Edexcel A Level (IAL) Biology -6.14 Core Practical 14: The Effects of Different Antibiotics- Study Notes- New Syllabus

Investigate the Effect of Different Antibiotics on Bacteria

🧪 Aim

To compare the effectiveness of different antibiotics on bacterial growth using the agar disk diffusion method.

🌱 Introduction

Antibiotics are chemicals that inhibit or kill bacteria. The agar disk diffusion method measures bacterial sensitivity by observing the zone of inhibition around an antibiotic disk. A larger zone indicates greater bacterial inhibition under the conditions used.

⚙️ Variables

• Independent variable: Type of antibiotic (or concentration).
• Dependent variable: Diameter of zone of inhibition (mm).
• Control variables: Agar type, inoculum density, incubation temperature & time, disk size, volume of antibiotic, plate thickness.

🧾 Materials

• Non-pathogenic bacterial culture (e.g., E. coli K-12)
• Sterile nutrient agar plates (90 mm)
• Antibiotic disks or filter paper disks soaked in antibiotic solutions
• Sterile saline for inoculum
• Sterile cotton swabs
• Sterile forceps / tweezers
• Ruler or calipers
• Incubator (~25°C for school-safe use)
• Marker and labels
• Disinfectant (10% bleach) and autoclave access
• Gloves, lab coat, eye protection

🔬 Method

Prepare bacterial lawn:

• Mix bacterial colonies in sterile saline and adjust turbidity.
• Dip sterile swab, remove excess, streak agar evenly in three directions.
• Let surface dry ~2 min.

Place antibiotic disks:

• Use sterile forceps to place disks evenly (3–4 per plate).
• Label underside of plate with antibiotic name/concentration.

Include controls:

• Negative control: sterile disk with no antibiotic.
• Optional positive control: antibiotic with known effect.

Incubate plates:

• Invert and incubate at 25°C for 24–48 h.
• Avoid 37°C unless approved and with proper containment.

Measure zones:

• Measure diameter of clear inhibition zones in two perpendicular directions; calculate mean.
• Repeat at least three replicates per antibiotic.

Dispose safely:

• Do not open plates after incubation.
• Autoclave or soak in 10% bleach before disposal.

📊 Example Data Table

🧮 Analysis & Calculations

Mean:
\[ \bar{x} = \frac{\sum x}{n} \]

Standard deviation to show variation. Rank antibiotics: larger mean → more effective. Consider diffusion rates, molecule size, or bacterial resistance.

🔎 Evaluation / Sources of Error

• Uneven inoculum → standardise turbidity and streak technique.
• Variable agar depth → pour plates to ~4 mm thickness.
• Disk placement or size errors → uniform disks and sterile forceps.
• Environmental factors → same temperature & incubation time.
• Measurement error → measure twice, use calipers.

🟢 Conclusion

• Compare zones of inhibition to determine relative effectiveness.
• Larger zones indicate stronger inhibition, but in vitro results may not reflect clinical effectiveness.
• Mention bacteriostatic vs bactericidal action if relevant.