Zones of Inhibition Virtual Lab: Antiseptics and Antibiotics

Updated 2026-10-07

This zones of inhibition virtual lab follows the AQA GCSE Biology required practical on antiseptics and antibiotics. Students prepare an agar plate with aseptic technique, place discs soaked in chlorhexidine at four concentrations, and incubate three plates. They measure each clear zone with an on-screen ruler, calculate its area with πr² and compare the means. Skipping a step on purpose makes contaminant colonies grow. Every number below was read from the simulation.

Zones of inhibition – antiseptics and antibiotics on bacteria

Curriculum links

AQA GCSE Biology: specification 4.1.1.6 (culturing microorganisms, Biology only) and its required practical on how antiseptics or antibiotics affect bacterial growth, measured as clear zones. It includes the maths skill of calculating a clear area with πr².
AP Biology: the same aseptic technique comes before the bacterial transformation investigation. See the biotech virtual lab.
Simulic is not affiliated with or endorsed by the College Board or AQA.

Before the lab (5 min)

Ask: "Four discs hold chlorhexidine at 0.2 %, 0.1 %, 0.05 % and 0.02 %. How will the clear zone change as the concentration falls?" Students choose an answer and give one reason. On a class link this is question 1; the simulation unlocks after they answer.

Method in the simulation

In the starting values below the simulation, choose Bacillus subtilis, Chlorhexidine at 4 concentrations, 25 °C and 48 h. Leave the random variation at 5 %. Disc E is soaked in sterile water: it is the control.
Plate 1: press the blue technique button for each of the eight steps, from "Flame, let cool" to "Invert, incubate at 25 °C". Read the reason shown under each step.
After incubation, set Zoom to Disc A. Drag the ruler so its edge passes through the centre of the disc, put 0 on one edge of the clear zone and read the other edge. Record the diameter including the 6 mm disc. If there is no zone, record 6.
Measure discs B to E the same way, type the diameters into the results table and press Check measurements. Readings within 1 mm are marked correct.
Press New plate (repeat) and then Quick run, every step correct. Measure plate 2, then do the same for plate 3.
Read the mean diameter d̄ and the area A for each disc in the table. Use Calculate for to see the πr² steps.
Contamination test: press Start again. Press "Flame, let cool", then Skip at "Work close to the Bunsen flame", then Quick run. Count the colonies that look different from the lawn.

Disc	Chlorhexidine (%)	Plate 1 d (mm)	Plate 2 d (mm)	Plate 3 d (mm)	Mean d̄ (mm)	Area πr² (mm²)
A	0.2
B	0.1
C	0.05
D	0.02
E	0 (sterile water)

Expected results

Without random variation, the zones are 20.3 mm (A), 18.6 mm (B), 16.7 mm (C) and 13.8 mm (D). Disc E always gives 6 mm: no zone. With 5 % variation each plate differs a little. In our test plates, disc A measured 20.7, 20.8, 21.3 and 21.5 mm. A mean of three plates almost always lands between 18.5 and 22 mm.

The mean areas are about 320, 270, 220 and 150 mm². Each time the concentration doubles, the area grows by roughly the same amount, about 50 mm². It does not double: going from 0.1 % to 0.2 % multiplies the area by only about 1.2. The agent diffuses outwards, so its concentration falls off steeply with distance from the disc.

When "Work close to the Bunsen flame" was skipped, the plate grew 2 or 3 contaminant colonies (fuzzy mould, yellow or pink), and the note under the plate said it was contaminated. The zones themselves did not change.

Questions for students

Prediction: how will the clear zone change as the chlorhexidine concentration falls from 0.2 % to 0.02 %?
Which is a control variable in this investigation?
Disc A (0.2 %), three plates: what is the mean diameter of its clear zone?
From 0.1 % to 0.2 % the concentration doubles. What happens to the mean zone area?
Skip "Work close to the Bunsen flame" on a new plate. Describe what grows and explain why the step matters.

Answers for teachers:

The zones get smaller.
The incubation temperature and time (also the bacteria, disc size and volume of culture).
About 20 mm; any mean from 17.5 to 23 mm is accepted.
It increases, but only about 1.2 times, not double.
Contaminant colonies (mould, yellow or pink) grow among the lawn. Rising hot air from the flame carries spores and dust away from the open plate. Without it, airborne microbes land on the agar, compete with the lawn, may be pathogens, and make the results unreliable.

Common misconceptions

"A bigger zone means the bacteria are bigger." The zone shows where the agent stopped growth. A larger zone means a more effective agent at that concentration.
"Double the concentration, double the zone." The area rises by about the same step for each doubling.
"Ethanol is weak because its zone is small." Ethanol evaporates from the disc and tea tree oil diffuses poorly in agar. Both kill bacteria well on contact.
"37 °C is better because bacteria grow faster." Schools never go above 25 °C, because human pathogens grow best at body temperature. In the simulation, zones are also slightly smaller at 37 °C.

Extension

Gram-negative bacteria. Choose E. coli K-12 and the preset Antibiotics and antiseptics. Penicillin gives about 30 mm on B. subtilis but no zone at all on E. coli. Ask students why.
Compare antiseptics. Use the Four antiseptics preset and rank them by mean area.

FAQ

Does this replace the required practical?

No. AQA required practicals must still be done by hand. Use the simulation to plan and practise aseptic technique, or to discuss results. See virtual labs vs physical labs.

Why are my zones different from my partner's?

Each plate has a little random variation, as real plates do. That is why the method uses three plates and a mean. To give everyone identical plates, set the random variation to 0 % in the starting values on your class link.