A2 Practicals

Question 1

TOPIC 5A (1) - What method can you use to determine/compare what pigments are present in the leaves of the plant?

Answer 1

Thin Layer Chromotography (TLC)

Question 2

TOPIC 5A (1) Describe how to use TLC to extract pigments

STEPS 1-3

Answer 2

1. Grind up several leaves from shade-tolerant plant you’re investigating, with some anhydrous sodium sulfate (ASS), then add few drops of propanone
2. (FUME CUPBOARD) Transfer liquid to test tube, and add some petroleum ether (PE). Gently shake tube so that 2 distinct layers form in liquid - top layer is pigments mixed in with PE
3. Transfer some of top layer into 2nd test tube with ASS

Question 3

TOPIC 5A (1) Describe how to use TLC to extract pigments

STEPS 4-6

Answer 3

4. Draw horizontal pencil line near bottom of TLC plate and draw your point of origin by applying several drops to line, ensuring each drop is dry before adding next (so that the spot is concentrated).
5. (FUME CUPBOARD) Once dry, place plate into small glass container with some prepared solvent - just enough so that the spot is a bit above solvent. Put a lid on container and leave.
   - As the solvent spreads up the plate, different pigments move with it, but at different rates, so they separate.
6. When solvent has nearly reached top, take plate out, mark solvent front with a pencil, and leave plate to dry in well ventilated place .

Question 4

TOPIC 5A (1) Describe how to use TLC to extract pigments

STEPS 7-9

Answer 4

7. As spots separate, calculate distance moved by spots
8. Calculate RF value = distance moved by spot/distance moved by solvent
9. Repeat process with shade-intolerant plant and compare pigments present in leaves.

Question 5

TOPIC 5A (1) How are shade-tolerant plants adapted for photosynthesis?

Answer 5

• They adapt to light conditions by possessing different proportion of photosynthetic pigments, which allows them to make the best use of the light available
• mixture of pigments is also likely to be different e.g. presence of anthocyanins

Question 6

TOPIC 5A (1) How do anthocyanins protect shade-tolerant plants?

Answer 6

As chloroplasts are adapted for photosynthesis in low light conditions, they are sensitive to higher levels of light. Dark red and purple pigments called anthocyacins, protect chloroplasts from brief exposure to higher levels of light

Question 7

Topic 5A (2) How is a dehydrogenase enzyme related to the light-dependent stage of photosynthesis?

Answer 7

In photosystem I, during the light-dependent stage of photosynthesis, NADP acts as an electron acceptor ad is reduced.
- The reaction is catalysed by dehydrogenase enzyme

Question 8

Topic 5A (2) How can dye be used to investigate the activity of the dehydrogenase enzyme?

Answer 8

1. If you add redox indicator dye to extracts of chloroplasts, like NADP, the dye acts as an electron acceptor and gets reduced by the dehydrogenase in the chloroplasts. As dye gets reduced, there would be a colour change from blue colourless
2. Rate of dehydrogenase acivity can be measured by measuring rate at which DCPIP loses its colour using a colorimeter

Question 9

Topic 5A (2) What’s a colorimeter?

Answer 9

A machine that measures how much light a solution absorbs when a light source is shone directly through it

Question 10

Topic 5A (2) Describe a method to investigate the effect of light intensity on dehydrogenase activity in the extracts of chloroplasts?

(STEPS 1-5)

Answer 10

1. Cut a few spinach leaves into pieces, removing stalks
2. Using a pestle and mortar, grind up the leaves with some chilled isolation solution (sucrose, Potassium Chloride, and phosphate buffer at pH 7). Filter the liquid into a beaker through a funnel lined with muslin cloth
3. Transfer liquid to centrifuge tubes and centrifuge at high speed for 10 mins. This will make chloroplasts gather as pellet. Get rid of the supernatant
4. Re-suspend pellets in fresh, chilled isolation solution (chloroplast extract) and store in cold ice for rest of time
5. Set up colorimeter with a red filter and zero it using a cuvette (cuboid shaped vessel) containing chloroplast extract and distilled water

Question 11

Topic 5A (2) Describe a method to investigate the effect of light intensity on dehydrogenase activity in the extracts of chloroplasts?

(STEPS 6-9)

Answer 11

6. Set up test tube rack at a set distance from becnch lamp and switch lamp on
7. Put a test tube in the rack, add a set volume of chloroplast extract to the tube and a set volume of DCPIP. Mix
8. Immediately take a sample of the mixture from the tube and add it to a clean curvette. Then place the curvette in a colorimeter and record absorbance for every 2 mins till you get to 10 mins.

9- Repeat steps 6-8 for each distance in investigation

Question 12

Topic 5A (2) What should you results tell you in this DCPIP experiment?

Answer 12

If dehydrogenase activity is taking place, absorbance decreases as DCPIP gets reduced and loses blue colour
- The faster the absorbance decreases, the faster the rate of dehydrogenase activity.

Question 13

Topic 5A (2) What can you do at the end of your DCPIP experiment to determine how light intensity affects the rate of dehydrogenase activity?

Answer 13

You can plot a graph of absorbance against time for each distance from light source then compare your results to determine how light intensity affects the rate of dehydrogenase activity

Question 14

Topic 5A (2) What controls should you include in this experiment and why are they important?

Answer 14

2 NEGATIVE CONTROLS
1- DCPIP & chilled isolation solution ONLY (no chloroplast extract)
2- DCPIP and chloroplast extract (wrapped in foil so no light gets to it)

• You need to ensure that the absorbance doesnt change at each distance in these 2 negative controls, as you are only trying to observe the effect of light intensity on dehydrogenase activity.

Question 15

Topic 5A (3) What single-celled organism can be used to investigate factors affecting respiration and why?

Answer 15

Yeast as it can respire aerobically and anaerobically

Question 16

Topic 5A (3) Describe a method to investigate factors affecting AEROBIC respiration in single-celled organisms?

(STEPS 1-4)

DIAGRAM

Answer 16

1. Put known volume and conc of substrate solution (glucose) in test tube. Add known volume of buffer to keep pH constant
2. Place test tube in water bath set temp being investigated. Leave for 10 mins to allow temp of substrate to stabilise
3. Add a known mass of dried yeast to test tube and stir for 2 mins
4. After yeast has dissolved into solution, put a bung, with a tube attached to a gas syringe (set at 0), in the top of test tube. Start stopwatch

Question 17

Topic 5A (3) Describe a method to investigate the effect of temp on AEROBIC respiration in single-celled organisms?

(STEPS 5-8)

DIAGRAM

Answer 17

5. As yeast respire, CO2 formed will travel up tube and into syringe, which is used to measure vol of CO2 released.
6. At regular time intervals (every min) record the vol of CO2 that is present in the gas syringe. Do this for set amount of time (10 mins)
7. A control exp should be set at each temp, where no yeast is present. No CO2 should be formed without yeast
8. Repeat exp 3 times at each temp. Use data to calculate mean rate of CO2 production at each temp.

Question 18

Topic 5A (4) Describe a method to investigate the effect of temp on ANAEROBIC respiration in single-celled organisms?

DIAGRAM

Answer 18

1. Set up apparatus according to steps 1-3 of the aerobic exp above.
2. After yeast has dissolve in solution, trickle some paraffin down inside of test tube so that it settles on and completely covers the surface of the solution. This will stop oxygen getting in, forcing yeast to respire anaerobically.
3. Put a bung, with tube attached to a gas syringe (set at 0), in top of test tube. Perform same steps as other exp.

Question 19

Topic 5A (3&4) What other variables could you investigate in these experiments?

Answer 19

The effects of:

1. substrate conc
2. The use of different respiratory substrates

-on respiration rate

Question 20

Topic 5A (5) What is a respirometer?

Answer 20

A device that can be used to indicate the rate of aerobic respiration of an organism by measuring the amount of O2 consumed by an organism over a period of time.

Question 21

**Topic 5A (5) Describe how a respirometer can be used to measure the respiration rate of woodlice?

(STEPS 1-3)

DIAGRAM

Answer 21

1. Set up apparatus in a water bath of 15 degrees, to provide optimum temp for the woodlice and thus, the optimum temp for the enzymes involved in their respiration
2. Ensure that glass beads in control tube are of the same mass as the woodlice
3. For 10 mins, leave tap open and syringe is removed to allow apparatus to equilibrate (accounting for any expansion that might cause the pressure to change inside) and the respiration rate of the woodlice to stabilise in their new environment

Question 22

**Topic 5A (5) Describe how a respirometer can be used to measure the respiration rate of woodlice?

(STEPS 4-6)

DIAGRAM

Answer 22

4. When 10 mins is up, tap is closed and syringe is attached
5. Syringe is used to reset manometer so that ends of fluid are on equal level and reading from vol scale of syringe is recorded
6. As respiration occurs, vol of air in woodlice test tube decreases, due to O2 consumed during process (all the CO2 produced is absorbed by K0H)

Question 23

**Topic 5A (5) Describe how a respirometer can be used to measure the respiration rate of woodlice?

(STEPS 7-9)

DIAGRAM

Answer 23

7. Decrease in vol of air will reduce pressure in test tube, causing coloured fluid in capillary tube to move towards it.
8. After leaving apparatus to run for a set period of time (e.g. 10 mins), the syringe is used to reset the manometer and the reading on the syringe’s volume scale is recorded again. The difference between this figure and the figure taken at the start of exp. is the oxygen consumption for this time period.
   - You can use this to calculate a rate of respiration.
9. To check the precision of the results, the experiment is repeated and a mean volume of 02 is calculated.

Question 24

(TOPIC 6A) (1) what is a choice chambers?

Answer 24

It is a container with different compartments, in which you can create different environmental conditions.
- It can be used to investigate how animals, e.g woodlice, respond to conditions like light intensity and humidity

Question 25

(TOPIC 6A) (1) Describe how to use a choice chamber? (steps 1-4) *DIAGRAM*

Answer 25

1- Construct a choice chamber using the equipment shown in the diagram 2. To investigate light effect on lice movement, cover 1 half of lid (including sides) with black paper to make it dark. Put damp filter paper in both sides of base. 3. Place 10 woodlice on mesh in centre of chamber and cover chamber with lid. 4. After 10 mins, take off the lid and record number of woodlice on each side of the chamber. (try to minimise amount of time lid is off, so that environmental conditions aren't disturbed).

Question 26

(TOPIC 6A) (1) Describe how to use a choice chamber? (steps 5-6)

Answer 26

5. Repeat experiment after gently moving the woodlice back to centre. You should find that most woodlice end up on dark side as tactic response to light. 6. To investigate humidity, place damp filter paper in one side of base and dessicating agent in other side. Put lid on and leave chamber for 10 mins to stabilise before carrying out steps 3-5.

Question 27

(TOPIC 7C) (1) What type of sample should you take when investigating populations?

Answer 27

A random sample

Question 28

(TOPIC 7C) (1) Describe how to take a random sample? | 5 steps

Answer 28

1. Choose an area to sample - small area within place being investigated 2. Divide field into grid, and use random number generator to produce co-ordinates for a site. 3. Use quadrats/transects/mark-release recapture, where appropriate 4. Repeat process as often as possible to reduce likelihood that results are down to chance 5. Calculate number of individuals for whole area by multiplying mean of data in each sample and multiplying by size of whole area.

Question 29

(TOPIC 7C) (2) What methods can you use to investigate the abundance of non-motile (moving) organisms?

Answer 29

Quadrats and belt transects

Question 30

(TOPIC 7C) (2) What is a quadrat?

Answer 30

It is a square frame, that you place onto the ground the different points within the area you're investigating.

Question 31

(TOPIC 7C) (2) What can you measure using a quadrat?

Answer 31

1. The species frequency 2. The number of individuals of each species 3. The % cover of a species (count a square if it's more than half-covered)

Question 32

(TOPIC 7C) (2) How can you cover large distances with a transect?

Answer 32

Using an interrupted belt transect = By placing quadrats at intervals along the line (with spaces in between them)

Question 33

(TOPIC 7C) (3) What method can you use to investigate the abundance of more motile species?

Answer 33

Mark-Release-Recapture method

Question 34

(TOPIC 7C) (3) Describe the mark-release-recapture method? | 5 steps

Answer 34

1. Capture a sample of species using an appropriate technique e.g. pitfall traps, and count them. 2. Mark them in a harmless way (e.g. paint) 3. Release them back into their habitat 4 After a week, take a 2nd sample from the population 5. Count how many of the 2nd sample are marked and use this equation: pop.size = (no. in 1st sample * no. in 2nd) / no. marked

Question 35

(TOPIC 7C) (3) What assumptions do you have to make with the mark-release-recapture method?

Answer 35

1. The marked sample have had enough time and opportunity to mix back in with the population 2. The marking hasn't affected the individuals chances of survival 3. The marking is still visible 4. There is no change in pop. size due to births, deaths and migration during the period of the study

Question 36

TOPIC 7C (4) Describe a method for how you could investigate the effect of soil pH on the distribution of marram grass (in a coastal ecosystem),

Answer 36

1. Place tape measure in straight line from shore (transect) 2. Take 1m^2 quadrat. 3. Starting from the shore, place quadrat next to tape measure. 4. Work out % cover of marram grass 5. At each sample point, measure pH and record results in table 6. Record observations every 10 metres along transect.

Question 37

TOPIC 7C (4) How can you measure pH?

Answer 37

1. With a pH probe 2. Take a sample of your substance, sieve it to remove debris, and place it in a test tube. Then add some barium sulfate, distilled water and pH indicator. - observe for a colour change