Microbiology

1 Plan and risk assessment

Each antiseptic diffuses out of its paper disc into the agar, getting weaker as it spreads. Visible bacterial growth is inhibited where the antiseptic is sufficiently concentrated, so a clear zone (a zone of inhibition) forms round the disc while a cloudy lawn grows everywhere else. Zone size depends on growth inhibition and diffusion through the agar; clear agar alone does not prove that bacteria were killed.

area = π r²

Prediction

2 Apparatus

• ✓Sterile nutrient agar plate The agar is food for the bacteria; the Petri dish keeps them in.
• ✓Culture of E. coli (a safe strain made for schools) in a bottle The bacteria you grow as a lawn.
• ✓Sterile plastic pipette Moves a few drops of culture onto the agar.
• ✓Glass spreader Spreads the culture evenly over the agar.
• ✓Small beaker of ethanol, with a lid The spreader is dipped in it, then flamed, to sterilise it.
• ✓Bunsen burner on a heatproof mat Sterilises the spreader and forceps; its updraught keeps microbes away from the plate.
• ✓Filter-paper discs (6 mm) Each one carries a liquid onto the agar.
• ✓Mouthwash, TCP, antiseptic cream and sterile water The antiseptics to compare, and water for the control disc.
• ✓Forceps Pick up and place the discs without touching them.
• ✓Clear sticky tape Holds the lid on with two short strips.
• ✓Wax pencil Labels the base of the plate.
• ✓Disinfectant spray and antibacterial hand wash Kills microorganisms on the bench and your hands.
• ✓Discard pot of disinfectant Used pipettes, spreader and forceps go in it.
• ✓Incubator set to 25 °C Keeps the plate warm for 48 hours so the bacteria grow.
• ✓30 cm ruler (resolution 1 mm) Measures each clear zone’s diameter through the base.
• ✓Safety goggles Protect your eyes from antiseptic splashes.

3 Method: your choices

• Which version are you doing?: AQA required practical 2: antiseptics and bacterial growthMouthwash, TCP and antiseptic cream on the discs.
• Disinfect the bench and wash your hands: DoneThe bench and your hands carry microorganisms. Disinfecting kills them, so they cannot get onto the agar and grow there.
• Put on safety gogglesSafety: DoneThe liquids on the discs can sting your eyes if they splash, and you are working next to a flame and ethanol.
• Light the Bunsen and work beside it: DoneThe flame heats the air above it, so the air rises. This updraught carries microorganisms in the air up and away from the open plate.
• Label the plate: On the base, underneath the agarThe labels stay with the agar, so each label is always next to its own disc.
• Open the plate: Lift the lid at an angle, open only on the Bunsen sideThe lid shields the agar from the air above it. Only a little air reaches the agar, and the updraught is on the open side.
• Spread the bacteria: Dip, flame, then hold it near the flame for 10 seconds to coolThe spreader is sterile and cool, so it spreads the bacteria without killing them: an even lawn.
• Soak the discs: Lift each disc out and touch it on the side of the dish so the extra drains offEvery disc holds about the same small volume of liquid: a fair test.
• Flame the forceps: DoneFlaming kills microorganisms on the tips, and stops one liquid being carried to the next disc.
• Place the discs: One in each quarter, about 2 cm from the edgeThe discs are about 3 cm apart. Every zone has room to form on its own, so each one can be measured.
• Tape the lidSafety: Two short strips across the edge, on opposite sidesThe lid cannot fall off, and small gaps let oxygen in, so the conditions stay aerobic.
• Set up the incubator: Upside down (lid at the bottom), at 25 °CCondensation forms on the lid, below the agar, and cannot drip onto it. At 25 °C, bacteria that cause disease in humans do not grow well.
• Where do you measure the zones?: Upside down on a sheet of black cardThe clear zones look black and the lawn looks cream, so each edge is sharp and easy to read.
• How many times do you measure each zone?: Twice: one diameter, then another at right angles to it, then the meanThe mean of two diameters at right angles allows for zones that are slightly oval, and shows up a reading that does not fit.

Anomaly: No anomalous reading.

6 Analysis

1. Area: Mouthwash

   area = π r² (r = mean diameter ÷ 2)

   Work out the area of the clear zone round the mouthwash disc, from its mean diameter.

   133 mm² Right first time

2. Area: TCP

   area = π r² (r = mean diameter ÷ 2)

   Work out the area of the clear zone round the TCP disc, from its mean diameter.

   241 mm² Right first time

3. Area: Antiseptic cream

   area = π r² (r = mean diameter ÷ 2)

   Work out the area of the clear zone round the antiseptic cream disc, from its mean diameter.

   70.9 mm² Right first time

4. Why a bar chart

   Why do you show these results on a bar chart, not a line graph?

   The type of antiseptic is a categoric variable: there is nothing in between mouthwash and TCP. Right first time

5. Most effective antiseptic

   From your results, which antiseptic was the most effective against these bacteria?

   TCP Right first time

6. The water disc

   The water disc had no clear zone. What does that show?

   The paper disc on its own does not stop bacteria growing, so the zones are caused by the antiseptics. Right first time

7. How a clear zone forms

   3 of 3 right first time Right first time

7 Conclusion and evaluation

Conclusion

Sources of error

Improvement

Repeatable or reproducible

9 Exam questions on your data

On your own readings, self-marked against the mark scheme, with error carried forward where it applies. Total 30/30.

1. 01.1[2 marks]

   A student prepared an agar plate of bacteria to test three antiseptics.

   Give two aseptic techniques the student should use when preparing the plate.

   Your answer

   Pass the forceps through a Bunsen flame before using them. Lift the lid of the dish only a little, at an angle.

   2/2 self-marked

   • Sterilise (flame) the equipment, e.g. pass the forceps / spreader / loop through a flame, or flame the neck of the bottle
   • Disinfect the bench / work surface (or hands); ignore clean
   • Lift the lid only a little / at an angle
   • Secure the lid with (adhesive) tape
   • Work near a (Bunsen) flame (the updraught carries microbes away)
   • Use sterilised agar / Petri dish

   Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

   Model answer: Pass the forceps through a Bunsen flame before using them. Lift the lid of the dish only a little, at an angle.

2. 01.2[2 marks]

   The student incubated the plate at 25 °C for 48 hours. Hospital laboratories often incubate bacteria at 37 °C.

   Explain why school laboratories incubate plates at 25 °C and not at 37 °C.

   Your answer

   37 °C is human body temperature, where bacteria that are harmful to humans grow best. At 25 °C pathogens are much less likely to grow, so it is safer.

   2/2 self-marked

   • 37 °C is (human) body temperature, where (pathogenic) bacteria grow best (fastest)
   • At 25 °C, bacteria that are harmful to humans (pathogens) grow less / are less likely to grow (it is safer)

   Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

   Model answer: 37 °C is human body temperature, where bacteria that are harmful to humans grow best. At 25 °C pathogens are much less likely to grow, so it is safer.

3. 01.3[1 mark]

   The student stuck the lid to the base with two short strips of tape.

   Give one reason why the lid should not be sealed with tape all the way round.

   Your answer

   So that oxygen can still get in. Without oxygen, anaerobic bacteria that could be harmful might grow.

   1/1 self-marked

   • Oxygen (air) can still get in, so anaerobic bacteria (which may be pathogens) do not grow; allow keeps conditions aerobic

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: So that oxygen can still get in. Without oxygen, anaerobic bacteria that could be harmful to humans might grow.

4. 01.4[1 mark]

   The student also put a disc soaked in sterile water on the plate.

   Give the reason for the disc soaked in sterile water.

   Your answer

   It is a control, to show the paper disc on its own does not stop bacteria growing.

   1/1 self-marked

   • It is a control: something to compare with, showing that a clear zone is caused by the antiseptic and not by the paper disc or the water. A “control variable” is a different idea and is not credited

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: It is a control: it shows that the paper disc itself does not stop the bacteria growing, so any clear zone is caused by the antiseptic.

5. 01.5[1 mark]

   The student compared three antiseptics by measuring the clear zone round a disc of each.

   Which is a control variable in this investigation?

   Tick (✓) one box.

   • The type of antiseptic
   • The diameter of the clear zone
   • ✓ The volume of antiseptic on each disc (your choice)
   • The number of colonies on the plate

   Your answer

   The volume of antiseptic on each disc

   1/1

   • The volume of antiseptic on each disc

   Full marks. Clear and precise.

   Model answer: The volume of antiseptic on each disc. (The type of antiseptic is the independent variable; the diameter of the clear zone is the dependent variable.)

6. 01.6[1 mark]

   The glass spreader is dipped in ethanol and then passed through a Bunsen flame.

   Give one reason why the beaker of ethanol must be kept away from the Bunsen flame.

   Your answer

   Ethanol is highly flammable, so it could catch fire and cause burns.

   1/1 self-marked

   • Ethanol is (highly) flammable / could catch fire / cause burns; ignore dangerous

   Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

   Model answer: Ethanol is highly flammable, so it could catch fire and cause burns.

7. 02.1[1 mark]

   Table 1 shows your two diameters of the clear zone round the TCP disc.

   Table 1

   Antiseptic	Diameter 1 in mm	Diameter 2 in mm
   TCP	17	18

   Calculate the mean diameter of the clear zone round the TCP disc.

   Your answer

   17.5

   Working: (17 + 18) ÷ 2

   1/1

   • (17 + 18) ÷ 2 = 17.5 (mm)

   Full marks. Your answer meets all the points for this question.

   Model answer: (17 + 18) ÷ 2 = 17.5 mm

8. 02.2[3 marks]

   Use your answer to Question 02.1 to calculate the area of the clear zone round the TCP disc.

   Your answer

   241

   Working: r = 17.5 ÷ 2 = 8.75 area = π r² = 3.142 × 76.56

   3/3

   • radius = 17.5 ÷ 2 = 8.75 (mm)
   • area = π r² (allow 3.14 for π)
   • = 241 (mm²) (allow π taken as 3.14: 240; allow correct use of an incorrect mean)

   Full marks. Your answer meets all the points for this question.

   Model answer: r = 17.5 ÷ 2 = 8.75 mm area = π r² = π × 8.75² = 241 mm²

9. 02.3[1 mark]

   Give your answer to Question 02.2 in cm².

   Your answer

   2.41

   1/1

   • 241 ÷ 100 = 2.41 (cm²) (allow their answer ÷ 100)

   Full marks. Well done.

   Model answer: 241 ÷ 100 = 2.41 cm²

10. 02.4[1 mark]

    Another student measured each clear zone twice, at right angles. Table 2 shows their results.

    Figure 1 shows the mouthwash zone as it looked when it was measured.

    Which measurement is anomalous?

    Use Table 2 and Figure 1. Select one row.

    Table 2

    Antiseptic	Measurement	Diameter in mm
    Mouthwash	Diameter 1	12
    Mouthwash	Diameter 2	18
    TCP	Diameter 1	16
    TCP	Diameter 2	16
    Antiseptic cream	Diameter 1	8
    Antiseptic cream	Diameter 2	9

    Your answer

    Mouthwash, diameter 1

    1/1

    • Mouthwash, diameter 1: it disagrees with diameter 2 of the same zone, and Figure 1 shows a colony on its line

    Full marks. Your answer meets all the points for this question. Compare the two diameters of each zone: the mouthwash zone's (12 and 18 mm) disagree. Figure 1 shows which line a colony lies on.

    Model answer: Mouthwash, diameter 1: it disagrees with the other diameter of the same zone, and Figure 1 shows a colony inside the zone on the line of that diameter, so it was not measured to the zone's edge.

11. 02.5[1 mark]

    Figure 1 shows the clear zone round the Mouthwash disc. A small round colony is growing inside the clear zone.

    Suggest why diameter 1 of the clear zone round the Mouthwash disc is smaller than diameter 2.

    Your answer

    A colony of another microorganism grew inside the clear zone, so the diameter was measured to the colony instead of the edge of the zone.

    1/1 self-marked

    • A colony of another microorganism (a contaminant) grew in the zone, and the diameter was measured to the colony (not to the edge of the zone); allow the plate was contaminated

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: A colony of another microorganism (a contaminant) grew inside the clear zone, and the diameter was measured to the colony instead of the edge of the zone.

12. 02.6[2 marks]

    Table 3 shows your mean diameters.

    Table 3

    Antiseptic	Mean diameter of clear zone in mm
    Mouthwash	13.0
    TCP	17.5
    Antiseptic cream	9.5

    Which antiseptic was the most effective against these bacteria? Give a reason for your answer.

    Your answer

    TCP, because it had the largest clear zone, so it killed the most bacteria.

    2/2 self-marked

    • TCP
    • It has the largest (clear) zone / mean diameter / area, so it killed the most bacteria (stopped most growing); allow largest zone of inhibition

    Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

    Model answer: TCP: it had the largest mean diameter of clear zone (17.5 mm), so it killed the most bacteria.

13. 02.7[1 mark]

    Suggest why each clear zone was measured twice, at right angles, and a mean calculated.

    Your answer

    The clear zones are not always perfect circles, so the mean of two diameters at right angles is more representative.

    1/1 self-marked

    • The zones are not (always) perfectly circular (they may be oval / irregular), so a mean is more representative; allow reduces the effect of random error

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: The clear zones are not always perfect circles, so the mean of two diameters at right angles is more representative of the zone’s size.

14. 02.8[1 mark]

    The ruler has a resolution of 1 mm. The clear zone round the antiseptic cream was much smaller than the zone round TCP.

    Suggest why the ruler is less suitable for measuring the zone round the antiseptic cream.

    Your answer

    The zone is small, so 1 mm is a large percentage of its diameter.

    1/1 self-marked

    • The zone is small, so 1 mm is a large percentage (proportion) of its diameter; allow the percentage uncertainty (error) is larger

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: The cream’s zone is small, so a 1 mm difference is a large percentage of its diameter: the percentage uncertainty is bigger.

15. 02.9[2 marks]

    Another student measured the radius of each clear zone from the edge of the paper disc to the edge of the clear zone.

    Explain how this affects the areas they calculated.

    Your answer

    Each radius is too small because the radius of the disc is left out, so every area would be too small.

    2/2 self-marked

    • Each radius is too small (short by the radius of the disc, 3 mm) / the disc is not included
    • So every area is too small (an underestimate); a systematic error

    Self-marked: 2 of 2 marks. This is your own judgement against the mark scheme.

    Model answer: Each radius is too small, because the radius of the paper disc (3 mm) is left out. So every area is too small: a systematic error.

16. 03.1[1 mark]

    A scientist tested four antibiotics, A, B, C and D, on the same species of bacteria. Figure 2 shows the plate after incubation.

    What does the result for antibiotic D show?

    Your answer

    The bacteria are resistant to antibiotic D, so it does not kill them.

    1/1 self-marked

    • The bacteria are resistant to antibiotic D / D does not kill the bacteria (they grow right up to the disc)

    Self-marked: 1 of 1 mark. This is your own judgement against the mark scheme.

    Model answer: The bacteria are resistant to antibiotic D: it does not kill them, so they grow right up to the disc.

17. 03.2[2 marks]

    In good conditions, a bacterium divides once every 40 minutes.

    Calculate the number of bacteria produced from one bacterium after 10 hours.

    Give your answer in standard form.

    Your answer

    3.28 × 10⁴

    Working: 10 × 60 ÷ 40 = 15 divisions, 2^15

    2/2

    • number of divisions = 10 × 60 ÷ 40 = 15
    • 2^15 = 3.3 × 10⁴ (allow 32768)

    Full marks. That is exactly what the mark scheme is looking for.

    Model answer: number of divisions = 10 × 60 ÷ 40 = 15 number of bacteria = 2^15 = 32 768 = 3.3 × 10⁴

18. 04[6 marks]

    A student wants to compare how well three antiseptics stop the growth of bacteria. They have an agar plate, a culture of bacteria, paper discs and the three antiseptics.

    Describe a method the student could use.

    Include how the student should keep the investigation safe and avoid contamination.

    Your answer

    1. Spray the bench with disinfectant and wash your hands. 2. Light a Bunsen burner and work close to the flame. 3. Flame the neck of the culture bottle and use a sterile pipette to put a few drops of bacteria on the agar, lifting the lid only a little at an angle. 4. Dip a glass spreader in ethanol, flame it, let it cool, then spread the bacteria evenly over the agar. 5. Soak paper discs of the same size in each antiseptic for the same time, and one in sterile water as a control. 6. Use flamed forceps to put the discs on the agar, evenly spaced apart. 7. Tape the lid on with two strips of tape and label the base. 8. Incubate upside down at 25 °C for 48 hours. 9. Measure the diameter of the clear zone round each disc twice at right angles, and calculate the mean. 10. Calculate each area using π r². The antiseptic with the largest clear zone is the most effective.

    6/6 self-marked

    • Disinfect the bench (and hands)
    • Sterilise equipment in a flame (forceps / spreader / loop / bottle neck) or use sterile equipment
    • Work near a Bunsen flame
    • Lift the lid only a little / at an angle
    • Spread the bacteria (culture) evenly over the agar
    • Soak paper discs (same size, same time) in each antiseptic
    • A control disc soaked in (sterile) water
    • Place the discs on the agar (with forceps), spaced apart
    • Tape the lid on (not sealed all the way round) / label the base
    • Incubate at 25 °C (not 37 °C), for about 48 hours (upside down)
    • Measure the diameter (or radius) of the clear zone round each disc
    • Two diameters at right angles, then a mean (or repeat plates and a mean)
    • Calculate the area (π r²) and compare: largest zone = most effective

    Self-marked: Level 3, 6 of 6 marks. You ticked 13 of 13 indicative points. This is your own judgement against the mark scheme.

    Model answer: 1. Spray the bench with disinfectant and wash your hands. 2. Light a Bunsen burner and work close to the flame. 3. Flame the neck of the culture bottle and use a sterile pipette to put a few drops of bacteria on the agar, lifting the lid only a little at an angle. 4. Dip a glass spreader in ethanol, flame it, let it cool, then spread the bacteria evenly over the agar. 5. Soak paper discs of the same size in each antiseptic for the same time, and one in sterile water as a control. 6. Use flamed forceps to put the discs on the agar, evenly spaced apart. 7. Tape the lid on with two strips of tape and label the base. 8. Incubate upside down at 25 °C for 48 hours. 9. Measure the diameter of the clear zone round each disc twice at right angles, and calculate the mean. 10. Calculate each area using π r². The antiseptic with the largest clear zone is the most effective.

Unseen data: the same skills, new apparatus

1. 01[2 marks]

   Use this new recorded mean diameter from the separate mouthwash investigation.

   Approximate the zone as a circle. Include the paper disc in the outer circle; do not subtract its area.

   Diameter used for the circular approximation

   Diameter in mm
   13

   Calculate the area in mm². Use area = π × radius².

   Your answer

   133

   Working: 13 ÷ 2 = 6.5 π × 6.5²

   2/2

   • Radius = 6.5 mm.
   • 133 mm².

   Full marks. Well done.

   Model answer: Radius = 13 ÷ 2 = 6.5 mm. Area = π × 6.5² = 133 mm².

2. 02[1 mark]

   In another investigation, a clear zone from an antiseptic reaches the water-only disc.

   Which action improves the control comparison? Tick (✓) one box.

   • ✓ Repeat with the discs far enough apart for their zones to remain separate. (your choice)
   • Call every clear patch next to the water disc an effect of water.
   • Remove the water control from the investigation entirely.

   Your answer

   Repeat with the discs far enough apart for their zones to remain separate.

   1/1

   • Repeat with the discs far enough apart for their zones to remain separate.

   Full marks. Well done.

   Model answer: Separate the zones so the water-only treatment can be interpreted independently.

3. 03[2 marks]

   Use the recorded mean diameters on this separate plate. All zones were measured through the closed plate.

   New recorded mean diameters

   Antiseptic	Mean diameter in mm
   Mouthwash	13
   TCP	15.5
   Antiseptic cream	8

   Which two statements are supported? Tick (✓) two boxes.

   • ✓ TCP had the largest recorded circular zone area on this plate. (your choice)
   • ✓ The clear zones show inhibited visible growth. Diffusion and the organism tested limit comparisons of general effectiveness. (your choice)
   • Every bacterium in every clear zone was certainly killed.
   • These readings prove which product is best against all microorganisms in every setting.

   Your answer

   TCP had the largest recorded circular zone area on this plate.; The clear zones show inhibited visible growth. Diffusion and the organism tested limit comparisons of general effectiveness.

   2/2

   • TCP had the largest recorded circular zone area on this plate.
   • The clear zones show inhibited visible growth. Diffusion and the organism tested limit comparisons of general effectiveness.

   Full marks. Clear and precise.

   Model answer: TCP had the largest recorded diameter and hence largest circular area. This is evidence of growth inhibition under these conditions, not proof of killing or a universal ranking.