Culturing microorganisms means growing bacteria or fungi under controlled conditions on a nutrient medium so that you can observe, count, or test them. In the GCSE required practical, you grow bacteria on agar plates using aseptic technique — a set of procedures designed to prevent contamination from the environment or from yourself.
What is the purpose of this required practical?
The culturing microorganisms required practical has two main aims:
- To practise aseptic technique — the procedures used in microbiology to prevent contamination of cultures and to protect the experimenter.
- To investigate the effect of antibiotics or antiseptics on bacterial growth by measuring the clear zone (inhibition zone) around a disc soaked in the substance.
You may be assessed on how you carry out the technique, how you control variables, and how you calculate and interpret the size of inhibition zones.
What equipment and materials do you need?
| Item | Purpose |
|---|---|
| Nutrient agar plates (pre-poured, sterile) | Growing medium providing nutrients for bacteria |
| Inoculating loop or cotton swabs | Spreading bacteria evenly across the plate |
| Bunsen burner | Sterilising the inoculating loop; creating a warm updraught to keep airborne microbes away |
| Ethanol (for dipping loops) | Killing any bacteria on the loop before flaming |
| Bacterial culture | Source of bacteria (e.g. Escherichia coli or Micrococcus luteus) |
| Antibiotic/antiseptic discs | Testing for inhibition of bacterial growth |
| Sticky tape | Sealing agar plates (but NOT airtight) |
| Incubator set to 25 °C | Incubating the plates safely |
School safety note: schools must incubate cultures at 25 °C or below (not 37 °C). At 37 °C, pathogens that grow best inside the human body (including potential harmful mutants) could thrive. The bacteria used are safe environmental strains, but this temperature limit is a standard safeguarding rule.
How do you carry out the aseptic technique step by step?
- Prepare your workspace. Clean the bench with disinfectant. Wash hands thoroughly. Tie back hair.
- Label the agar plate on the base (not the lid) with your name, the date, and the bacterium being used. Work near a lit Bunsen burner.
- Sterilise the inoculating loop by holding it in the blue flame of the Bunsen burner until it glows red-hot. Allow it to cool without touching anything.
- Dip the cooled loop into the bacterial broth or use a sterile swab dipped in the suspension.
- Open the agar plate as little as possible — lift the lid at a slight angle, never fully remove it. Streak or spread the bacteria across the agar surface.
- Replace the lid immediately. Do not breathe directly over the open plate.
- Place antibiotic discs (if investigating inhibition) onto the inoculated agar using sterile forceps. Gently press each disc so it makes contact with the agar.
- Seal the plate by taping two strips of sticky tape around the edge. Do not seal all the way around — this prevents anaerobic (airborne contaminant) growth that would occur if oxygen were excluded, but more importantly follows lab safety guidelines.
- Incubate upside-down at 25 °C for 24–48 hours. Inverting the plate prevents condensation from dripping onto the colonies.
- Do not open the incubated plate. Examine and record results through the lid, then dispose of the sealed plate by autoclaving or placing it in a biohazard bag for sterilisation.
How do you measure and calculate inhibition zones?
If you placed antibiotic or antiseptic discs on the plate, bacteria-free circles (inhibition zones) will appear around any disc that was effective. Measure the diameter of each zone using a ruler (measuring through the lid without opening the plate), then calculate the area:
Area of inhibition zone (mm²) = π × (diameter ÷ 2)²
Worked example: an antibiotic disc produces an inhibition zone with a diameter of 18 mm.
- Radius = 18 ÷ 2 = 9 mm
- Area = π × 9² = π × 81 = 254 mm² (to 3 significant figures)
A larger inhibition zone means the antibiotic is more effective against that bacterium. A zone of zero means the bacterium is resistant to that antibiotic. Comparing zone sizes allows you to rank antibiotics by effectiveness — useful information in clinical settings where doctors need to match antibiotic to pathogen.
What variables should you control?
To make the experiment a fair test:
- Use the same concentration of each antibiotic/antiseptic solution on each disc
- Use the same volume of solution to soak each disc
- Spread bacteria using the same technique each time (same inoculation method)
- Use plates from the same batch of agar (same nutrient concentration)
- Incubate all plates at the same temperature for the same duration
- Use the same bacterium for all plates being compared
The only variable you should change is the type (or concentration) of antibiotic or antiseptic being tested.
Frequently asked questions
Why must you use aseptic technique?
Aseptic technique prevents two types of contamination: (1) environmental microbes getting onto your culture, which would produce misleading results and make the culture unsafe; and (2) the cultured bacteria escaping into the environment or onto your skin, posing a (small, but preventable) health risk. A Bunsen burner flame creates a warm updraught that deters airborne particles from settling, and sterilising equipment ensures no bacteria survive on the tools you use.
Why are agar plates incubated upside-down?
Plates are incubated lid-down to prevent condensation from forming on the lid and dripping onto the bacterial colonies. Drops of water falling from the lid would cause colonies to merge and run together, making it impossible to distinguish individual colonies or measure inhibition zones accurately. Inverting the plate keeps the agar surface dry and the colonies well-separated.
Why is 25 °C used instead of 37 °C for school experiments?
At 37 °C, bacteria that grow best inside the human body (including potentially harmful species) would thrive. Even with safe environmental strains, maintaining a 25 °C incubation temperature is a precautionary measure that prevents any harmful organisms — including rare mutations — from reaching their optimum growth conditions. Most environmental bacteria still grow at 25 °C; the colonies simply take a little longer to develop.
What does a clear zone around an antibiotic disc mean?
The clear (inhibition) zone is an area where bacteria have been killed or prevented from growing by the antibiotic diffusing out of the disc into the agar. The larger the clear zone, the more effective the antibiotic is against that particular bacterium. If no clear zone forms, the bacterium is resistant to that antibiotic — its enzymes or cell wall structure are unaffected by the drug. Comparing zone sizes from different antibiotics is a standard way to assess antibiotic sensitivity in clinical microbiology.
For GCSE biology explained as living systems — not bullet points — try Professor Darwin at aitutors.me.