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Better Equipped Practical Teaching Guides
A curriculum-linked GCSE Biology practical guide to investigating the effectiveness of antibiotics and antiseptics using bacterial cultures, with aseptic technique, zones of inhibition, data analysis, evaluation, troubleshooting, antibiotic resistance and examination support.
Teacher note: This resource is provided as practical support to accompany laboratory equipment. Teachers should adapt procedures and risk assessments to suit their curriculum requirements, examination-board specifications and local laboratory policies. Use only approved school-safe bacterial strains and the school's approved microbiological handling and disposal procedures.
In this investigation, students compare the effectiveness of different antimicrobial substances by measuring the clear zones of inhibition that develop around treated discs placed onto a bacterial lawn grown on nutrient agar. The practical develops aseptic microbiology technique, accurate quantitative measurement and evidence-based evaluation while reinforcing the importance of antibiotic resistance.
Core enquiry question: How can the diameter of a zone of inhibition be used to compare how effectively different antimicrobial treatments prevent bacterial growth under controlled conditions?
Antibiotics kill bacteria or prevent them from reproducing. Examples in the guide include penicillin, streptomycin and tetracycline.
Different bacteria respond differently because antibiotics target specific cellular structures or processes.
Antiseptics are chemicals used on living tissue to reduce the number of microorganisms. Examples include iodine, chlorhexidine and alcohol.
Unlike antibiotics, antiseptics are not usually taken internally.
Disinfectants are used on non-living surfaces to kill or inhibit microorganisms. They are distinct from antibiotics and antiseptics and should be used according to product instructions and local safety procedures.
An antibiotic may kill bacteria (bactericidal) or stop bacterial growth and reproduction (bacteriostatic), depending on the antibiotic and target.
When an antimicrobial substance prevents bacterial growth, a clear area appears around the paper disc. This is the zone of inhibition.
A larger zone shows that bacterial growth was prevented over a larger area for the bacterium tested under those experimental conditions.
Important: zone size is also affected by diffusion, disc loading, inoculum, agar depth and incubation conditions, so different antimicrobial substances cannot always be compared directly.
Overall risk level: Low to Medium with correct microbiological procedures. Schools should complete their own task-specific risk assessment in accordance with local procedures. Only approved school-safe bacterial strains should be used, and cultures should be treated as potentially hazardous.
| Hazard | Possible harm | Example control measures |
|---|---|---|
| School-safe bacterial culture | Exposure to microorganisms if aseptic technique is not followed | Use only approved school-safe strains. Keep Petri-dish exposure to a minimum. Do not reopen cultures after incubation. |
| Antibiotic discs | Skin irritation or allergic reaction in sensitive individuals | Handle with sterile forceps, avoid direct skin contact, wash hands after the practical and check for known allergies where appropriate. |
| Antiseptic solutions | Skin or eye irritation depending on the product | Follow the manufacturer's instructions, wear eye protection and use only the recommended concentration. |
| Disinfectants used for cleaning | Skin, eye or respiratory irritation | Use the correct disinfectant and dilution, allow the required contact time, and never mix incompatible cleaning chemicals. |
| Alcohol-based products, if used | Highly flammable | Keep away from naked flames and ignition sources. Allow alcohol to evaporate completely before introducing any heat source. |
| Iodine, chlorhexidine, hydrogen peroxide or bleach products, if used | Irritation, staining or chemical burns depending on concentration | Use only products approved for school laboratories, follow manufacturer safety instructions, wear eye protection and avoid skin contact. |
| Glassware or broken equipment | Cuts | Handle carefully and clear breakages using appropriate equipment, never with bare hands. |
| Biological waste | Exposure to microorganisms | Use the school's approved biological-waste or sterilisation procedure. Do not place unsterilised cultures into general waste. |
Three non-negotiable practical controls: incubate at 25°C or below, secure the lid with two small pieces of tape rather than sealing it completely, and do not reopen the plate after incubation.
Label the underside of the agar plate into equal sections. Add the required treatment labels and group details.
Dip a sterile cotton swab into the approved bacterial culture.
Spread bacteria evenly across the entire agar surface in three directions to produce a uniform bacterial lawn. Keep the lid open only as much and for as long as necessary.
Allow the agar surface to dry for around one minute.
Using sterile forceps, place the antibiotic or antiseptic discs onto the agar. Space them well apart and away from the edge. Include a control disc.
Lightly press each disc onto the agar with sterile forceps so that it has full contact with the surface.
Replace the lid and secure it with two small pieces of tape. Do not seal the entire plate.
Turn the plate upside down for incubation.
Incubate at 25°C or below for 24–48 hours, following the school's microbiological procedures.
Do not reopen the plate after incubation. Observe through the lid and measure the full diameter of each clear zone in millimetres.
| Treatment | Zone diameter (mm) | Rank by mean zone diameter under these conditions |
|---|---|---|
| Antibiotic A | ||
| Antibiotic B | ||
| Antibiotic C | ||
| Antiseptic A | ||
| Control disc | Exclude from treatment ranking |
| Treatment | Zone (mm) |
|---|---|
| Antibiotic A | 28 |
| Antibiotic B | 18 |
| Antibiotic C | 10 |
| Antiseptic | 22 |
| Water control | Record 0 mm, but state clearly that the disc is excluded from treatment ranking |
Interpreting the result: a larger mean zone indicates that growth was prevented over a larger area for the bacterium tested under those conditions. This does not by itself prove that one antimicrobial is universally more powerful because diffusion, loading, inoculum, agar depth and incubation conditions can also affect zone size.
Type of antibiotic or antiseptic used.
Diameter of the zone of inhibition in millimetres.
| Possible source of error | Effect | Improvement |
|---|---|---|
| Uneven bacterial spreading | Produces patchy growth and unfair zone comparisons | Standardise the swabbing pattern and amount of inoculum. |
| Discs too close together | Zones may overlap | Space discs evenly before incubation. |
| Agar drying unevenly | May affect growth and diffusion | Use fresh, consistently prepared agar plates. |
| Measuring from the wrong edge | Produces inaccurate zone diameters | Measure outer edge to outer edge through the centre. |
| Different amounts of bacteria | Changes bacterial-lawn density | Use identical bacterial concentrations and a standardised inoculation method. |
| Different incubation times | Makes results less comparable | Incubate all plates for the same time under the same conditions. |
| Human measurement error | Increases spread between readings | Measure twice, use two diameters at 90°, or use digital calipers / image analysis where appropriate. |
The investigation is more likely to be valid if only the antimicrobial treatment is changed while the other experimental conditions remain constant.
Antibiotic resistance occurs when bacteria have or acquire features that allow them to survive or grow despite an antibiotic. The guide links resistance to natural variation, selection by antibiotic exposure and the survival and multiplication of resistant variants.
Practical interpretation: in real-world medicine, a small or absent zone may indicate resistance, but laboratory methods use standardised procedures and interpretation criteria. Classroom results should be interpreted only within the conditions of the investigation.
| Misconception | Correction |
|---|---|
| A larger zone always proves that an antibiotic is more powerful. | A larger zone means bacterial growth was inhibited over a larger area under those conditions. Diffusion and other experimental factors also affect zone size. |
| Antibiotics kill viruses. | Antibiotics work against bacteria, not viruses. |
| All bacteria respond equally to antibiotics. | Different bacteria can have different levels of susceptibility or resistance. |
| Antiseptics and antibiotics are the same. | Antibiotics are medicines used to treat bacterial infections; antiseptics are used on living tissue externally to reduce microorganisms. |
| Mistake | Likely consequence |
|---|---|
| Spreading bacteria unevenly | Uneven growth makes zones difficult to compare fairly. |
| Placing discs too close together | Zones overlap and cannot be measured accurately. |
| Opening the Petri dish after incubation | Increases exposure risk and breaks safety procedures. |
| Measuring radius instead of diameter | Produces inaccurate results. |
| Touching discs with fingers | Introduces contamination. |
| Forgetting the control disc | Removes evidence that inhibition is due to the antimicrobial treatment rather than the disc. |
| Incubating above 25°C | Increases the risk of growing harmful pathogens and does not follow school safety guidance. |
| Measuring from the paper-disc edge rather than the clear-zone edge | Produces incorrect zone measurements and invalid conclusions. |
| Problem | Possible cause | Action |
|---|---|---|
| Little or no bacterial growth | Culture not viable or bacteria not spread evenly | Use a fresh approved culture and spread it evenly across the entire agar surface. |
| No inhibition zones visible | Discs inactive or placed incorrectly | Check that discs are suitable, use sterile forceps and press them gently onto the agar. |
| Zones overlap | Discs placed too close together | Space discs evenly before incubation. |
| Uneven or patchy bacterial lawn | Incomplete swabbing | Swab in several directions to create an even lawn. |
| Contaminated agar plate | Poor aseptic technique or lid left open too long | Keep the lid closed as much as possible and work quickly with sterile equipment. |
| Zone difficult to measure | Fuzzy edge or wrong measurement point | Measure two diameters at 90° and calculate a mean. |
| Agar plate dries out | Lid not secured or plate stored incorrectly | Use two small pieces of tape and incubate the plate inverted. |
| Water control shows a clear zone | Cross-contamination | Use sterile forceps and avoid transferring contamination between discs. |
To reduce the growth of harmful human pathogens.
Bacterial growth was prevented over a larger area for the bacterium tested under those experimental conditions. Other factors such as diffusion and agar conditions also affect zone size.
To prevent contamination by unwanted microorganisms.
Harmful microorganisms may have grown.
To show that any inhibition is caused by the antimicrobial rather than the paper disc itself.
They improve reliability and allow calculation of a mean.
Question: How does antibiotic concentration affect the size of the zone of inhibition?
Different substances diffuse through agar at different rates. A larger zone does not always mean an antimicrobial is intrinsically more powerful.
Repeats reduce the influence of anomalous results and improve reliability.
Uneven bacterial lawns can change inhibition-zone size and reduce validity.
Zone edges are not always perfectly sharp. Multiple measurements or independent observers can improve accuracy.
A clear zone shows that growth was prevented, but does not show whether bacteria were killed or simply prevented from reproducing.
A small or absent zone may indicate resistance in a standardised test, but classroom conclusions must remain tied to the organism and conditions used.
Thinking like a scientist: do not judge an antimicrobial from one result alone. Repeat investigations, control variables carefully, compare mean values, consider diffusion and measurement uncertainty, and recognise that a laboratory result is only one part of deciding how well an antibiotic will work in a patient.
| Activity | Time |
|---|---|
| Introduction and theory | 10 min |
| Safety briefing | 5 min |
| Plate preparation | 15 min |
| Applying discs | 10 min |
| Incubation | 24–48 hours |
| Measuring zones | 15 min |
| Analysis | 15 min |
| Evaluation | 10 min |
| Question | Expected answer |
|---|---|
| Why does a larger zone indicate a more effective antibiotic or antiseptic under these conditions? | Bacterial growth has been prevented over a larger area. |
| Why must school agar plates be incubated at 25°C or below? | To reduce the risk of growing harmful human pathogens and carry out the investigation safely. |
| Why is aseptic technique important? | To prevent contamination and help produce reliable, valid results while working safely. |
| What should be changed, and what should be kept the same? | Change the type of antibiotic or antiseptic; keep factors such as bacterial species, agar type, incubation time, temperature and disc size constant. |
| If two antibiotics produced 15 mm and 27 mm zones, what can be concluded? | The 27 mm treatment inhibited growth over a larger area under the conditions of the investigation. |
Examiner advice summary: measure the full diameter through the centre in millimetres, use appropriate control variables, explain why aseptic technique is necessary, remember the 25°C-or-below school incubation rule, and never reopen the plate after incubation.
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This guide was written and reviewed by Better Equipped's technical team and further reviewed by former A Level Science Teachers. Our technical team draw on experience supplying practical science equipment to schools, colleges, laboratories and science departments throughout the UK. They include ex-school laboratory technicians and are here to support schools, colleges and laboratories. If you have feedback on this guide, we'd love to here it so please contact us. Don't forget we will be regularly updating our guides and resources on our Better-Resources Hub. If you would like us to cover a particular subject matter in these guides or have some top tips you'd like to share then again we'd love to hear from you.
Last reviewed and updated: August 2026
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