Manipulation, Measurement and Observation

As you carry out each test to identify the presence or absence of the biological molecule in S1, S2 and S3, complete the following:

Decide which biological molecule to identify in the first test.

First test: Test for ______

Describe how you used the reagents to carry out this test.

Carry out the first test and record your observations.

Use these observations to complete the sentence.

Solution(s) ______ contain(s) the biological molecule ______ .

Decide which biological molecule to identify in the second test.

Second test: Test for ______

Describe how you used the reagents to carry out this test.

Carry out the second test and record your observations.

Use these observations to complete the sentence.

Solution(s) ______ contain(s) the biological molecule ______ .

Decide which test you will use to check the identity of the third biological molecule.

Third test: Test for ______

Describe how you used the reagents to carry out this test.

Carry out the third test and record your observation.

Use this observation to complete one of the following sentences.

Solution(s) ______ contain(s) the biological molecule ______ .

OR

Solution ______ does not contain any of these biological molecules, glucose, starch or sucrose.

You are required to identify which of the other two biological molecules is hydrolysed by the enzyme, E using the procedure shown in Fig. 1.1 on each solution.

Set up two beakers as shown in Fig. 1.1.
Leave the mixtures for 5 minutes so that E can carry out the hydrolysis.
After 5 minutes, test the mixtures to find out whether E has hydrolysed the biological molecule to its products.

Prepare the space below to record:

• the biological molecule tested for
• the observations.

Describe how you would modify this procedure to investigate the effect of temperature on the enzyme, E.

Complete Fig. 1.1 to show how you will make three further concentrations of glucose solution, G.

Complete Table 1.1 to show the volumes of solutions you intend to use in your investigation.

Table 1.1

State one variable, other than the volume of each solution, which needs to be kept the same in this investigation. Describe how you will keep this variable the same.

Prepare the space below and record your results.

State the hazard with the greatest level of risk when using the apparatus and reagents in step 1 on page 4.

State the level of risk of the procedure: low or medium or high.

hazard ______

level of risk ______

You are required to make a serial dilution of the 0.3% solution of X which reduces the concentration of X by a factor of 10 between each successive dilution.

You will need to prepare $10\ \text{cm}^3$ of each concentration of solution X.

You should use the beakers shown in Fig. 1.1 to show how you will prepare the serial dilutions.

You will need to use $9\ \text{cm}^3$ of each different concentration of X in the investigation.

For each beaker, complete Fig. 1.1 to show how you will dilute the solution by:

• stating, under the beaker, the concentration and volume of the solution available for use in the investigation
• using one arrow, with a label above the beaker, to show the concentration and volume of the solution X added to prepare the concentration
• using another arrow, with a label above the beaker, to show the volume of W added to prepare the concentration.

You are required to investigate the effect of different concentrations of X on the activity of catalase by finding the number of bubbles of oxygen released in two minutes.

Proceed as follows:

1. Prepare the concentrations of X as shown in (b)(i). Note: syringe labelled X should be used for solution X only.
2. Put $10\ \text{cm}^3$ of P into each of the concentrations of X, including 0.3% X. Shake gently to mix.
3. Put $20\ \text{cm}^3$ of P and $18\ \text{cm}^3$ of W into a separate beaker.
4. Leave for at least three minutes.

Read step 5 to step 13 before proceeding.

1. Put $10\ \text{cm}^3$ of H into each of five test-tubes.
2. Put $10\ \text{cm}^3$ of the mixture of P and W into one of the test-tubes.
3. Put the bung (with the delivery tube attached) into this test-tube.
4. Put the end of the delivery tube into the large beaker containing water labelled T.
5. Start timing and count the number of bubbles of oxygen released in 2 minutes.
6. Record the result in (b)(ii).

Note: if no bubbles are released then make sure the bung is securely fitted into the test-tube. You may ask for petroleum jelly if necessary.

1. Put $10\ \text{cm}^3$ of the mixture of P with the lowest concentration of X into another test-tube containing H.
2. Repeat steps 7 to 10.
3. Repeat steps 11 and 12 with each of the other concentrations of X, including 0.3% X.

Consider how you will obtain results which are as accurate as possible.

Prepare the space below and record your results.

You are required to find the rate of activity of the catalase by measuring the time taken to collect $2\ \text{cm}^3$ of oxygen produced by the hydrolysis of H.

You are going to collect the oxygen released by displacement of water as shown in Fig. 1.2.

The sealed syringe is full of water and is upside down over the end of the delivery tube.

You need to time how long it takes for the bubbles of oxygen to push (displace) $2\ \text{cm}^3$ of the water out of the syringe.

1. Put $5\ \text{cm}^3$ of H into a clean test-tube.
2. Put $10\ \text{cm}^3$ of the mixture of P and W into this test-tube.
3. Put the bung (with the delivery tube attached) into this test-tube.
4. Fill the sealed syringe with water from the beaker and turn it upside down keeping the open end of the syringe under the water as shown in Fig. 1.2.
5. Immediately put the end of the delivery tube into the beaker of water so that the bubbles of oxygen pass into the syringe.
6. Start timing.

Record the time for $2\ \text{cm}^3$ of oxygen to be collected.

time = ______

Using your recorded time, calculate the rate of activity of the catalase in $\text{cm}^3\,\text{s}^{-1}$.

You may lose marks if you do not show your working and do not use the appropriate units.

rate of activity = ______ $\text{cm}^3\,\text{s}^{-1}$

This first procedure investigated the effect of the concentration of X on the activity of catalase in the plant extract.

To modify this procedure for investigating another variable, the independent variable (concentration of X) would need to be standardised.

Describe how the independent variable (concentration of X) will be standardised.

______

Consider how you would modify this procedure to investigate the effect of temperature on the activity of the catalase in the plant extract.

Describe how the independent variable, temperature, will be investigated.

Prepare the space below and record your observations.

The rate at which enzyme E catalyses a reaction with S can be investigated by measuring the rate at which the products of the reaction are produced.

You are required to identify the type of biological molecule produced (the product) when S and E are mixed.

1. Put $4\text{ cm}^3$ of Benedict’s into the test-tube containing the mixture of S and E. This will inhibit enzyme E.
2. Put this test-tube into the boiling water-bath and immediately start timing.

Record the time taken for the first appearance of any colour change. If there is no colour change after 180 seconds remove the test-tube from the water-bath and record your result.

1. Turn off the Bunsen burner.

Explain the effect that enzyme E has on starch, using your observations from (a)(i) and the result from (a)(ii).

When carrying out a practical procedure, the hazards of the use of all the apparatus and all of the reagents need to be considered, then the level of risk needs to be assessed as low or medium or high.

State the hazard with the greatest level of risk when carrying out steps 8 to 10.

State the level of risk of the procedure: low or medium or high.

hazard = ______

level of risk = ______

You are required to make a serial dilution of the 1% starch solution, S, which reduces the concentration of the starch solution by half between each successive dilution.

You will need to prepare $20\text{ cm}^3$ of each concentration.

You should use the beakers shown in Fig. 1.1 to show how you will prepare the serial dilutions.

For each beaker, complete Fig. 1.1 to show how you will dilute the solution by:

• stating, under the beaker, the concentration and volume of the starch solution available for use in the investigation
• using one arrow, with a label above the beaker, to show the concentration and volume of starch solution added to prepare the concentration
• using another arrow, with a label above the beaker, to show the volume of W added to prepare the concentration.

Fig. 1.1

Proceed as follows:

1. Prepare the concentrations of starch solution as stated in (b)(i).
2. Label test-tubes with the concentrations of starch solution you have prepared.
3. Put $3\text{ cm}^3$ of the lowest concentration of starch solution into the labelled test-tube.
4. Repeat step 13 with each of the other concentrations of starch solution.
5. Put $1\text{ cm}^3$ of E into each of the test-tubes.
6. Shake each test-tube gently to mix the contents.
7. Leave the test-tubes for 5 minutes, shaking them occasionally. Relight the Bunsen burner to prepare a boiling water-bath ready for step 19.
8. After five minutes, put $4\text{ cm}^3$ of Benedict’s into each of the test-tubes.
9. Put the test-tubes containing the two lowest concentrations of starch solution into the boiling water-bath and immediately start timing.
10. Record in (b)(ii) the time taken for the first appearance of any colour change.
    If there is no colour change after 180 seconds, remove the test-tubes from the water-bath and record ‘more than 180’.
11. Repeat steps 19 and 20 with the remaining test-tubes.

Prepare the space below and record your results, including the result from (a)(ii).

Fruit juice X contains an inhibitor of enzyme E. Consider how you would modify the procedure you have just carried out to investigate the effect of the concentration of X on the activity of enzyme E.

Describe how the independent variable in the procedure you have just carried out would be standardised in the new investigation.

Describe how the independent variable, concentration of X, will be investigated.

State which biological molecule(s) might diffuse through the wall of the Visking tubing.

[1]

You are provided with:

Fig. 1.1 shows the set-up of the apparatus.

You must now read up to the end of step 11 before proceeding.

Samples of the water surrounding the Visking tubing will be removed at 5 minute intervals for 15 minutes.

To compare the diffusion of any single biological molecule at each 5 minute interval the test for the biological molecule needs to be standardised.

For example, if you carried out the test for reducing sugars:

• one standardised variable is the volume of sample removed from water, e.g. $2\ \text{cm}^3$
• the dependent variable is measuring the time taken for the first colour change to appear.

State the other variables which would need to be standardised for the reducing sugars test, using only the reagents and apparatus provided.

Describe how you will standardise each variable.

[3]

Decide:

• the test (or tests) you will carry out on the water
• the volume of the water you will need to sample for the test (or tests) at each time interval.

You may find it helpful to calculate the total volume of water needed for all the tests.

Draw on Fig. 1.1 (on page 4) the level of the water:

• before you remove any samples (label this 'before')
• after the total volume of water needed to sample for all the tests has been removed (label this 'after').

[2]

Proceed as follows:

1. Tie a knot in the Visking tubing as close as possible to one end so that it seals the end.
2. To open the other end, wet the Visking tubing and rub the tubing gently between your fingers.
3. Put $5\ \text{cm}^3$ of P into the open end of the Visking tubing.
4. Rinse the outside of the Visking tubing by dipping it into the water in the container labelled V.
5. Put the Visking tubing into a small beaker or container as shown in Fig. 1.1.
6. Make sure the open end of the Visking tubing is held in place by a paperclip.

You will start timing as soon as you add W.

Read steps 7 to 11 before proceeding.

1. Put W into the small beaker to the level you decided in (iii).
2. Immediately start timing and remove the first sample of water into a separate container to keep for the tests.
3. After 5 minutes, remove the next sample into a different container.
4. Repeat step 9 for two more samples.
5. Use the reagents and apparatus provided to identify the biological molecule(s) that you decided in (a)(i) may be present in the samples.

Prepare the space below and record your results.

[5]

Suggest how you would modify this investigation to investigate the concentration of reducing sugars in P.

[3]

When carrying out a practical procedure, the hazards of using the solutions need to be considered. Then the level of risk needs to be assessed as low or medium or high.

State the hazard with the greatest level of risk when using the solutions, then state the level of risk of the procedure: low or medium or high.

hazard ______

level of risk ______

Table 1.1 shows how to make up two of the concentrations of protein solution you will use.

Decide which concentrations of protein solution to prepare using simple dilution of the 1.0% protein solution, P.

Complete Table 1.1 to show how you will prepare the other concentrations.

Table 1.1

Proceed as follows:

1. Prepare the concentrations of protein solution, as shown in Table 1.1, in the beakers provided.
2. Put $1\ \text{cm}^3$ of the 1.0% protein solution into a test-tube.
3. Put $1\ \text{cm}^3$ of K into the same test-tube. Shake gently to mix.
4. Using the syringe labelled C, put $1\ \text{cm}^3$ of C into the same test-tube. Shake gently to mix.

You are required to standardise this test (step 2 to step 4) to be able to use the results to estimate the concentration of protein in U.

State which variable you will need to standardise when testing the other protein solutions and U.

Read step 5 to step 8 before proceeding.

1. Carry out the standardised test for each of the protein solutions prepared in step 1.
2. Record your observations of the colour of each solution in (b)(iii).
3. In a test-tube rack, put the test-tubes in order of palest blue to darkest purple.
4. Record in (b)(iii) each colour as a number using the scale shown in Fig. 1.3.

Prepare the space below and record your observation for each test-tube as a colour and its number, using the scale in Fig. 1.3.

State two of the variables which need to be standardised when using this method to compare different samples of urine.

Describe a method which can be used to standardise each of these variables.

variable 1 ______
description ______

variable 2 ______
description ______

Complete Table 1.2 to show three other temperatures that you will use to show the effect of temperature on the activity of enzyme E.

Table 1.2

Carry out step 1 to step 14.

step 1 Set up a water-bath ready for step 6. The starting temperature of the water-bath should be $30^\circ\text{C}$, as shown in Table 1.2.
step 2 Put $10\,\text{cm}^3$ of M into a test-tube.
step 3 Repeat step 2 so that you have two test-tubes containing M.
step 4 Put $1\,\text{cm}^3$ of C into each test-tube.
step 5 Gently shake the test-tubes to mix M and C.
step 6 Put the test-tubes into the water-bath and leave for 3 minutes.

Explain why the test-tubes are left in the water-bath for 3 minutes in step 6.

step 7 Remove one of the test-tubes from the water-bath.

The process of coagulation will start when E is added to the test-tube.

step 8 Put $1\,\text{cm}^3$ of E into the test-tube, so that it runs down the side of the test-tube and forms a layer on the surface of the mixture, as shown in Fig. 1.2.

step 9 Gently shake the test-tube to mix the solutions and start timing.
step 10 Rotate the test-tube and continue to rotate it while observing the mixture until the end-point is reached. Stop timing when the end-point is reached.

If the end-point has not been reached by 180 seconds, stop timing and record this as 'more than 180'.

step 11 Record in (a)(iii) the time to reach the end-point.
step 12 Repeat step 7 to step 11 with the other test-tube in the water-bath.
step 13 Set up the water-bath at the next temperature after $30^\circ\text{C}$ stated in Table 1.2.
step 14 Repeat step 2 to step 13 for each temperature stated in Table 1.2.

Record your results in an appropriate table.

State the independent variable in this experiment.

Suggest a suitable control for this investigation to show that it is the enzyme E that coagulates the milk.

The procedure described by step 1 to step 14 investigated the effect of temperature on the activity of enzyme E, using the time taken to reach the end-point.

Describe how you would modify the procedure to investigate the effect of changing the concentration of milk on the time taken to reach the end-point.

Decide which three further concentrations of triglycerides to make, then complete Table 1.1.

• The difference between each concentration should be 1%.
• You will need to make up $20\ \text{cm}^3$ of each concentration.

Table 1.1

1. Prepare all the concentrations of triglycerides as in Table 1.1 in the containers provided.
2. Put $20\ \text{cm}^3$ of A into each of the concentrations which you prepared in step 1 and mix well.
3. Put $3\ \text{cm}^3$ of each mixture, as made in Step 2, into separate test-tubes.
4. Put five drops of P into each of the test-tubes and gently shake so that all the mixture turns pink. (Note that each of the concentrations might not be the same shade of pink).
5. Set up a water-bath and adjust the temperature of the water to between $45\ ^\circ\text{C}$ and $50\ ^\circ\text{C}$. You will need to add hot water/cold water to maintain the temperature of the water-bath between $45\ ^\circ\text{C}$ and $50\ ^\circ\text{C}$ for steps 6 to 9.
6. Put the test-tubes from Step 4 into the water-bath for five minutes.
7. Put $3\ \text{cm}^3$ of M in a test-tube to act as a standard to help you recognise the end-point. Put this test-tube in the water-bath.

When you start the timer after adding E to the first test-tube in Step 8, you must not stop the timer at any of the end-points, just record the time.

The reaction will start as soon as you add E, so read steps 8 to 13 before proceeding.

Continue as follows:

1. Put $2\ \text{cm}^3$ of E into the test-tube containing the lowest concentration of triglycerides and mix well, then return it to the water-bath.
2. Start timing and record start time on Fig. 1.2.
3. Immediately, put $2\ \text{cm}^3$ of E into the next test-tube containing next highest concentration of triglycerides and mix well, then return it to the water-bath.
4. Record start time on Fig. 1.2.
5. Immediately repeat steps 10 and 11 for the remaining concentrations.
6. Observe the four test-tubes and record the time on Fig. 1.2 when each end-point is reached. This is your raw data.

Using the colour of M may help you recognise the end-point.

If the time taken to reach the end-point for any one concentration is longer than 5 minutes record 'more than 300'.

Fig. 1.2

From your timer readings you will be required to calculate the time taken to reach the end-point in each test-tube.

1. Process your raw data to find the time taken to reach the end-point for each concentration (these are your processed results).

If you have time check your results.

Prepare the space below to record your processed results.

Describe how you could set up a control for this investigation using the apparatus provided.

A student carried out an investigation into the effect of immobilised lipase on the hydrolysis of triglycerides in milk. The student used the apparatus in Fig. 1.3.

Fig. 1.3

Identify two variables that the student would need to standardise to compare the activity of different concentrations of lipase immobilised in alginate beads.

Describe how one of these variables would be standardised.

Complete Table 1.2 to show the sizes of agar cubes you will investigate.

The first row, for the largest cube, has been completed for you.

Table 1.2

Carry out step 1 to step 13.

Use the blunt forceps and paper towel to handle the pieces of agar.

1. Draw a grid on the graph paper as shown in Fig. 1.2.

2. Put the graph paper into the plastic wallet and put it on the white tile.

3. Cut a small piece of agar from block B. Put the small piece of agar on the plastic wallet so that it is on top of the grid (drawn in step 1).

4. Cut the small piece of agar so that each side is $10\text{ mm}$, using the grid as a guide, as shown in Fig. 1.3.

You will need to turn the piece of agar to make sure that each side is cut to $10\text{ mm}$.

1. Label a beaker 10 x 10 x 10 and put the agar cube you have cut into this beaker.
2. Put any waste pieces of agar into the container labelled For waste.
3. Remove the graph paper from the plastic wallet.
4. Draw on the graph paper a grid for each of the sizes of agar cube as stated in Table 1.2.
5. Repeat step 2 to step 6 with each of the sizes of agar cube as stated in Table 1.2.
6. Put $20\text{ cm}^3$ of A into the beaker labelled 10 x 10 x 10.
7. Repeat step 10 with each of the other beakers you have labelled.
8. Start timing.
9. Record the time taken for each agar cube to reach the end-point (the agar cube has decolourised).

Do not stop the timer – keep it running continuously.

If any agar cube remains blue after 900 seconds, stop timing and record ‘more than 900’.

Record your results in an appropriate table.

State one way in which you could improve the confidence in your results.

Describe a suitable control for this investigation.

The procedure, described by step 1 to step 13, investigated the effect of surface area to volume ratio on diffusion, using the time taken to reach the end-point.

Think about how you could modify this procedure to investigate the effect of ascorbic acid concentration on the time taken to reach the end-point.

The size of the agar cube should be standardised.

Look at your results in (a)(ii) and state a size of agar cube that is appropriate to use.

size of agar cube = ______

Describe how you would modify the procedure to investigate the effect of ascorbic acid concentration on the time taken to reach the end-point.