Triple Science Only - Required Practical - bacterial growth on agar plates

Aim

To investigate the effect of different antibiotics or antiseptics on bacterial growth by measuring the zones of inhibition on an agar plate.

When bacteria are grown on nutrient agar, they multiply to form a continuous layer. If discs soaked in antibiotics or antiseptics are placed on this layer, any bacteria that are killed or inhibited will leave clear circular zones, known as zones of inhibition.

The size of these clear zones provides a visual indication of how effective each antibiotic is at stopping bacterial growth. The larger the zone of inhibition the more effective the antibiotic / antiseptic.

Equipment

• Sterile Petri dishes and nutrient agar

• Inoculating loop (for transferring bacteria)

• Bacterial culture (safe, non-pathogenic strain e.g. E. coli)

• Paper discs soaked in different antibiotics or antiseptics

• Sterile forceps, adhesive tape, bunsen burner, incubator (set to 25°C)

Method Summary

1. Prepare and inoculate an agar plate with bacteria using a sterile loop to create a thin, even bacterial layer.

2. Place paper discs soaked in different antibiotics (and one control disc soaked in sterile water) onto the agar using sterile forceps.

3. Secure and label the Petri dish using adhesive tape at 2–3 points (not fully sealed) and incubate it upside down at 25°C for 24–48 hours.

4. Observe and measure the zone of inhibition (clear zone) around each disc where bacteria have not grown.

5. Calculate the area of each zone using the formula:

Where r = the radius of the zone of inhibition.

The larger the area, the more effective the antibiotic.

Zone of inhibition around different antibiotics.

Antibiotic C is the most effective as the zone of inhibition is the largest.

• The clear zones around the antibiotic or antiseptic discs show where bacteria have been killed or growth has been inhibited.

• The larger the zone, the more effective the antibiotic or antiseptic.

• A control disc should show no clear zone, confirming that any effect is due to the antibiotic and not another factor.

• Results can be compared quantitatively by calculating the area of each clear zone.

• Inconsistent bacterial spreading can lead to uneven growth and unreliable results.

• Discs not pressed firmly onto agar may reduce contact and inhibit diffusion.

• Temperature fluctuations could affect bacterial growth rates. It is also important that the concentration of antibiotic, bacteria and any nutrient broth is kept the same.

• Measurement errors when determining zone diameters can affect area calculations.

• To improve reliability:

• Repeat the experiment several times.

• Take the mean of multiple measurements for each disc.

Practice Question