2010 SPA Planning Compilation

2010 CJC
5 You are required to plan, but not carry out, an investigation into the effect of light intensity on the rate of a light dependent reaction of photosynthesis in a leaf extract using the dye DCPIP. When DCPIP is reduced, it changes from a blue colour to colourless.
DCPIP (blue) + electrons → reduced DCPIP (colourless)

A leaf extract can be made by mixing finely ground leaf with cold buffer solution. This leaf extract should be kept cold and in the dark except when taking samples.
When capillary tubes are dipped into this leaf extract, the solution rises up into the capillary tube and remains there.
The leaf extract should be placed into the capillary tubes to conduct the experiment.
Your planning must be based on the assumption that you have been provided with the following equipment and materials which you must use:
• capillary tubes
• fresh spinach leaves
• sharp knife
• mortar and pestle
• cold buffer solution
• ice
• a light-proof container with a removable lid
• light-proof aluminium foil
• DCPIP solution (freshly made)
• lamp (chosen so it does not generate heat)
• ruler
• fine sharp sand
• a room which can be made dark
• cloths
• a variety of different sized beakers, measuring cylinders, syringes and pipettes for measuring volumes

Your plan should have a clear and helpful structure to include:
• an explanation of theory to support your practical procedure
• a description of the method used including the scientific reasoning behind the method
• an explanation of the dependent and independent variables involved
• relevant, clearly labelled diagrams
• how you will record your results and ensure they are as accurate and reliable as possible
• proposed layout of results, tables and graphs with clear headings and labels
• the correct use of technical and scientific terms
[12 marks]

Outline [1]
1. Theoretical consideration or rationale of the plan to justify the practical procedure, including source of electrons for reduction of DCPIP; [1]

Procedure [10]
2. Chloroplast extraction - use of knife, cloths, pestle and mortar, and sharp sand;
3. For storage of chloroplast – use cold (e.g. water bath with ice) and dark (e.g. light-proof container) conditions;
4. To create leaf extract - use of buffer solution;
5. Method of filling capillary tube - capillary tubes are dipped into this leaf extract, the solution rises up into the capillary tube;
6. Description of control capillary tube - leaf extract and DCPIP wrapped in aluminium foil to keep out the light;
7. Labelled diagram of equipment;
8. Independent Variable: Five distances from the light source to vary intensity;
9. Propose three replicates;
10. Equilibration procedure – e.g. allow each capillary tube to rest on the tile for 5 mins prior to experiment so that it is at room temperature.
11. Measurement of colour change - measure time taken to decolourise completely;
12. Tabulation of data with correct headings;
13. Processing of data – mean figures;

Distance from lamp / cm Time taken to decolorise DCPIP completely Rate / s-1
Replicate 1 Replicate 2 Replicate 3 Average
10
20
30
40
50
14. Graphs with clear headings and labels;

Language [1]
15. The correct use of technical and scientific terms. [1]








2010 AJC
4 Write your answers to this question on the separate answer paper provided.

You are required to plan, but not carry out, an investigation into the effect of temperature on the permeability of the cell membrane of beetroot tissue. Beetroot cells contain a water-soluble red pigment in their vacuoles. This pigment cannot pass through membranes unless the membrane is damaged.

Your planning must be based on the assumption that you have been provided with the following equipment and material which you must use:

• A large piece of beetroot tissue with the skin removed
• Sharp knife
• White tile
• Ruler
• Waterbath
• Ice
• Thermometer
• Test-tubes
• Stopwatch
• Distilled water
• Colorimeter
• A variety of different-sized beakers , measuring cylinders and syringes for measuring volume

Your plan should have a clear and helpful structure to include:
• an explanation of the theory to support your practical procedure
• a description of the method used, including the scientific reasoning behind the method
• an explanation of the steps you have taken to ensure that the investigation is reliable and provides accurate quantitative data.
• the type of data generated and how the results will be analysed
[Total:12]
½ mark for each point

An explanation of the theory to support your practical procedure (4m)

• Pigment molecule is large/hydrophilic
• Cannot pass through hydrophobic core of phospholipid layer
• Increasing temperature increases the kinetic energy of molecules in the membrane/ phospholipid bilayer more fluid
• Above a certain temperature, there is denaturation / disruption of tertiary structure of
• membrane proteins
• disrupting the integrity/ regular arrangement of the membrane
• allowing pigment to diffuse
• through the vacuole membrane/tonoplast AND cell surface membrane
• down a concentration/diffusion gradient
[any 6]

• Membrane permeability can be measured by measuring the extent of leakage of pigment from beetroot tissue under different temperature
• Below room temperature, no leakage of color
• As temperature increases (above room temperature) , more leakage, more intense red coloration detected
[any 2 ]
A description of the method used, including the scientific reasoning behind the method (4m) – apparatus must be mentioned
• Beetroot tissue cut to same dimension into cubes/cylinders - white tile, sharp knife,ruler
• Cubes/cylinders washed and rinsed in distilled water
• to remove any pigments which may leak out during cutting
• Place cube/cylinder into separate test tubes containing equal volume of distilled water
• Each test-tube placed in a waterbath maintained at certain temperature – use of thermometer to check temperature
• for a fixed time interval (accept 5-20mins) – use of stopwatch
• At least 5 temperatures used
• A range of temperature should at least cover 10oC – 80oC
• Mention use of ice to achieve temperatures which are lower than room temperature
• Solution measured for intensity of color using colorimeter
[any 8]
An explanation of the steps you have taken to ensure that the investigation is reliable and provides accurate quantitative data. (3m)

Control experiment
• Place cube/cylinder in a test tube containing distilled water at room temperature
• Subject control to same experimental conditions
• There should be no leakage of color from the beetroot tissue
• Purpose: to ensure that any leakage of color from the beetroot tissue is due to the change in temperature and not any other external factors

Replicates and Repeats
• Replicate at least 3 times for each temperature;
• Repeat entire experiment at least once;
• to obtain the average absorbance;
[any 6]
The type of data generated and how the results will be analysed (1m)
• Plot graph of absorbance/color intensity(dependent variable) against temperature (independent variable);
• Determine range of temperature where leakage occurs

2010 JJC
You are given two extracts, A and B. One of these is an apple extract and the other one is a potato extract.

You are required to plan, but not carry out, an investigation to identify the two extracts by using the reagents with which you have been provided to determine the presence of reducing sugars, starch, and proteins in these extracts.

Your planning must be based on the assumption that you have been provided with the following equipment and materials which you must use:

• 2 small beakers with solutions labeled A and B
• Benedict’s solution
• 1 % of KOH (potassium hydroxide)
• 1 % of CuSO4 (copper(II) sulfate)
• sodium hydrogen carbonate powder
• 1 mol dm-3 HCl (hydrochloric acid)
• Iodine solution
• boiling-tubes
• test-tubes
• test-tube rack
• test-tube holder
• 5 cm3 syringe
• 1 cm3 syringe
• glassrod
• waterbath
• 500 cm3 beaker
• White-tile
• Dropper
• Stopwatch
• Filterfunnel
• Filterpaper
• Labels

You plan should have a clear and helpful structure to include:
• an explanation of theory to support your practical procedure
• a description of the method used including the scientific reasoning behind the method
• proposed layout of results tables with clear headings and labels
• the correct use of technical and scientific terms


Mark Scheme
1 Theoretical consideration or rationale of the plan to justify the practical procedure
2 Volume / number of drops of reagent to be kept the same
3 Temperature of water bath for Benedict’s test is at boiling point
4 Time of heating kept constant (not more than 2 minutes)
5 Labeling of the test tubes
6 Method of starch test
7 Method of Benedict’s test
8 Method of Biuret test
9 Correct identification of biological molecules present in apple extract.
10 Correct identification of biological molecules present in potato extract.
11 Tabulation of results with correct headings
12 The correct use of technical and scientific terms
[Total: 12]

The presence of starch indicates that extract is potato extract1.
This is because potato is a plant storage1 and reproductive organ1 and its main role is to provide food for the growth of the plant. Hence, it will contain a large amount of the storage polysaccharide – starch hence positive result for starch test 1 and 10 (blue-black coloration).

The higher amount of brick red ppt observed1 during the Benedict's test and the absence of starch indicates that extract is the apple extract1.
This is because during the process of fruit ripening1, apple being a fruit will breakdown starch into glucose to be ready for animal seed dispersers. As ripening proceeds and starch is converted to sugar1 and 9, starch will be absence when iodine's test is performed and a large amount of brick red ppt will be expected during Benedict's test.

1. Using only the reagents provided, carry out the appropriate tests to identify the contents of each of the extracts A and B.

Test Method for test
Reducing sugar7 Add 2 cm3 2 of extract A into a test tube labeled A5. Add equal volume2 of Benedict’s solution into the test tube. Shake and mix well. Place tube A into a boiling water bath3 for 2 minutes4. Repeat the procedure for extract B.
Protein8 Add 2 cm3 2 of extract A into a test tube labeled A5. Add equal volume2 of 1% KOH into the test tube. Shake and mix well. Add 3 drops2 of 1% copper sulphate solution. Shake and mix well. No heating is required. Repeat the procedure for extract B.
Starch6 Add 2 cm3 2 of extract A into a test tube labeled A5. Add 3 drops2 of iodine solution. Repeat the procedure for extract B.

2. Carry out the tests on all the solutions.
3. Record your observations of the tests on the solutions.
11
Test Observations
Extract A Extract B
Benedict’s test
Biuret test
Starch test
2010 PJC
You are required to plan, but not carry out, an investigation into the effect of light intensity on the rate of light dependent reaction of photosynthesis in a leaf extract using the dye DCPIP. When DCPIP is reduced, it changes from blue colour to colourless.

DCPIP (blue) + electrons  reduced DCPIP (colourless)

A leaf extract can be made by mixing finely ground leaf with cold buffer solution. This lead extract should be kept cold and in the dark except when taking samples.

When capillary tubes are dipped into this leaf extract, the solution rises up into the capillary tube and remains there.

The leaf extract should be placed into the capillary tubes to conduct the experiment.

Your planning must be based on the assumption that you have been provided with the following equipments and materials which you must use:

• capillary tube
• fresh spinach leaves
• sharp knife
• mortar and pestle
• cold buffer solution
• ice
• a light-proof aluminium foil
• DCPIP solution (freshly made)
• lamp (chosen so it does not generate heat)
• ruler
• fine sharp sand
• a room which can be made dark
• cloths
• a variety of different sized beakers, measuring cylinders, syringes and pipettes for measuring volumes

Your plan should: have a clear and helpful structure to include

• an explanation of theory to support your practical procedure
• a description of the method used including the scientific reasoning behind the method
• an explanation of the dependent and independent variables involved
• relevant, clearly labeled diagrams
• how you will record your results and ensure they are as accurate and reliable as possible
• proposed layout of results tables and graphs with clear headings and labels
• the correct use of technical and scientific terms

Aim:
To investigate the effect of light intensity on the rate of light dependent reaction of photosynthesis in a leaf extract using the dye DCPIP;

Introduction:
A1 (photoactivation): In light dependent rxn, when PSII in the chloroplast of leaf extract absorbs light, and light energy is transferred to a pair of special chlorophyll a of the reaction centre P680 where e- is excited to higher energy level. This e- will then be captured by primary e- acceptor (photoactivation).
A2 (flow of electrons): Each photoexcited e- passes from primary e- acceptor of PSII to PSI via e- transport chain. When e- reaches the ‘bottom’ of e- transport chain, it fills the e- hole in P700. The primary e- acceptor of PSI passes the photoexcited electron to second electron transport chain (photophosphorylation)
A3 (DCPIP accepts electrons): DCPIP accepts electrons from ETC and is being reduced. It turns from blue to colourless. DCPIP competes with NADP+ for electrons.
A4 (measuring rate of PS): rate of PS is measured by the time needed for decolorisation of DCPIP.
Hypothesis:
A5: The rate of photosynthesis increase linearly with light intensity;
Max 3


Procedure:
B1 (obtaining leaf extract): Fresh spinach green leaves are cut with sharp knife and homogenize with cold buffer solution with mortar and pestle and fine sand.
B2 (cold and dark): The chloroplast extract is covered by a light-proof aluminium foil/place in dark room and placed in a petri dish. Extract has to be maintained at low temperature using ice. (2 underlined points)
B3 (DCPIP and mix): Using pipette, add 3 drops of DCPIP to the leaf extract in the petri dish. Rock the petri dish to mix the liquids and replace the foil cover

B4 (filling capillary tubes): Take five capillary tubes. Stand one end of each tube in the leaf extract/DCPIP mixture to collect some of the liquid and cover the capillary tube quickly in aluminium foil to prevent exposure to light.

B5 (control): Prepare a control capillary tube wrapped with aluminium foil to keep out from the light

B6 (experiment procedure): Place the first capillary tube 5cm away from the lamp (measure by ruler) on a white tile, remove the aluminium foil from capillary tube and immediately turn on the lamp. Record the time taken for DCPIP to decolorize.
B7 (at least 5 distances): Steps 7 and 8 are repeated for the subsequent four capillary tubes with distance of 10cm, 15cm, 20cm and 25cm respectively.

(diagram – 1 mark)
B8 (replicates and repeats): Set up in replicates of 3
Repeat the whole experiment twice

5 max

Variables:
i) Independent variable: light intensity (distance between the lamp & capillary tube/cm)
ii) Dependent variable: time taken to decolorize DCPIP/s
iii) Other variables to be kept constant:
• Carbon dioxide concentration (atmospheric CO2)
• pH of solution (buffer is added to ensure constant pH throughout the experiment)
• temperature (room temperature)

1 max

Table: tabulation of data with correct heading

Distance from capillary tube to the lamp (cm) Time taken for decolorization (s) Rate of photosynthesis, 1/time (S-1)
1st trial 2nd trial 3rd trial Average
5
10
15
20
25

Table: Processing data to get mean results
2 marks
Graph:


• Graph of 1/time (S-1) against distance from the capillary tube to the lamp (cm) is drawn. OR graph of time (s) against distance (cm)

Evaluation:
• a negative slope graph should be obtained
• As the distance increases (light intensity decreases), the rate of light dependent reaction of photosynthesis decrease.


2010 SAJC
Respiration begins when a seed absorbs water. The enzymes inside seeds have to be in suspension to function. Activated enzymes start respiration and use organic matter stored in the seed during respiration to make ATP molecules needed for growth. Respiration continues in the seed after the sprout has emerged until the seed’s endospermic material is used up and the cotyledons have produced the sprout.
[Source: http://www.ehow.com/about_6695705_respiration-germinating-seeds.html]

The respiratory quotient (RQ) is the ratio of the amount of carbon dioxide produced, to the amount of oxygen used. RQ is used in the calculations of basal metabolic rate (BMR).

Variety radiata and variety sublobata are two varieties of Vigna radiata (commonly known as green beans), an importance source of vegetable protein of the human diet in the tropics.

You are required to compare respiration of this two varieties of germinating green bean seedings.

Your planning must be based on the assumption that you have been provided with the following equipment and materials.

• Syringe
• Rubber connecting tube
• Glass capillary tube with bore diameter of 0.4 mm
• Soda lime granules
• Germinating green bean seedlings (Variety radiata and Variety sublobata)
• Glass beads
• Coloured liquid (manometer fluid)
• Ruler / Graph paper
• Marker pen
• Stop watch
• Weighing balance
• Paper towels

• an explanation of the theory to support your practical procedure
• a description of the method used, including the scientific reasoning behind the method
• the type of data generated by the experiment
• how the results will be analysed

[Q4 Total: /12]


Theoretical consideration and rationale of the plan
Rate of respiration is estimated by measuring the rate of gas exchange. In aerobic respiration, oxygen is used as the final electron acceptor in the ETC. The protons and electrons recombine with oxygen to form water. Carbon dioxide is produced during oxidative decarboxylation during the link reaction and the Krebs cycle.

In a closed vessel containing the respiring organism, when the organism takes in O2, it also gives off an equal volume of CO2. Hence, volume of gases remains constant.
When a compound that absorbs CO2 is placed in the closed vessel, eg. soda lime, the pressure in the vessel decreases due to the uptake of O2 during respiration and air is sucked from the manometer to keep the pressure constant. The oxygen uptake is thus detected by the displacement of the manometric fluid.

In the first experiment, volume of the O2 consumed by the germinating green beans is determined. In the second experiment, the soda lime in the respirometer is replaced by water.

Change in volume = volume of CO2 produced + the volume of O2 consumed.
Volume of CO2 produced = volume change in second experiment - volume change in first experiment.

RQ can then be calculated. RQ =


Procedure
Remove the plunger from the syringe and place soda lime granules inside the syringe. Take 4 or 5 of the green bean seedlings and carefully remove and discard its testa (seed coat). Place the seedlings in the syringe barrel and replace the plunger by pushing it in until it is about 0.5 cm from the seedling.

Connect the glass capillary tube securely to the syringe using a rubber connecting tubing. Dip the end of the glass capillary tube into the coloured liquid (also known as manometer fluid) so that a drop enter the capillary tube. Remove any excess liquid with paper towelling.

Place the respirometer horizontally on a piece of graph paper. Wait for 3 minutes to ensure that the manometer fluid is moving smoothly towards the syringe. Without handling the apparatus, measure the distance travelled by the manometer fluid in 6 consecutive time intervals of 1 min. Do this by marking the position of the fluid on the graph paper and reading off the distances. (Alternatively, a piece of white paper can be used, and the markings measured using a ruler).

Record these results in the following table.
Experiment 1
Minutes
1 2 3 4 5 6 Average
Distance travelled in each minute / mm

Calculate the volume of oxygen (in mm3) consumed by the respiring green beans:
Volume of O2 = x r2 x d
r = internal radius of capillary tube
d = distance moved by meniscus of manometer fluid

Detach the syringe from the capillary tube by pulling it gently from the rubber connecting tube. Fit another empty syringe to the capillary tube and flush out the manometer fluid onto a piece of filter paper so that the bore of the capillary tube is empty.

Compensation tube
Return to the syringe containing the green bean seedlings and soda lime. Remove the syringe plunger, and keeping the syringe more or less horizontal, remove the seedlings and replace with an equal mass of glass beads. Replace the plunger to its original position in the syringe. Connect the glass capillary tube securely to the syringe and introduce the manometer fluid as before. Place the respirometer on a piece of graph paper. Wait for 3 minutes before measuring the distance travelled by the manometer fluid in 6 consecutive time intervals of 1 min.

Record these results in the following table.
Compensation tube experiment
Minutes
1 2 3 Average
Distance travelled in each minute / mm

For the second experiment, set up the same respirometer with the green bean seedlings, but replace the soda lime granules with water. Wait for 3 minutes before measuring the distance travelled by the manometer fluid in 6 consecutive time intervals of 1 min.

Experiment 2
Minutes
1 2 3 4 5 6 Average
Distance travelled in each minute / mm

Change in volume = volume of CO2 produced + the volume of O2 consumed.
Volume of CO2 produced = volume change in second experiment - volume change in first experiment.
2010 YJC
Homo sapiens (wise man) is the only extant (living) species of the genus Homo. There were other species that used to exist such as Homo erectus (upright man), Homo heidelbergensis (Heidelberg man) and Homo neanderthalis (Neanderthal man). Methods of classification have depended very much on morphological assessment of the skull and skeletal structures, as well as the analysis of bone and soil materials, so as to determine relationships between these groups.

In 2003, a group of fossils were discovered in Indonesia, and were named Homo floresiensis (Flores man). The group was nicknamed “the hobbit” for the small size of the adult fossils. Controversy over the status of the group arose, with some scientists arguing that Flores man was actually a tribe of dwarf Homo sapiens or a population suffering from thyroid disorders.

Plan an investigation to examine if the Flores man should be regarded as a different species, or to be regarded as a subspecies of modern man, based on molecular tools available, assuming that Homo sapiens and Homo neanderthalis are to be regarded as two different species.

Your planning must be based on the assumption that you have been provided with the following equipment and materials.
• tissue samples (skin) from museum specimens of Flores man and Homo neanderthalis, as well as that from human volunteers.
• pestle and mortar
• DNA extraction buffer solution
• glass rods
• microcentrifuge tubes and centrifuge
• restriction enzyme
• agarose or polyacrylamide gel plate and a suitable source of electric current
• radioactive probe and autoradiography equipment
• nitrocellulose membrane

Your plan should have a clear and helpful structure to include:
• an explanation of the theory to support your practical procedure
• a description of the method used including the scientific reasoning behind the method
• the type of data generated by the experiment
• how the results will be analysed, including how the status of Flores man (as a species or subspecies) can be determined.
[Total: 12]

Mark Scheme for Specimen Planning Question
1 Theoretical consideration or rationale of the plan to justify the practical procedure
• What biological principles can we depend on?
• Brief explanation of which overall technique (s) to use: RFLP analysis / VNTR analysis. Certain RFLP / VNTR are conserved within a species (DNA fingerprinting at the species level) but different between species. Differences in size / length of DNA sequence reflects distance in phylogeny / ancestry.
• Good suggestion: mitochondrial DNA / essential genes and neutral theory;
2 Method of DNA extraction including homogenisation and use of buffers
• Mechanical breakage of tissues with pestle and mortar
• Use of DNA extraction buffer solution
o water
o detergent (to dissolve membrane)
o salt (prevent DNase activity)
o protease (to digest histones and other enzymes)
• Temperature treatment at 60 °C
• Filtration with coarse paper
• Ice-cold ethanol (to precipitate DNA – make insoluble, salt removal)
3 Preparation of samples for electrophoresis, including use of centrifuge?
• Re-dissolve DNA in water and centrifuge to remove pellet?
• Mixing of DNA solution with loading dye
• Preparation of agarose gel (…)
4 Selection of restriction enzyme and reasons for the selection
• Designed restriction enzyme must be able to cleave the RFLP / VNTR / DNA sequence at different sites / number of sites for all species, generating different sized fragments between species.
5 Amplification of DNA fragments using PCR including detail of PCR
• Primer design must flank gene to be amplified
• Denaturation, annealing and elongation steps with temperature
• 20 to 30 cycles
6 Separation of fragments by gel electrophoresis and the principles behind the separation.
• Loading of DNA into separate lanes for Dodo, unknown bird and several outgroups (e.g. pigeons, cuckoos)
• 100V for 30min
• Why DNA migrates toward the positive electrode (anode)
7 Transfer of DNA onto nitrocellulose membrane
• The gel is immersed in alkaline solution (sodium hydroxide) to denature double-stranded DNA
• Pressing nitrocellulose membrane and several layers of paper towels using a heavy weight with a flat surface to transfer DNA onto membrane
8 Hybridisation with radioactive labelled DNA probe
• The nitrocellulose membrane is incubated with radioactive labelled DNA probe that hybridises to target sequence
• Excess probes are washed off
9 Autoradiography method
• X-ray film laid over nitrocellulose blot
• Expose the film to autoradiography to visualise the DNA position in the X-ray film relative to the position in the gel
10 Method of visualisation
• Positions of bands represent the sizes (lengths) of target sequences
11 Significance of matching bands
• Compare the fragment sizes of the Homo floresiensis to Homo sapiens and Homo neanderthalis – it could be closer to either one or different from both
• Repeat steps 4-10 if necessary to analyse other genetic loci
• Conclude to see if it is sufficiently different from Homo sapiens (at least as dissimilar as Homo neanderthalis) in terms of the DNA profile to conclude
12 The correct use of technical and scientific terms
• Cathode / anode, lanes, DNA fragments / sequence
• Buffer solution
• No confusion of terms (e.g. primers and probes)
• Terms such as nucleic acid hybridisation, double-stranded / single-stranded DNA
13 Bonus: Valid safety or reliability concerns;