Culturing Microorganisms (Required Practical)

This lesson covers the techniques used to culture microorganisms safely, as required by the AQA GCSE Combined Science Trilogy specification (8464). This is a Required Practical — you need to know the method, understand aseptic technique and be able to calculate the areas of bacterial colonies and zones of inhibition.

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Why Culture Microorganisms?

Scientists grow (culture) microorganisms for several important reasons:

• To investigate the effect of antibiotics or antiseptics on bacterial growth.
• To study bacterial reproduction and growth rates.
• To produce useful substances (e.g. antibiotics, enzymes, insulin using genetically modified bacteria).

In schools, bacteria are grown on agar plates (Petri dishes containing a nutrient jelly) at controlled temperatures.

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What Bacteria Need to Grow

To grow and reproduce, bacteria need:

Requirement	Provided By
Nutrients (carbon source, nitrogen source, minerals)	Nutrient agar or nutrient broth
Warmth	An incubator set to a specific temperature
Moisture	Provided by the agar jelly
Time	Bacteria reproduce rapidly — populations can double every 20 minutes

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Aseptic Technique

Aseptic technique is a set of procedures designed to prevent contamination of cultures by unwanted microorganisms from the environment (e.g. airborne bacteria, skin bacteria). Contamination could:

• Produce unreliable results (unwanted bacteria interfere with the experiment).
• Allow pathogenic (disease-causing) organisms to grow, which would be a health risk.

Key Aseptic Techniques

Step	Reason
Sterilise the Petri dish and agar before use (e.g. by autoclaving at 121°C)	Kills any existing bacteria on the equipment
Sterilise the inoculating loop by passing it through a Bunsen burner flame until it glows red	Kills bacteria on the loop before transferring the culture
Briefly open the lid of the Petri dish — do not remove it completely	Reduces the chance of airborne microorganisms falling into the dish
Seal the lid with adhesive tape in a few places (not all the way around)	Prevents microorganisms from entering or leaving, but allows air to enter so dangerous anaerobic bacteria cannot grow
Work near a lit Bunsen burner	Creates an updraught of warm air that carries airborne microorganisms away from the work area
Wash hands before and after handling cultures	Removes bacteria from the skin

Exam Tip: The Petri dish lid must not be sealed completely. If all the air is excluded, anaerobic bacteria could grow — some of which are dangerous pathogens. Leaving gaps in the tape allows oxygen to enter.

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Incubation Temperature

Setting	Maximum Temperature	Reason
School laboratory	25°C	At higher temperatures, harmful pathogens are more likely to grow rapidly. 25°C allows sufficient growth for investigation without significant risk.
Industrial laboratory	Higher temperatures (e.g. 37°C)	Trained professionals work in controlled environments with additional safety measures. Higher temperatures produce faster growth.

Exam Tip: A common exam question asks why school cultures are incubated at 25°C rather than 37°C. The answer is: to reduce the risk of growing harmful pathogens, which are more likely to thrive at body temperature (37°C).

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Required Practical — Investigating Antibiotics

Aim

To investigate the effect of antibiotics (or antiseptics) on bacterial growth.

Method

1. Sterilise all equipment using aseptic technique.
2. Pour molten nutrient agar into a sterilised Petri dish and allow it to set.
3. Use a sterilised inoculating loop to spread bacteria evenly across the surface of the agar (this creates a bacterial lawn).
4. Place paper discs soaked in different concentrations of antibiotic (or different types of antibiotic) onto the agar surface using sterilised forceps.
5. Include a control disc — a disc soaked in distilled water only (no antibiotic).
6. Seal the lid with tape and label the dish.
7. Incubate upside down at 25°C for 24–48 hours.
8. After incubation, observe the results. Do not open the dish after incubation.

Expected Results

• Where bacteria have grown, the agar will appear cloudy or have visible colonies.
• Around effective antibiotics, there will be a clear zone called a zone of inhibition — this is where the bacteria have been killed or prevented from growing.
• The larger the zone of inhibition, the more effective the antibiotic.
• The control disc should show no zone of inhibition (bacteria grow right up to the disc).

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Calculating the Area of a Zone of Inhibition

The zone of inhibition is approximately circular, so its area can be calculated using:

$A = \pi r^2$A=πr2

where:

• $A$A = area of the clear zone (in mm² or cm²)
• $r$r = radius of the clear zone (in mm or cm)
• $\pi \approx 3.14$π≈3.14

Worked Example

Question: The diameter of a zone of inhibition is 18 mm. Calculate the area of the zone. Give your answer to 2 significant figures.

Solution:

Step 1: Calculate the radius.

$r = \frac{\text{diameter}}{2} = \frac{18}{2} = 9 \text{ mm}$r=2diameter​=218​=9 mm

Step 2: Calculate the area.

$A = \pi r^2 = \pi \times 9^2 = \pi \times 81 = 254.47 \text{ mm}^2$A=πr2=π×92=π×81=254.47 mm2

Step 3: Round to 2 significant figures.

$A \approx 250 \text{ mm}^2$A≈250 mm2

Exam Tip: Always measure the diameter across the widest point of the clear zone, then halve it to get the radius before using $\pi r^2$πr2. A very common error is to use the diameter instead of the radius in the formula.

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Worked Example — Comparing Antibiotics

Question: Two antibiotics are tested. Antibiotic A produces a zone of inhibition with a diameter of 12 mm. Antibiotic B produces a zone with a diameter of 20 mm. Calculate the area of each zone and determine which antibiotic is more effective.

Solution:

Antibiotic A:

$r = \frac{12}{2} = 6 \text{ mm}$r=212​=6 mm

$A = \pi \times 6^2 = \pi \times 36 \approx 113 \text{ mm}^2$A=π×62=π×36≈113 mm2

Antibiotic B:

$r = \frac{20}{2} = 10 \text{ mm}$r=220​=10 mm

$A = \pi \times 10^2 = \pi \times 100 \approx 314 \text{ mm}^2$A=π×102=π×100≈314 mm2

Antibiotic B is more effective because it has a larger zone of inhibition (314 mm² compared to 113 mm²), meaning it killed or inhibited bacteria over a greater area.

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