A2 Biology: Paper 5

Dilutions
The student used the 250mmoldm–3 KCl solution to make 100cm3 of four other concentrations by
reducing the concentration by 50mmoldm–3 each time.
Describe a procedure that the student could use to prepare these four concentrations. (3M)

Conc / mmoldm-3 Vol of 250mmol-3 KCl solution Vol of distilled water added / cm-3
/cm-3

250 100 0

200 80 20

150 60 40

100 40 60

50 20 80

Measure volume of solution and distilled water to be added by using a measuring cylinder.
Carry out a serial dilution to achieve the following concentrations.
Pour measured volume into a beaker and stir using a glass rod.

Effect of concentrations on ____

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 and should
not repeat the details from (a)(ii) of how to dilute the 250 mmol dm–3 solution of KCl.

1. Put epidermal strips into solutions in Petri dishes. Use distilled water for one of the Petri dish to
act as a control.
2. Cut them to equal length by using a 15 cm ruler and a scalpel
3. Keep them in the dark when in solution and use a water bath to prevent evaporation. Cover Petri
dish with a lid to prevent evaporation
4. Mount them on a slide and use a light microscope with the same magnification to count the
number of stomata.
5. Record the number of stomata that are open.
6. Make 3 counts on each leaf strip and take a mean to identify anomalies.
7. It is a low risk experiment but wear gloves to avoid contact with leaves in case of allergy to
plants.

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The students were provided with a 3mmoldm−3 GA solution.
Describe how the students could use the method from their preliminary investigation to test their
hypothesis.

1. Carry out a serial dilution to give 5 dilutions of <within range>

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.

Chromatograms

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.

(Draw a diagram)
1. Use a pencil to draw the base line
2. Use a capillary tube to give a spot of hydrolysed extract from endoprotease around 5 cm apart
on the chromatography paper. Allow the spot to dry and repeat to build up a small,
concentrated spot of sample.
3. Repeat 1 and 2 for exoprotease.
4. Concentrate the extract by drying between adding spots. Keep the number of spots added for
each extract.
5. Place the filter paper in a beaker and pour in the solvent such that the solvent line is just below
the base line.
6. Cover the beaker to prevent evaporation and maintain a saturated environment
7. Run all chromatograms for 10min.
8. Find the Rf value of each spot by measuring the distance travelled by the spot / solvent front.
9. Compare the Rf values of both chromatograms of different proteases.
10. Run at least 3 chromatograms for both enzyme and take a mean of Rf values for each spot to
identify anomalies
11. Safety: Wear gloves to avoid contact with dye in case of allergies to dyes.

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Plant chromatogram
1. Cut the leaves into small pieces and place them in a mortar. Add some propanone and a little
clean sand. Grind the leaves with the pestle until you have a dark green solution of chlorophyll.
Filter the mixture and collect the solution in a beaker.
2. Draw a pencil line 2.5cm from one end of the paper. Secure each end of the paper with a pin
3. Use a capillary tube to give a spot of extract on the chromatography paper. Allow the spot to
dry and repeat to build up a small, concentrated spot of sample.
4. Place the filter paper in a beaker and pour in the solvent such that the solvent line is just below
the base line.
5. Cover the beaker to prevent evaporation and maintain a saturated environment
6. Run all chromatograms for 10min.
7. Find the Rf value of each spot by measuring the distance travelled by the spot / solvent front.
8. Compare the Rf values of both chromatograms of different proteases.
9. Run at least 3 chromatograms for different leaves and take a mean of Rf values for each spot
to identify anomalies

Immobilised enzymes

Outline how the student could immobilise the enzyme ethanol dehydrogenase.
1. Mix enzyme with sodium alginate solution in a beaker. Stir well to prevent setting
2. Use a syringe to transfer droplets of this solution into a beaker containing calcium chloride
solution
3. The jelly beads formed contains the immobilised enzyme
4. Separate the beads from CaCl2 by filtration and wash the beads with distilled water.

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Describe a method the student could use to find the activity of the immobilised and free
ethanol dehydrogenase.
Assume that the immobilisation traps all of the available enzyme from the solution.

Your method should be detailed enough for another person to use.

(Draw diagram)

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. Add 3
drops of methylene blue into each beaker.
3. Keep pH constant by adding a buffer to this beaker.
4. Keep temperature constant by placing beakers in water bath. Wait for 20min to reach
temperature equilibrium before mixing enzyme and substrate.
5. Mix enzymes and substrates and start stop watch immediately.
6. Use a stopwatch to measure time taken for methylene blue to decolourise
7. Repeat at least 3 times and find mean time to avoid anomalies
8. Safety: Alcohol is flammable hence avoid any open flames. Wear gloves to avoid contact with
enzymes in case of allergies to enzymes.

Hill reaction (ref to practical sheet)

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Respirometer

1.
2. Use a funnel to pour 5 cm3 of potassium hydroxide solution into each respirometer vessel.
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. Make sure that the seeds or
invertebrates are not touching the potassium hydroxide or the wick.
5. Add water to vessel A to match the volume of respiring material in vessel B
6. Fit vessel A with a bung holding two connecting tubes – one with a screwclip on a flexible hose.
7. Fit vessel B with a bung holding a 1 cm3 syringe and a connecting tube
8. Draw some coloured fluid into the manometer U-tube using a syringe. The fluid must be free of
bubbles and come to about the middle of the scale on each side.
9. Open the screw clip and remove the syringe, then connect the manometer U-tube.
10. 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.
11. Record new positions of the manometer fluid at regular intervals for 30 minutes. When it nears
the end of the scale on one side, restore it to its original position and note the new position of
the syringe piston.
12. Find the amount of oxygen absorbed by germinating seeds in a period of 30 minutes at 20 °C.
This is Vol1.
13. Remove the potassium hydroxide solution from both vessels and wash them out with water.
14. Replace the basket containing seeds or invertebrates in vessel B, an equivalent volume of
water in the other vessel and the bungs in both. 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.
15. Calculate the volume of carbon dioxide produced.
16. Calculate the respiratory quotient.

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The volume of oxygen absorbed is Vol1. This is recorded in the first part of the investigation.
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.

Potometer

Suggest a hypothesis the student could test about the transpiration of a mesophyte (a plant
adapted to a moist environment) and a xerophyte (a plant adapted to a dry environment).

Transpiration rate in mesophyte is higher than xerophyte.

Using this potometer, outline a procedure that the student could use to test this hypothesis

1. Cut a leafy shoot from the mesophyte underwater to prevent air bubbles using a scissors.
2. Cut another leafy shoot of the same size from the xerophyte underwater.
3. Dry leaves
4. Insert a rubber rung to ensure airtight seal around shoot
5. Use syringe to set water level in capillary
6. Wait for the water level to equilibrate
7. Using a stopwatch, measure the time taken for the air bubble in the capillary tube to move
10cm for each plant
8. Repeat the experiment for each plant 2 more times and take a mean to avoid anomalies.
9. Keep the temperature constant by using a water bath at 30oC and keep the light intensity
constant by using the same lamp of same light intensity on the plant in a dark room
10. This is a low risk experiment but wear gloves to avoid contact with plants in case of allergies.

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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.

Number of individuals of each type of species present and total number of individuals of all species

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 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
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

Line transect- ref to booklet

Mark and recapture

1. Trap 100 water voles using longworth small mammal trap with bait in it
2. Mark trapped water voles with fur clipping / felt tip pen ensuring that it does not have adverse
effect on mammal
3. Release marked mammals and recapture another 100 again after 3 days. This is to allow
mixing to occur and reduce chances of migration.
4. <formula>

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Electrophoresis

1. Put 0.32g powdered agarose into conical flask

2. Add 50ml of TBE (0.5mol) into conical flask. Place a sponge above conical flask to prevent
loss of solution
3. Heat it using a microwave to make it dissolve for around 30s to prevent boiling. Stir and put it in
for another 15s. Cool it down.
4. Put 2 carbon electrodes at each end of the plastic tank
5. Slot combs into plastic tank which will make wells
6. Pour agarose gel solution into tank
7. Wait for solution to set to form gel
8. Add TBE until the gel is covered which will act as buffer and to remove comb
9. Stain each sample with a dye.
10. Use a micropipette set to 20mml to transfer sample into each well. Use a separate micropipette
to transfer difference samples
11. Connect the end at which samples are to the negative electrode and apply a potential
difference across both ends of the tank
12. Keep the current running for about 1h or till the dyes are 1cm from the end
13. Observe bands using UV light
14. Safety: wear gloves to avoid contact with stains in case of allergy, do not touch electrical
conductors with wet hands to prevent electric shock

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