fair test
needed Perform the experiment as planned to get a fair test Collect data - either experimental
needed Perform the experiment as planned to get a fair test Collect data - either experimental

Collect data - either experimental or observational Make accurate measurements

experimental or observational Make accurate measurements Analyse and interpret the data to draw a valid conclusion

Analyse and interpret the data to draw a valid conclusion Use statistical tests (A 2 only) or simple calculations of percentage or means for analysis and comparison of data (AS ) Consider standard deviation (error bars), range bars, systematic or random errors, outliers, correlation or causation and conflicting evidence, while interpreting the data

systematic or random errors, outliers, correlation or causation and conflicting evidence, while interpreting the data 38

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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 ques tion 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 work

State the variable s and any other relevant information

Identify the independent and the dependent variables and state them

Det ailed 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 encourag es 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

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are writing the experiment you are not sure which variables are awarded so it is b etter 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 monitored

Measuring the dependent variable

The dependent variable is to be measured to gi ve 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 t he 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 plan

A hypothetical graph is drawn; you should label the axes appropriately and state th e 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 s ignificant level used

Limitations They are genuine sources of errors that cannot be controlled

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