Culturing microorganisms — effect of antiseptics or antibiotics
GCSE Biology (8461) · Required practical 2 — method, variables, the marks examiners report students losing.
Investigate the effect of a range of antiseptics or antibiotics on the growth of bacteria, using aseptic technique, by measuring the zone of inhibition.
Apparatus
- Sterile agar plate inoculated with bacteria
- Paper discs soaked in different antiseptics/antibiotics (and a control disc in sterile water)
- Sterile forceps and an inoculating loop
- Bunsen burner (for aseptic technique) and adhesive tape
- Ruler for measuring zones, and a marker pen
Method
- 1Work near a Bunsen flame and sterilise the inoculating loop by passing it through the flame until red hot, then let it cool.
- 2Spread the bacterial culture evenly over the sterile agar using aseptic technique, briefly lifting the lid only as far as needed.
- 3Use sterile forceps to place the soaked discs onto the agar, well spaced, including one control disc soaked in sterile water.
- 4Tape the lid on (do not seal completely), label the base, and incubate at 25 degrees C for 48 hours (school labs keep below 25 degrees C to limit growth of pathogens).
- 5Measure the clear zone of inhibition around each disc, where bacteria have not grown.
Variables
Independent
Type (or concentration) of antiseptic/antibiotic
Dependent
Size (area) of the zone of inhibition
Control
- Type and amount of bacteria
- Volume of chemical on each disc and disc size
- Incubation temperature and time
Results & processing
- Calculate the area of each clear zone using area = pi x r squared (measure the diameter, halve it for the radius).
- A larger zone of inhibition means the antiseptic/antibiotic is more effective at killing or inhibiting the bacteria.
- The control disc should show no zone, confirming it is the chemical (not the disc) causing the effect.
Where students lose marks
Not sterilising equipment or leaving the lid open too long.
Fix: Flame the loop and work by the Bunsen; lift the lid only slightly to keep out unwanted microbes.
Forgetting the control disc.
Fix: A disc in sterile water shows the disc itself has no effect, so any zone is caused by the chemical.
Comparing diameters instead of areas.
Fix: Use area = pi r squared so a fair comparison accounts for the circular zone.
Improve the method
- Repeat with several plates for each chemical and take a mean zone size.
- Incubate at the same temperature and time for every plate for a fair test.
- Tape the lid rather than sealing it, so no anaerobic pathogens are cultured.
Try it — exam-style
A clear zone of inhibition has a diameter of 20 mm. Calculate its area. Use area = pi x r squared and give your answer to 3 significant figures.
Explain why a student passes the inoculating loop through a Bunsen flame before spreading the bacteria.
Questions are written in the style of past AQA papers — never copied from them.
Drill it properly
Stuck on culturing microorganisms — effect of antiseptics or antibiotics?
Aseptic technique and the zone-of-inhibition maths are classic mark-droppers — I drill the method and the calculation, and your first lesson is free.