Core practical 14: the effect of antibiotics on bacteria

Method

1. Use aseptic technique throughout.

2. Spread a known volume of bacterial broth culture evenly over an agar plate with a sterile spreader to make a lawn.

3. Soak filter-paper discs in each antibiotic (and one in sterile water as a control), or use ready-made antibiotic discs, and place them on the agar with sterile forceps, spaced apart.

4. Tape the lid with two strips, label the base, and incubate upside down at 25 °C for about 48 hours.

5. Measure the diameter of the clear zone of inhibition around each disc.

Results

Where the antibiotic has diffused into the agar and killed or inhibited bacteria, there is a clear zone. Calculate its area:

area = πr², with r = diameter / 2.

A larger zone suggests a more effective antibiotic against that bacterium. The control disc should have no clear zone.

Control variables: same bacterial culture and volume, same agar depth, same disc size, same concentration of antibiotic, same soaking time, incubation time and temperature.

Evaluation

Zone size also depends on how fast each antibiotic diffuses through agar (related to molecule size) and its concentration, not only on how effective it is. Repeat with several plates and calculate means. Safety: disinfect the bench, never open plates after incubation, and autoclave plates before disposal.

Worked example

A clear zone has a diameter of 16 mm. Calculate its area.

1. r = 16 / 2 = 8 mm
2. Area = π x 8² = 201 mm²

Answer: 201 mm²

Check yourself

1. A zone of inhibition has a diameter of 22 mm. Calculate its area in mm² to 3 significant figures.
   Show answer

   380 mm². r = 11 mm; π x 121 = 380 mm².

2. A zone of inhibition has a radius of 6.5 mm. Calculate its area in mm² to 3 significant figures.
   Show answer

   133 mm². π x 6.5² = 133 mm².

3. What is the purpose of the disc soaked in sterile water?
   • It speeds up bacterial growth
   • It kills all the bacteria
   • It is a control, showing the disc itself does not stop bacterial growth
   • It measures the temperature
   Show answer

   It is a control, showing the disc itself does not stop bacterial growth. Any clear zone around test discs must then be due to the antibiotic.

4. What does a large zone of inhibition suggest?
   • The bacterium is resistant to the antibiotic
   • The antibiotic is effective at killing or inhibiting this bacterium
   • The disc was contaminated
   • The agar was too thin to grow bacteria
   Show answer

   The antibiotic is effective at killing or inhibiting this bacterium. Bacteria could not grow in a wide area round the disc.

5. Why might zone size not give a fair comparison of antibiotic effectiveness?
   • All antibiotics are the same molecule
   • The bacteria grow on top of the discs
   • Zones are always identical
   • Different antibiotics diffuse through agar at different rates
   Show answer

   Different antibiotics diffuse through agar at different rates. Larger molecules diffuse more slowly, giving smaller zones.

6. Why are agar plates incubated upside down?
   • So condensation does not drip onto the agar and spread colonies
   • So the bacteria fall off
   • To let oxygen in
   • To keep the plate warmer
   Show answer

   So condensation does not drip onto the agar and spread colonies. Drips would smear the lawn and colonies.

7. Which variable must be controlled when comparing antibiotics?
   • The type of antibiotic
   • The size of the zone of inhibition
   • The effectiveness of the antibiotic
   • The concentration of each antibiotic on the discs
   Show answer

   The concentration of each antibiotic on the discs. The type is the independent variable; concentration must be fixed.

Practise with instant feedback Print this lesson

Sources

• Pearson Edexcel International Advanced Level in Biology specification (XBI11/YBI11, first teaching 2018)
• Physics & Maths Tutor: Edexcel IAL Biology CP14, Effect of different antibiotics

Last updated 7 November 2026. Spotted a mistake? Tell us.