Required Practical 6, Aseptic Technique: AQA A Level Biology Exam Questions by Topic

AQARequired PracticalsExam Questions
Written by Tom Whitburn

Required practical 6 is the use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth. It sits in section 3.4.4 of the specification and turns up on both AS and A-level papers. You need to describe how to move a culture from a broth bottle onto an agar plate without contaminating it, explain why each step works, interpret the clear zones around discs or wells of disinfectant, antibiotic or plant oil, and use a dilution series to work back to the number of bacteria in the original culture.

The examiners' reports show where the marks go. Students list steps without saying why they matter, think the air rising from a Bunsen burner kills bacteria, write 'work quickly' instead of lifting the lid at an angle, and give 'fair test' when the answer is the same number of bacteria on each plate. Very few mix each dilution, and in the calculations only about 15% carry the dilution factor and the volume plated all the way through.

Start with the aseptic technique questions, then the clear zones and evaluations, and finish with the dilution series and calculations. Answer each question on paper, with a calculator, before you open the mark scheme. If you'd like help with this topic, my AQA masterclasses run online on Tuesdays at 5.15pm for Year 12 and on Wednesdays or Thursdays at 6.30pm for Year 13.

What you need from GCSE and Year 12

Each question on this page assumes you're secure on these GCSE and Year 12 ideas. If any of them feels shaky, revise it before you start.

  • GCSE: growing bacteria on nutrient agar plates and in nutrient broth, sterilising Petri dishes and inoculating loops, and why school cultures are incubated at 25 °C.
  • GCSE: the effect of antiseptics or antibiotics on bacteria, measured from the size of the clear zone around a disc using πr².
  • Year 12 (AQA 3.2.1.2): prokaryotic cells divide by binary fission. See the Prokaryotic cells and viruses page.
  • Year 12 (AQA RP 3 and AT c): making a dilution series by transferring a fixed volume into fresh liquid and mixing at each step. See the Required practicals 3 and 11 page.
  • Year 12 (AQA 3.4.4): antibiotic resistance arises by mutation and spreads by natural selection. See the Genetic diversity and adaptation page.

Key terms

These words come up in the questions and mark schemes. Examiners expect you to use them exactly.

  • Aseptic technique: working methods that stop unwanted microorganisms getting into a culture and stop the culture escaping into the lab.
  • Sterile: free of all living microorganisms, for example a pipette from an unopened pack or a loop flamed until red hot.
  • Agar plate: a Petri dish of solid nutrient agar on which bacteria grow as colonies or as a lawn.
  • Broth: a liquid nutrient medium; a liquid culture is bacteria growing in broth.
  • Colony: a visible group of bacteria on agar, assumed to have grown from one cell, so counting colonies estimates the number of cells plated.
  • Clear zone: the area around a disc or well where the antimicrobial substance has killed the bacteria; a larger area means a more effective substance.
  • Antimicrobial substance: a substance that kills or inhibits microorganisms, such as a disinfectant, antiseptic or antibiotic.
  • Upward air movement: the convection current above a lit Bunsen burner that carries airborne microorganisms away from the open plate; it does not kill them.
  • Positive control: a treatment known to work, such as a named antibiotic or disinfectant, to show the method can detect an effect.
  • Negative control: the same set-up without the antimicrobial substance, such as sterile water or oil without cinnamon.
  • Dilution factor: how many times a culture has been diluted; multiply the count by it, and by the volume conversion, to find the number in the undiluted culture.

What these questions cover (AQA required practical 6)

The questions come from the sixth required practical in the AQA AS and A-level Biology specification (7401/7402), and test these statements and practical skills:

  • Required practical 6: Use of aseptic techniques to investigate the effect of antimicrobial substances on microbial growth (every question on this page; the antimicrobial substances are disinfectants, antibiotics, cinnamon oil and two unnamed substances)
  • AT i use microbiological aseptic techniques, including the use of agar plates and broth
  • AT c use laboratory glassware apparatus for a variety of experimental techniques to include serial dilutions (one question asks you to describe a dilution series; the calculations use the dilution factor to work back to the undiluted culture)
  • PS 2.1 Comment on experimental design and evaluate scientific methods
  • PS 2.4 Identify variables including those that must be controlled
  • PS 3.3 Consider margins of error, accuracy and precision of data
  • MS 0.2 Recognise and use expressions in decimal and standard form
  • MS 0.3 Use ratios, fractions and percentages

How to use this page

Write your answer on paper before you open the mark scheme. Allow about 1.3 minutes per mark: the 18 questions are worth 39 marks, so about 51 minutes in total. Then mark your answer strictly against the mark scheme and read the examiners' tip underneath it. If you'd rather print the questions, download the PDF pack.

Aseptic technique

Question 1

A level Paper 1 · June 2022 · Q02.3 · 3 marks · Easy

Describe the aseptic techniques for transferring 0.3 cm³ of a liquid culture from a bottle onto an agar plate.

Figure 2: bar chart of the growth rate of non-resistant and antibiotic-resistant C. difficile on agar with no carbohydrate or with trehalose added. Question asks how the scientists could use aseptic techniques to transfer 0.3 cm³ of C. difficile in liquid culture from a bottle onto an agar plate, 3 marks.
Show the mark scheme Mark scheme, any three: wash hands with soap or disinfect surfaces; use a sterile pipette or syringe; remove the bottle lid and flame the neck of the bottle; lift the lid of the plate at an angle; work close to upward air movement; use a sterile spreader; place the pipette or spreader into disinfectant straight after use. Reject a loop for transferring the culture.

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From the examiners' report: Two-thirds got 2 or 3 marks. A loop can't transfer a measured volume, so use a sterile pipette. Hold the lid at an angle rather than just working quickly.

Question 2

AS Paper 2 · June 2019 · Q06.1 · 3 marks · Medium

Describe and explain three ways to keep the technique aseptic when moving agar cubes of a fungus onto a new plate.

Question text: a scientist crossed pink-spored and white-spored strains of the fungus Neurospora by moving a small agar cube of each strain onto a new agar plate using aseptic techniques; describe and explain three ways he would ensure he used aseptic techniques, 3 marks.
Show the mark scheme Mark scheme, any three, each with its reason: wash hands or wear gloves to prevent contamination; Bunsen burning close by to create an upward current of air; disinfect the bench to kill microbes; flame the instrument to sterilise it; lift the lid slightly to prevent entry of microbes.

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From the examiners' report: About a third scored nothing because they didn't explain each step, or said the upward air current kills bacteria. Say how the loop or scalpel is sterilised, and keep to moving the cubes.

Question 3

AS Paper 2 · June 2025 · Q08.2 · 2 marks · Medium

Suggest one more aseptic step a trainee microbiologist took when culturing a bacterium, and justify it.

Question text: a trainee microbiologist disinfected the bench, used a sterile pipette to transfer S. pneumoniae from a broth bottle to an agar plate, spread it with a glass spreader and sealed the lid with four strips of tape; suggest one other action to make the technique aseptic and justify it, not referring to gloves, masks or safety glasses, 2 marks.
Show the mark scheme Mark scheme, mark as a matching pair: lift the lid at an angle, to prevent contaminating the dish; flame the spreader or the neck of the culture container, to keep a pure culture; work close to a Bunsen flame, to create a convection current that carries contaminants away; put the used pipette or spreader into disinfectant, so no bacteria reach the bench. Ignore wash hands.

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From the examiners' report: About a third got both marks. Don't repeat a step already listed. If you choose the Bunsen burner, say the rising air carries contaminants away from the dish.

Question 4

AS Paper 1 · June 2017 · Q02.1 · 2 marks · Hard

Explain why the agar was boiled and why the same volume of culture went onto each plate.

Question text: a student investigated the effect of three disinfectants on Lactobacillus; she boiled the agar before pouring the plates, transferred 0.5 cm³ of diluted liquid culture onto each plate, left some plates as controls and added disinfectants to others; explain the purpose of boiling the agar and of transferring the same volume of culture onto each plate, 2 marks.
Show the mark scheme Mark scheme: so no contamination or other bacteria (accept sterilisation or kills all bacteria); so the same number of bacteria is transferred to allow comparison.

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From the examiners' report: Only 7.6% got both marks. 'So it could be poured' copies the stem, and 'fair test' is too vague. Say the same volume gives the same number of bacteria on each plate.

Question 5

AS Paper 1 · June 2022 · Q08.1 · 2 marks · Medium

Explain why the E. coli count could have been lower if the pipette hadn't been sterilised.

Question text: a student used a sterilised pipette to transfer E. coli into three identical cultures, one with no antimicrobial and two with substances J and K, and counted the cells per mm³ after 24 hours; suggest and explain why the number of E. coli cells might have been lower if she had not used a sterilised pipette, 2 marks.
Show the mark scheme Mark scheme: unknown or different microorganisms introduced (ignore chemical contaminant); these use food or space, or produce toxins.

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From the examiners' report: 63% got at least one mark. Name new microorganisms, not chemicals, then say what they compete for, food or space, or that they make toxins.

Clear zones and controls

Question 6

AS Paper 2 · June 2016 · Q08.1 · 1 mark · Easy

Explain why there's a clear zone around each disc of disinfectant on a lawn of bacteria.

Figure 8: drawing of a Petri dish of bacteria grown on nutrient agar with four filter paper discs, A to D, soaked in different concentrations of disinfectant; each disc has a clear zone around it, smallest at A and largest at D. Question asks why there is a clear zone around each disc, 1 mark.
Show the mark scheme Mark scheme: bacteria killed (accept destroyed or no living bacteria). Ignore no growth or growth prevented.

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From the examiners' report: About two thirds got it. The bacteria had already grown before the discs went on, so 'growth prevented' didn't score: say the bacteria were killed.

Question 7

AS Paper 2 · June 2016 · Q08.2 · 1 mark · Medium

Use the clear zones to explain why the student used no more than 40% disinfectant.

Question text: on the basis of his research, the student used a maximum disinfectant concentration of 40%; use Figure 8 to explain why, 1 mark.
Show the mark scheme Mark scheme: the clear zone would be too large, or the clear zones would overlap or merge, or it could kill all the bacteria on the plate.

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From the examiners' report: Slightly over half got this. Use the figure: the zone around D is already large, so a stronger solution would make zones merge. Vague answers about concentration or safety didn't score.

Question 8

AS Paper 2 · June 2016 · Q08.3 · 2 marks · Easy

Suggest two variables to control when using the filter paper discs.

Question text: suggest two variables the student should control in using the filter paper discs in this investigation, 2 marks.
Show the mark scheme Mark scheme, any two: same size of disc (any measure, such as diameter or thickness); same material or absorbency; soaked in the solution for the same time. Ignore same shape and volume of disinfectant.

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From the examiners' report: Almost 90% got one mark, usually for disc size, but only about 40% added soaking time. The discs were soaked, so 'same volume of disinfectant' didn't score.

Question 9

AS Paper 2 · June 2016 · Q08.4 · 2 marks · Medium

Use the areas of the clear zones to find how many times more effective concentration D is than B.

Question text: use the areas of the clear zones in Figure 8 to determine how many times more effective concentration D is than concentration B, showing your working, 2 marks.
Show the mark scheme Mark scheme: any answer between 2.5 and 3.2 for 2 marks; 1 mark for a method that divides diameters or radii instead of areas (giving about 1.6 to 1.7).

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From the examiners' report: 30% got both marks. Most who got one mark divided the diameters or radii. Square the radius (πr²) for each zone before you divide.

Question 10

A level Paper 1 · June 2023 · Q05.3 · 2 marks

Suggest what was added to the wells for the positive and negative controls.

Information from earlier in this question:

Earlier part of the same exam question, giving the information this part uses. Figure 5: drawing of an agar plate with bacterial culture growing on the agar, a central well containing cinnamon oil and a clear zone with no growth around it. Table 1: diameter of the clear zone in mm for five bacteria with cinnamon oil (15, 20, 18, 16, 14), the positive control (14, 17, 12, 12, 12) and the negative control (all 0), with standard deviations. Question asks exactly what the student added to the wells for the positive and negative controls, 2 marks.
Show the mark scheme Mark scheme: positive control, an antimicrobial or antibacterial solution, an antibiotic, or an antiseptic or disinfectant; negative control, sterile water or oil without cinnamon.

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From the examiners' report: 'Doing nothing to the well' was the commonest wrong negative control. The positive control is a known antimicrobial; the negative is the liquid without cinnamon, such as sterile water.

Evaluating antimicrobial investigations

Question 11

AS Paper 1 · June 2017 · Q02.3 · 2 marks · Hard

Describe how to get a more accurate minimum concentration of propan-2-ol that stops growth.

Information from earlier in this question:

Earlier part of the same exam question, giving the information this part uses. Earlier part of the same exam question, giving the information this part uses. Table 1: number of colonies of Lactobacillus at disinfectant concentrations of 0 to 20 arbitrary units for Lysol, propan-2-ol and ammonia; propan-2-ol gives 300, 290, 195, 0 and 0 colonies. Question: the student concluded the minimum concentration of propan-2-ol needed to stop growth was 15 units, which is incorrect; describe how to obtain a more accurate estimate, 2 marks.
Show the mark scheme Mark scheme: use several concentrations between 10 and 15 units; repeat each concentration.

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From the examiners' report: Over half got the first mark for testing between 10 and 15 units, but very few added that each concentration should be repeated. It wasn't wrong, just left out.

Question 12

AS Paper 2 · June 2024 · Q06.3 · 4 marks · Hard

Evaluate how well disinfectant X kills microorganisms on non-living surfaces.

Information from earlier in this question:

Earlier part of the same exam question, giving the information this part uses. Table 2: percentage light absorbance by tubes of B. subtilis after 24 hours at 25 °C with 0, 20, 40, 60, 80 and 100% disinfectant X, giving 100, 100, 87, 52, 10 and 10. Question: use Table 2 to evaluate the effectiveness of disinfectant X at killing microorganisms on non-living surfaces, 4 marks.
Show the mark scheme Mark scheme, any four: as effective at 80% as at 100%; not effective at 20%; absorbance may not show the number of living cells, as dead cells absorb light; 100% isn't totally effective as 10% absorbance remains; only tested on one species; only tested in a test tube, not on surfaces; only tested at 25 °C. Ignore statistical tests and repeats.

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From the examiners' report: Many rambling answers just restated the table. Use separate points: what the data show, then the limits, such as dead cells also absorbing light and testing in tubes, not on surfaces.

Question 13

A level Paper 3 · June 2023 · Q03.4 · 2 marks · Medium

Suggest two reasons why bacteria still grew with chloramphenicol, when M. luteus isn't resistant to it.

Information from earlier in this question:

Earlier part of the same exam question, giving the information this part uses. Question text and Table 1: a student added chloramphenicol, novobiocin or no antibiotic to 9 cm³ of M. luteus culture, diluted each by 1 in 100 000 and plated three 0.25 cm³ samples of each; chloramphenicol plates had 2, 4 and 6 colonies, novobiocin 238, 263 and 261, and no antibiotic 276, 258 and 324. Question: M. luteus is not resistant to chloramphenicol; suggest two reasons why bacteria grew in the culture containing it, 2 marks.
Show the mark scheme Mark scheme, any two: the chloramphenicol concentration was too low, or there were too many bacteria to kill; contamination with other or resistant bacteria; a mutation occurred; chloramphenicol wasn't evenly mixed in the culture.

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From the examiners' report: 45% got one mark and 14% both. Read the method: the antibiotic was mixed into the bottle, not spread on the plate. Bacteria become resistant by mutation, never 'immune'.

Dilution series and counting bacteria

Question 14

AS Paper 2 · June 2018 · Q08.1 · 3 marks · Hard

Describe how to make a 1 in 10 dilution of a bacterial culture and use it to make a 1 in 1000 dilution.

Question text: a student used a dilution series to investigate the number of cells in a liquid culture of bacteria; describe how he made a 1 in 10 dilution and then used this to make a 1 in 1000 dilution of the original culture, 3 marks.
Show the mark scheme Mark scheme: add 1 part culture to 9 parts sterile liquid to make a 10⁻¹ dilution; mix well; repeat with 1 part of the 10⁻¹ and then 10⁻² dilutions in 9 parts fresh sterile liquid, or add 1 part of 10⁻¹ to 99 parts liquid, to make 10⁻³. Reject 1 part undiluted culture to 999 parts liquid.

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From the examiners' report: Only 3.2% got all three, mostly because they didn't mix. 1 in 10 is 1 part culture to 9 parts liquid, not 10, and the 1 in 1000 must be made from the 1 in 10 dilution.

Question 15

AS Paper 2 · June 2018 · Q08.2 · 2 marks · Hard

Calculate the number of cells in 1 cm³ of undiluted culture from a count in 0.004 mm³ of a 1 in 1000 dilution.

Question text: using an optical microscope, the student found 15 cells in 0.004 mm³ of the 1 in 1000 dilution; calculate the number of cells in 1 cm³ of undiluted liquid culture, 2 marks.
Show the mark scheme Mark scheme: 3.75 × 10⁹ for 2 marks; 1 mark for 3.75 × 10⁶ cells per mm³, 3.75 × 10¹² (wrong volume conversion), 3750 per mm³ of diluted culture, or using both × 1000 conversions.

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From the examiners' report: Many missed one step and got one mark, with the number of zeros after 375 varying wildly. Set your working out step by step: per mm³, then × 1000 for the dilution, then × 1000 for cm³.

Question 16

AS Paper 2 · June 2018 · Q08.3 · 2 marks · Hard

Suggest why the student didn't use the 1 in 10 dilution to count the cells.

Question text: the student looked at cells in the 1 in 10 dilution during preliminary work but decided not to use it to determine the number of cells in the undiluted culture; suggest an explanation, 2 marks.
Show the mark scheme Mark scheme: the count is unlikely to be accurate, repeatable or reliable; because there are too many cells, or the cells overlap and aren't spread out.

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From the examiners' report: Many thought a 1 in 10 dilution would have too few cells. It's only slightly diluted, so there are too many to count accurately. 'Time-consuming' didn't score.

Question 17

AS Paper 1 · June 2017 · Q02.2 · 2 marks · Medium

Calculate the number of bacteria in 1 cm³ of undiluted culture from a 1 in 10 000 dilution.

Information from earlier in this question:

Earlier part of the same exam question, giving the information this part uses. Table 1: number of colonies of Lactobacillus at disinfectant concentrations of 0 to 20 arbitrary units for Lysol, propan-2-ol and ammonia, with 300 colonies on every control plate. Question: the 0.5 cm³ of liquid culture transferred was diluted by 1 in 10 000 (10⁻⁴); calculate how many bacteria were present in 1 cm³ of undiluted culture, 2 marks.
Show the mark scheme Mark scheme: 6 000 000 or 6 × 10⁶ for 2 marks; 1 mark for 3 × 10⁶ or for 600 in 1 cm³ of diluted culture.

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From the examiners' report: Only 15.6% got both marks, though it only needs three numbers multiplied together: 300 colonies, × 2 for 1 cm³, × 10 000 for the dilution.

Question 18

A level Paper 3 · June 2023 · Q03.2 · 2 marks · Hard

Calculate the mean number of bacteria in the undiluted culture containing novobiocin, in standard form.

Question text and Table 1: 9 cm³ of M. luteus culture in each of three bottles, with chloramphenicol, novobiocin or no antibiotic; after 24 hours each was diluted 1 in 100 000 (10⁻⁵) and three 0.25 cm³ samples of each were plated; novobiocin plates had 238, 263 and 261 colonies. Question: calculate the mean number of bacteria in the undiluted bottle of culture containing novobiocin, in standard form, showing working, 2 marks.
Show the mark scheme Mark scheme: 9(.144) × 10⁸ for 2 marks; 1 mark for 914 400 000 not in standard form, wrong standard form, 1.016 × 10⁸ (not multiplied by 9), 2.286 × 10⁸ (0.25 cm³ plating ignored) or 9.432 × 10⁸ (238 discounted).

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From the examiners' report: Only 15% scored 2 marks and 77% scored 0. Work through every step: mean colonies, ÷ 0.25 for 1 cm³, × 100 000 for the dilution, × 9 for the whole bottle.

Where students lose marks (from AQA examiners' reports and mark schemes)

Each point below is followed by wording that gained marks.

  • Give the reason for each aseptic step: About one third of students scored nothing on describing and explaining three aseptic techniques, because they didn't explain why each step worked or gave a wrong reason. Some described flaming a lid or the agar plate, or didn't say how a loop or spreader was sterilised.

    ✔ Gains credit: "Flame instrument/equipment to sterilise/kill microbes/prevent contamination"; "Disinfect bench/work on disinfected cloth to kill microbes/prevent contamination."

  • Lift the Petri dish lid at an angle: Many students were vague, saying they reduced the time the lid was off the plate instead of holding it at an angle. 'Work quickly (with the lid off)' gained no marks.

    ✔ Gains credit: "Lift lid of (agar) plate at an angle" (lift lid slightly or keep lid over plate accepted).

  • The Bunsen's air current carries microbes away; it doesn't kill them: Many students mentioned working near a Bunsen burner but few could say why. Answers saying upward air movements kill bacteria or sterilise the air were rejected.

    ✔ Gains credit: "To create a convection current to carry other contaminants away/prevent them landing on the dish."

  • Contaminant microbes compete for food and space: 63% gained at least one mark. Chemical contamination didn't score, and many said the bacteria would compete without saying for what, or 'kill' the E. coli without saying how.

    ✔ Gains credit: "Unknown/new/different microorganisms ... (introduced)"; "(these bacteria) use food/space OR (these bacteria) produce toxins."

  • Same volume of culture, same number of bacteria: Only 7.6% scored both marks. Students copied the stem ('so it could be poured'), and gave generic answers such as 'to make it a fair test' or 'to control variables'.

    ✔ Gains credit: "So no contamination/other bacteria"; "So same number of bacteria transferred to allow comparison."

  • A clear zone on a grown lawn means bacteria were killed: About two thirds got this. Answers that only said growth of bacteria was prevented weren't credited, because the bacteria had already been grown on the agar.

    ✔ Gains credit: "Bacteria killed" (bacteria destroyed or no living bacteria accepted).

  • Mix at every step of the dilution series: Only 3.2% scored all three marks, mainly because most didn't mix the diluted suspension. Others added 10 parts liquid to 1 part culture, or 1 part of the 1 in 10 dilution to 999 parts water.

    ✔ Gains credit: "Add 1 part (bacteria) culture to 9 parts (sterile) liquid"; "Mix (well)"; "Add 1 part 10⁻¹ (suspension) to 99 parts (sterile) liquid."

  • A negative control well still gets a liquid: 'Doing nothing to the well' was a frequent wrong negative control, and 'adding bacteria to the well' was invalid. Some put water as the positive control and the antimicrobial as the negative.

    ✔ Gains credit: (Positive control) "Antimicrobial/antibacterial (solution) OR Antibiotic OR Antiseptic/disinfectant"; (Negative control) "(Sterile) water OR Oil (without cinnamon)."

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Download the PDF pack (18 questions, mark schemes at the end)

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Questions, mark schemes and examiners' report extracts © AQA.

Tom Whitburn
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