Culturing microorganisms (RP2) – GCSE Biology exam questions with mark scheme

1Grade 6–7Triple only5 marks

A student investigated the effect of antiseptics A, B and C on the growth of bacteria. Paper discs soaked in each antiseptic were placed on a nutrient agar plate covered with bacteria. Disc D was soaked in sterile water. The diagram shows the plate after incubation.

Disc	A	B	C	D
Diameter of clear zone in mm	14	9	21	0

(a) Calculate the area of the clear zone around disc A. Use: area = $\pi r^2$. Give your answer to 3 significant figures. [2]

(b) Which antiseptic was most effective? Give a reason. [1]

(d) Explain why disc D was included. [1]

(a) $\pi \times 7^2$ (radius = 7 mm)(1)
(a) 154 mm² (correct answer with no working scores 2 marks)(1)
(b) C and it has the largest clear zone (allow most bacteria killed / growth prevented over largest area)(1)
(c) to reduce the chance of growing pathogens that are harmful to humans (allow pathogens grow at body temperature / 37 °C); ignore 'for safety' alone(1)
(d) to show that the paper disc / water alone does not affect the growth of bacteria, so any clear zone is due to the antiseptic; allow as a control / for comparison(1)
Answer: (a) 154 mm² (b) C, largest clear zone (c) higher temperatures may grow human pathogens (d) control
Examiner tip: Use the radius (half of 14 mm) and give units of mm² — 616 mm² comes from using the diameter.

2Grade 6–7HigherTriple only3 marks

A bacterium divides once every 20 minutes. A culture starts with 150 bacteria.

Calculate the number of bacteria in the culture after 3 hours, assuming all the bacteria keep dividing at this rate.

Give your answer in standard form. [3]

number of divisions = 180 ÷ 20 = 9(1)
150 × 2⁹ (= 76 800)(1)
$7.68 \times 10^{4}$ (standard form required for this mark; 76 800 not in standard form scores 2)(1)
Answer: $7.68 \times 10^{4}$ bacteria
Examiner tip: Convert 3 hours to 180 minutes first, and remember the number doubles each time, so multiply by 2⁹ — not 2 × 9.

3Grade 1–3FoundationTriple only4 marks

Bacteria can be grown in the laboratory.

(a) Name the process bacteria use to divide and reproduce. [1]

(b) Bacteria can be grown on agar gel plates. Name the liquid that bacteria can also be grown in. [1]

Give two conditions that bacteria need to divide this quickly. [2]

(a) binary fission; allow fission; do not accept mitosis / meiosis(1)
(b) nutrient broth; allow broth (solution) / nutrient solution; ignore water alone(1)
(c) (enough / plenty of) nutrients; allow food / named nutrient e.g. glucose / sugar / amino acids(1)
(c) a suitable / warm temperature; allow (optimum) temperature; ignore heat alone
also allow: moisture / water; oxygen (for aerobic bacteria); space — max 2 marks for (c)(1)
Answer: (a) binary fission (b) nutrient broth (c) enough nutrients; a suitable temperature
Examiner tip: Students write 'mitosis' for bacteria — bacteria have no nucleus, so the process must be named binary fission.

4Grade 4–5Triple only4 marks

A student prepared an agar plate of bacteria using aseptic technique.

Give the reason for each step the student took.

(a) The inoculating loop was passed through a Bunsen burner flame until it glowed red, then allowed to cool before use. [1]

(b) The lid of the Petri dish was lifted only slightly while the bacteria were spread onto the agar. [1]

(d) The Petri dish was stored upside down. [1]

(a) to kill (any) microorganisms / bacteria on the loop; allow to sterilise the loop; ignore 'to clean the loop'
allow (cooling) so the loop does not kill the bacteria being transferred(1)
(b) to stop / reduce microorganisms from the air getting in / contaminating the plate; ignore 'to stop germs escaping' alone(1)
(c) so oxygen can (still) get in; allow to prevent the growth of anaerobic bacteria / anaerobic pathogens; (tape is to stop the lid coming off / microorganisms getting in — no mark for this alone)(1)
(d) so that condensation / water droplets do not drip onto the agar / bacteria; allow so the culture does not get disturbed by condensation; ignore 'so the agar does not fall out'(1)
Answer: (a) kill microorganisms on the loop (b) stop microbes from the air contaminating it (c) lets oxygen in so anaerobic pathogens do not grow (d) condensation cannot drip onto the agar
Examiner tip: For (c), 'to stop it being contaminated' does not score — the question is why it is NOT sealed all round, so the answer must be about oxygen and anaerobic bacteria.

5Grade 8–9HigherTriple only5 marks

A scientist grew bacteria in nutrient broth. At the start there were 400 bacteria in the broth. After 2.5 hours there were 12 800 bacteria.

(a) Calculate the mean division time of the bacteria in minutes. [3]

(b) A single bacterium on an agar plate grew into a circular colony with a diameter of 2.4 mm.

Calculate the cross-sectional area of the colony. Use the equation: area = $\pi r^2$

Give your answer to 2 significant figures. [2]

(a) 12 800 ÷ 400 = 32 (times as many bacteria)(1)
(a) 32 = 2⁵ so 5 divisions; allow 2 → 4 → 8 → 16 → 32 shown as a doubling sequence / 400 doubled 5 times = 12 800(1)
(a) 150 ÷ 5 = 30 (minutes); an answer of 30 with no working scores 3; 0.5 (hours) not converted to minutes scores 2 only(1)
(b) radius = 1.2 (mm) and $\pi \times 1.2^2$; allow 4.52 / 4.523…(1)
(b) 4.5 (mm²); correct answer to 2 s.f. with no working scores 2; 4.52 scores 1 only(1)
Answer: (a) 30 minutes (b) 4.5 mm²
Examiner tip: Students divide the time by 32 (the ratio) instead of by the number of doublings (5) — find how many times the population has doubled first.

6Grade 6–7HigherTriple only5 marks

The mean division time of a species of bacterium is 24 minutes.

A flask of nutrient broth is inoculated with 300 of these bacteria.

(a) Calculate the number of bacteria in the flask after 6 hours.

Give your answer in standard form to 3 significant figures. [3]

(b) When the scientist counted the bacteria after 6 hours, there were far fewer than the number calculated in part (a).

Give two reasons why. [2]

(a) number of divisions = (6 × 60) ÷ 24 = 15(1)
(a) 300 × 2¹⁵ (= 9 830 400)(1)
(a) $9.83 \times 10^{6}$; 9 830 400 not in standard form scores 2; allow ecf from an incorrect number of divisions for this mark only if in standard form to 3 s.f.(1)
(b) any two from: nutrients run out / are used up; (toxic) waste products build up; bacteria die (so not all keep dividing); allow oxygen runs out; allow lack of space; ignore 'conditions change' unqualified(1)
(b) second reason from the list above; allow the temperature may not be the optimum / not all bacteria divide at the mean rate(1)
Answer: (a) $9.83 \times 10^{6}$ bacteria (b) e.g. nutrients run out; toxic waste builds up
Examiner tip: Students multiply 300 by 15 or by 2 × 15 — the population doubles 15 times, so it is 300 × 2¹⁵.

7Grade 6–7Triple only4 marks

A student investigated the effect of antibiotic X on bacteria. She placed a paper disc soaked in antibiotic X on each of four agar plates covered with bacteria.

The table shows the diameter of the clear zone around the disc on each plate after incubation.

Plate	1	2	3	4
Diameter of clear zone in mm	18	20	31	19

(a) Identify the anomalous result. [1]

(b) Calculate the mean area of the clear zone for antibiotic X.

Do not include the anomalous result. Use the equation: area = $\pi r^2$

Give your answer to 3 significant figures. [3]

(a) 31 (mm) / plate 3(1)
(b) mean diameter = (18 + 20 + 19) ÷ 3 = 19 (mm); or alternative method: radii 9, 10 and 9.5 (mm) used to find each area (254, 314, 284 mm²)(1)
(b) radius = 9.5 (mm) and $\pi \times 9.5^2$; or alternative method: (254.5 + 314.2 + 283.5) ÷ 3; allow ecf from an incorrect mean diameter (halved and used in $\pi r^2$)(1)
(b) 284 (mm²) (both methods give 284 to 3 s.f.); correct answer with no working scores 3; allow 283.5 / 283.53 / 284.05 for 2 marks (not 3 s.f.)
note: using diameter as radius ($\pi \times 19^2$ = 1134) scores 1 for the mean only(1)
Answer: (a) 31 mm (plate 3) (b) 284 mm²
Examiner tip: The most common slip is putting the diameter straight into πr² — always halve the diameter to get the radius first.

8Grade 6–7Triple only6 marks

A student wants to find out which of three antibiotics, P, Q and R, is most effective at stopping the growth of a species of bacterium.

She is given a culture of the bacteria, sterile agar plates, sterile paper discs, solutions of each antibiotic and normal laboratory equipment.

Describe a method the student could use to investigate this. Your method should produce valid results and use aseptic technique. [6]

sterilise the inoculating loop / spreader by passing it through a flame (and let it cool); or use a sterile pipette / sterile spreader(1)
spread the bacteria evenly over the surface of the agar / pour plate with an even lawn of bacteria(1)
lift the Petri dish lid only slightly / work near a Bunsen flame to reduce contamination from the air(1)
soak paper discs in antibiotic P, Q and R; use sterile forceps to place them on the agar, evenly spaced(1)
include a control disc soaked in sterile water(1)
use the same concentration / volume of each antibiotic; same size discs; same soaking time(1)
tape the lid with two strips (not sealed all round) and label the plate on the base(1)
incubate upside down at 25 °C (or below) for the same time, e.g. 48 hours(1)
measure the diameter of the clear zone around each disc (with a ruler, across the base)(1)
calculate the area of each clear zone using $\pi r^2$(1)
repeat (e.g. three plates) and calculate a mean for each antibiotic(1)
the antibiotic with the largest clear zone is the most effective(1)

Level	Marks	What it looks like
Level 3	5–6	A coherent, logically ordered method that could be followed by another person to give valid results. It includes aseptic steps, placing antibiotic discs, incubation conditions, measuring the clear zones and comparing them, with at least two control variables or a control disc and repeats.
Level 2	3–4	A method with most of the key steps in a sensible order but with some omissions, e.g. little detail on aseptic technique, or no control variables, or the measurement is described only vaguely.
Level 1	1–2	Some relevant steps are given, e.g. putting antibiotics on a plate of bacteria or 'looking for a clear area', but the method lacks structure and could not be followed to give valid results.
No relevant content	0	Nothing worthy of credit.

Answer: Plan: sterilise equipment; spread bacteria evenly; discs of P, Q, R and a water control placed with sterile forceps; tape lid, incubate upside down at 25 °C; measure clear-zone diameters, calculate πr², repeat and compare means.
Examiner tip: Many answers miss the measurement step — a method must say what is measured (diameter of the clear zone) and how it is used to compare the antibiotics.

9Grade 4–5Triple only5 marks

A student tested different concentrations of an antiseptic on bacteria grown on agar plates. The graph shows the results.

(a) Describe the relationship between the concentration of antiseptic and the mean diameter of the clear zone. [2]

(b) Use the graph to determine the mean diameter of the clear zone at a concentration of 4%.

Then calculate the area of this clear zone. Use the equation: area = $\pi r^2$ [2]

(a) as the concentration increases the (mean) diameter (of the clear zone) increases(1)
(a) (but) it levels off / stays the same from about 5%; allow from 5–6% no further increase; allow the increase gets smaller at higher concentrations(1)
(b) 18 mm read from graph and radius = 9 (mm) used in $\pi \times 9^2$(1)
(b) 254 (mm²); allow 254.5 / 254.47; allow 254.3 if $\pi$ = 3.14 used; correct answer with no working scores 2(1)
(c) any one from: type / species of bacteria; volume of antiseptic (on each disc); size of paper disc; incubation temperature; incubation time; volume / depth / type of agar; ignore 'amount' unqualified; ignore concentration of antiseptic(1)
Answer: (a) diameter increases as concentration increases, then levels off from about 5% (b) 18 mm; 254 mm² (c) e.g. incubation temperature
Examiner tip: For a 2-mark 'describe' always give the overall trend AND the change in pattern (here, levelling off) — a single sentence only scores 1.

10Grade 8–9HigherTriple only4 marks

A student tested two antibiotics, P and Q, on the same species of bacterium.

One paper disc soaked in antibiotic P was placed on plate 1. Plate 1 was incubated for 48 hours.
One paper disc soaked in antibiotic Q was placed on plate 2. Plate 2 was incubated for 24 hours.
The concentration of antibiotic P was 10 µg/cm³ and the concentration of antibiotic Q was 5 µg/cm³.

Antibiotic	P	Q
Diameter of clear zone in mm	24	15

The student concluded: ‘Antibiotic P is more effective than antibiotic Q at killing these bacteria.’

Evaluate the student’s conclusion. [4]

(supports) P has a larger clear zone (24 mm compared with 15 mm), so P killed bacteria / stopped growth over a larger area; allow areas calculated (452 mm² vs 177 mm²)(1)
(against) plate 1 / P was incubated for longer, so P had more time to diffuse (through the agar) / more time to act; allow different incubation times so not a fair test(1)
(against) P was at a higher / double the concentration of Q, so the comparison is not fair; or only one disc / no repeats so cannot tell if the results are repeatable / anomalous; or different plates so the bacteria may not have been spread evenly / the same(1)
(judgement) conclusion is not valid / cannot be made because it was not a fair test; must be linked to at least one reason given; max 3 if no judgement given(1)
Answer: Not a valid conclusion: P has the larger clear zone, but P was incubated for twice as long, at double the concentration, with no repeats — not a fair test.
Examiner tip: 'Evaluate' needs both sides AND a final judgement — listing only the problems caps you at 3 marks.

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