The bacterium Streptococcus pneumoniae is commonly found in the throat. S. pneumoniae produces hydrogen peroxide as it grows. A sample can be taken from a patient's throat and tested to measure the concentration of hydrogen peroxide. This can be used as a measure of the growth of the bacteria. You will determine the growth of bacteria by measuring the concentration of hydrogen peroxide in a solution that represents a sample taken from a patient. You will do this by measuring how long it takes for a sample of hydrogen peroxide to cause a colour change in a reaction mixture. The faster the mixture changes to a blue-black colour, the higher the concentration of hydrogen peroxide, and the greater the growth of bacteria. You will use a range of known concentrations of hydrogen peroxide to estimate the concentration of hydrogen peroxide in a sample. You are provided with the materials shown in Table 1.1. Table 1.1 labelled | contents | hazard | volume/cm³ ---|---|---|--- R1 | dilute sulfuric acid | irritant | 100 R2 | starch solution | low | 10 R3 | potassium iodide solution | low | 10 R4 | sodium thiosulfate solution | low | 10 H | 2.0% hydrogen peroxide solution | irritant | 25 U | solution representing patient sample | irritant | 10 W | distilled water | low | 100 If any solution comes into contact with your skin, wash off immediately with cold water. It is recommended that you wear suitable eye protection. You will need to carry out a serial dilution of the 2.0% hydrogen peroxide solution, H, to reduce the concentration by half between each successive dilution. You will need to prepare four concentrations of hydrogen peroxide solution in addition to the 2.0% hydrogen peroxide solution, H. After the serial dilution is completed, you will need to have 10cm³ of each concentration available to use.

1. (a)(i)Complete Fig. 1.1 to show how you will prepare your serial dilution. Each beaker should have: • a labelled arrow to show the volume of hydrogen peroxide solution transferred • a labelled arrow to show the volume of distilled water, W, added • a label under the beaker to show the concentration of the hydrogen peroxide solution. [Figure 1.1][3]
2. (a)(ii)Carry out step 1 to step 17. step 1 Prepare the concentrations of hydrogen peroxide solution as shown in Fig. 1.1. step 2 Label test-tubes with the concentrations prepared in step 1. step 3 Put 10 cm³ of R1 into each test-tube. step 4 Put 1 cm³ of R2 into each test-tube. step 5 Put 1 cm³ of R3 into each test-tube. step 6 Put 1 cm³ of R4 into each test-tube. step 7 Stir the contents of each test-tube with a glass rod. step 8 Put 1 cm³ of the 2.0% hydrogen peroxide solution into the appropriately labelled test-tube. step 9 Stir the contents of the test-tube and immediately start timing. step 10 Record in (a)(ii) the time taken for a blue-black colour to appear. If the blue-black colour does not appear after 180 seconds, stop timing and record as 'more than 180'. step 11 Repeat step 8 to step 10 with the other concentrations of hydrogen peroxide solution prepared in step 1. Record your results in an appropriate table.[5]
3. (a)(iii)A sample can be taken from a patient's throat and tested to measure the concentration of hydrogen peroxide. You will be testing a solution, U, that represents a sample taken from a patient's throat. You will need to estimate the concentration of hydrogen peroxide in U. This can be used as a measure of the growth of bacteria. step 12 Label 3 test-tubes U1, U2 and U3. step 13 Repeat step 3 to step 7. step 14 Add 1 cm³ of U to test-tube U1. step 15 Stir the contents of the test-tube and immediately start timing. step 16 Record in (a)(iii) the time taken for a blue-black colour to appear. step 17 Repeat step 14 to step 16 using test-tubes U2 and U3. Record the time taken for a blue-black colour to appear. U1 = U2 = U3 =[1]
4. (a)(iv)Calculate the mean time taken for the blue-black colour to appear for sample U. Show your working. mean time =[1]
5. (a)(v)Use your results in (a)(ii) and (a)(iv) to estimate the concentration of hydrogen peroxide in sample U. concentration of hydrogen peroxide in sample U =[1]
6. (a)(vi)Explain why repeating the measurement for sample U allows you to have more confidence in your estimate.[1]
7. (a)(vii)With reference to your estimate for sample U, describe one other modification to the procedure that would allow a more accurate estimate of the concentration of hydrogen peroxide in sample U.[1]
8. (b)(i)Scientists investigated the effect of temperature on hydrogen peroxide production in a different species of bacterium, Streptococcus pyogenes. Cultures of the bacterial cells were incubated at 2 different temperatures for 168 hours. The percentage of bacterial cells that were able to produce hydrogen peroxide was measured. The results are shown in Table 1.2. Table 1.2 time/hours | percentage of bacterial cells able to produce hydrogen peroxide | bacterial cells at 20°C | bacterial cells at 37°C 24 | 6.0 | 3.0 48 | 10.0 | 3.0 72 | 12.0 | 3.2 96 | 16.0 | 5.2 168 | 19.8 | 5.4 Plot a graph of the data in Table 1.2 on the grid in Fig. 1.2. [Figure 1.2][4]
9. (b)(ii)State two conclusions from the results of the investigation at the 2 temperatures.[2]
10. (b)(iii)A sample was taken at 20°C that showed 14.5% of the bacteria were able to produce hydrogen peroxide. Use your graph in Fig. 1.2 to estimate when the sample was taken. Show on your graph how you obtained your estimate, and give your answer to the nearest hour. sample taken hours[2]
11. (b)(iv)State one variable that the scientists would need to keep constant so that the results at the 2 temperatures could be compared.[1]

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