Required practical 6: aseptic technique and antimicrobials - AQA A-Level Biology (Paper 3) Exam Questions

Practise 16 exam-style questions on Required practical 6: aseptic technique and antimicrobials for AQA A-Level Biology (Paper 3). Write your answer as you would in the exam and get it marked in seconds against the mark scheme, point by point, with the Study Helper on hand if you get stuck.

16 questions
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16 questions, marked as you go

Sample questions with model answers

Here are 4 of the questions in this topic, each with a model answer, an explanation of why it earns the marks and the mark scheme an examiner would use.

Question 1

2 marks

A student suggested testing a disinfectant on bacteria collected by wiping a sterile swab over a door handle, instead of using the culture of Escherichia coli (strain K12) provided.

Explain why her teacher did not allow this.

Model answer

Bacteria from a door handle are of unknown species and could include pathogens. Incubating them would multiply them into very large numbers, which would be a serious risk of infection. The E. coli K12 strain provided is known not to cause disease, so it is safe to use.

Explanation

School investigations use only cultures of microorganisms known to be safe, such as E. coli K12. Samples from the environment may contain pathogens, and culturing them produces huge numbers, so the risk is much greater than touching the original surface.

Mark scheme

1 mark
The swab could pick up pathogens / harmful microorganisms, and the species collected would be unknown
Max 2. Accept 'the bacteria might cause disease'.
1 mark
Incubating them would produce very large numbers (of any pathogens present), increasing the risk of infection
Accept 'culturing increases the numbers of any pathogens present'.
1 mark
The strain provided (E. coli K12) is known not to cause disease / is safe for school use

Question 2

3 marks

Two groups of students tested the same disinfectant on the same species of bacterium, using paper discs on lawns of bacteria. The clear zones of one group were much smaller than those of the other group. The group with the smaller zones had poured 30 cm³ of agar into each Petri dish; the other group had poured 15 cm³.

Suggest and explain how the volume of agar in each dish could affect the diameter of the clear zone.

Suggest one other variable, apart from the volume of agar, that could explain the difference between the groups' results.

Model answer

In the dishes with 30 cm³ the agar is deeper, so the disinfectant diffuses into a larger volume of agar, downwards as well as outwards. Its concentration therefore falls more quickly with distance from the disc, so it inhibits growth over a smaller area and the clear zone is smaller.

Another variable is the density of bacteria in the culture each group spread: a denser lawn needs more disinfectant to inhibit it, giving a smaller clear zone.

Explanation

The size of a clear zone depends on how far the substance diffuses through the agar at a concentration high enough to inhibit growth, so anything that changes that diffusion or the number of bacteria to be inhibited affects the result. Agar depth, culture density, the volume on each disc and incubation conditions must all be standardised before results from different plates can be compared.

Mark scheme

1 mark
In deeper / thicker agar the disinfectant diffuses into a larger volume of agar (downwards as well as outwards)
Accept 'the disinfectant is spread through more agar'.
1 mark
So its concentration falls more quickly with distance from the disc, so it inhibits growth over a smaller area / the clear zone is smaller
1 mark
The number / density of bacteria in the culture spread (e.g. the age of the broth culture) OR the volume of culture spread OR the volume of disinfectant on each disc / the time the discs were soaked OR the incubation temperature / time OR the time between spreading the lawn and adding the discs
One variable is needed. Ignore 'type of bacteria' or 'type of disinfectant' (the same in both groups).

Question 3

4 marks

A student placed a disc containing antibiotic A on a lawn of bacteria. After incubation there was a clear zone around the disc. Inside the clear zone, close to the disc, four small colonies had grown.

Suggest an explanation for the four colonies inside the clear zone.

Describe how the student could use the colonies to test whether these bacteria are resistant to antibiotic A.

Model answer

The bacteria in these colonies probably had a random mutation, giving an allele for resistance to antibiotic A. The antibiotic killed or stopped the growth of the other bacteria, but these resistant bacteria survived and divided to form colonies. (Alternatively, the colonies could be a contaminating species that A does not affect.)

Using a sterile loop, she could transfer bacteria from one colony to make a new lawn on an agar plate and place a disc of antibiotic A on it, alongside a plate of the original bacteria with the same disc. If there is no clear zone, or a much smaller one, around the disc on the new lawn, the bacteria are resistant.

Explanation

Resistance arises by random mutation before the bacteria meet the antibiotic; the antibiotic then selects the resistant bacteria, which survive and reproduce. This is directional selection. To test an explanation, culture the bacteria aseptically and expose them to the antibiotic again, comparing them with the original strain.

Mark scheme

1 mark
The bacteria in these colonies had a (random) mutation / an allele / a gene (e.g. on a plasmid) that gives resistance to antibiotic A
Points 1 to 3: max 2. Points 4 and 5: max 2. Reject 'the antibiotic caused the mutation'.
1 mark
These bacteria survived and divided (by binary fission) to form colonies, while the bacteria without resistance were killed / could not divide (selection)
Accept 'the resistant bacteria had no competition'.
1 mark
The colonies could be contaminants (a different species of microorganism) that antibiotic A does not affect
Accept as an alternative explanation.
1 mark
Use aseptic technique (e.g. a sterile loop) to transfer bacteria from one of these colonies to a new lawn / broth AND test them with a disc of antibiotic A / grow them on agar containing antibiotic A
Both the transfer and the test with antibiotic A are needed.
1 mark
Compare with the original bacteria treated in the same way: no clear zone / a smaller clear zone / growth on agar with A shows they are resistant
The comparison or the expected result is needed.

Question 4

6 marks

You are given a broth culture of a bacterium, a disinfectant, bottles of sterile water, sterile pipettes, sterile spreaders and plates of sterile nutrient agar.

Describe how you would use aseptic technique to find out the effect of the disinfectant on the number of living bacteria in the broth culture.

Do not describe how to dispose of the cultures.

Model answer

Add 1 cm³ of disinfectant to 9 cm³ of the broth culture, and 1 cm³ of sterile water to another 9 cm³ as a control, and leave both for 30 minutes at 25 °C. Working close to a Bunsen flame and flaming the neck of each bottle, use a new sterile pipette to transfer 1 cm³ of the treated culture into 9 cm³ of sterile water and mix to give a 10⁻¹ dilution; repeat to make 10⁻² to 10⁻⁶ dilutions. Do the same for the control. Lifting each lid only slightly, spread 0.1 cm³ of each dilution over an agar plate with a sterile spreader. Tape each lid on with two strips of tape and incubate the plates upside down at 25 °C for 48 hours. Count the colonies on plates with 30 to 300 separate colonies, and calculate bacteria per cm³ = colonies ÷ 0.1 × dilution factor. Use three plates for each dilution, calculate a mean and compare the treated culture with the control.

Explanation

This is the quantitative part of required practical 6: the disinfectant's effect is measured as the number of living bacteria left, found by serial dilution and counting colonies. Serial dilution is needed because a culture contains far too many bacteria to count. Aseptic technique keeps the cultures pure and protects the user, a control without disinfectant gives the comparison, and the calculation uses the volume spread and the dilution factor.

Mark scheme

1 mark
Add the disinfectant to a sample of the broth culture AND add the same volume of sterile water to another sample (as a control)
Max 4 if no serial dilution is described. Max 6.
1 mark
Leave both for the same time at the same temperature (e.g. 30 minutes at 25 °C)
1 mark
Make a serial dilution, e.g. transfer 1 cm³ into 9 cm³ of sterile water, mix, and repeat to give 10⁻¹, 10⁻², 10⁻³ and so on
Accept any correct description of serial dilution.
1 mark
Use a new sterile pipette for each transfer
1 mark
Flame the necks of the bottles / work close to a Bunsen flame / lift the lid of each plate only slightly / at an angle
Any one of these aseptic techniques for this mark.
1 mark
Spread a known volume (e.g. 0.1 cm³) of each dilution over an agar plate with a sterile spreader
1 mark
Tape the lid on (not sealed all round) and incubate the plates (upside down) at 25 °C (for 24-48 hours)
Reject incubation at 37 °C.
1 mark
Count the colonies on a plate with separate colonies (e.g. 30 to 300)
1 mark
Calculate the number of bacteria per cm³ = number of colonies ÷ volume spread × dilution factor, and compare the treated culture with the control
1 mark
Repeat (several plates for each culture) and calculate a mean

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