GCSE Biology Required Practical: Microbiology and Antiseptics

In 1867 the surgeon Joseph Lister published his results in The Lancet. He had been treating wounds and dressings with carbolic acid (phenol), following Louis Pasteur's idea that microorganisms cause decay, and fewer of his patients' wounds became infected. Carbolic acid was one of the first antiseptics. In this practical you test antiseptics the same way scientists still do: grow bacteria, add the chemical, and see where the bacteria fail to grow.

This lesson covers the microbiology required practical for AQA GCSE Biology Topic 1 (Cell biology). It's Biology only, so it isn't part of Combined Science.

What the practical asks you to do

Investigate the effect of antiseptics or antibiotics on bacterial growth, using agar plates and measuring zones of inhibition. A zone of inhibition is the clear area around a disc where bacteria haven't grown.

How bacteria grow

Bacteria multiply by binary fission, a simple cell division. With enough nutrients and a suitable temperature, they can divide as often as once every 20 minutes. You can grow them in a nutrient broth solution or as colonies on an agar gel plate.

To find the number of bacteria, work out how many divisions happen (total time ÷ mean division time), then multiply the starting number by 2 for each one. One cell dividing every 20 minutes becomes 64 cells after 2 hours. (Higher tier) Large answers go in standard form.

Aseptic technique

You need an uncontaminated culture, so the only thing growing is the bacterium you're testing.

1. Use Petri dishes and agar that have been sterilised before use.
2. Pass the inoculating loop through a Bunsen flame, then let it cool so it doesn't kill the bacteria.
3. Lift the lid only slightly and briefly while you spread the bacteria over the agar.
4. Secure the lid with a few pieces of tape so it can't come off and let microbes from the air in. Don't seal it all round: leaving gaps lets air in, so anaerobic bacteria are less likely to grow.
5. Store the dish upside down, so condensation doesn't drip onto the agar.
6. Incubate at a maximum of 25°C in school. That's well below body temperature, so pathogens that grow best at 37°C are less likely to grow.

Testing the antiseptics

Place equal-sized paper discs, each soaked in a different antiseptic, well apart on the agar. Add a control disc soaked in sterile water: it shows that a clear zone comes from the antiseptic, not the paper. After incubation, measure the diameter of each clear zone through the lid.

• Independent variable: the antiseptic (or antibiotic) on each disc.
• Dependent variable: the size of the clear zone.
• Control variables: the species of bacteria, disc size, volume of antiseptic, incubation temperature and time.

Calculating the area

Halve the diameter to get the radius, then use area = πr². A clear zone 14 mm across has a radius of 7 mm, so its area is π × 7² ≈ 154 mm². The bigger the area, the more effective the antiseptic was against that bacterium.

Safety

Never open an incubated dish. Your teacher sterilises the cultures, usually in an autoclave, before they're thrown away. Disinfect the bench and wash your hands at the end. Never culture microorganisms at home: you can't control which ones grow.

Common exam mistakes

• Using the diameter in πr². Halve it first.
• "Seal the lid all the way round." Tape it with a few strips so air can get in.
• "Incubate at 37°C." In school it's 25°C at most.
• Forgetting the control disc. Without it you can't show the antiseptic caused the clear zone.
• Mixing up the terms. Antiseptics go on living tissue, disinfectants on surfaces, and antibiotics are medicines that work inside the body.

Frequently asked questions

Why are agar plates incubated at 25°C?

It's below body temperature, so harmful bacteria that grow best at 37°C are less likely to grow in a school lab.

Why is a Petri dish stored upside down?

So condensation forming on the lid doesn't drip onto the agar and spread the colonies.

How do you calculate the area of a zone of inhibition?

Measure the diameter, halve it to get the radius, and use area = πr².

What does a bigger clear zone mean?

The antiseptic or antibiotic stopped more bacteria from growing, so it was more effective against that bacterium.

Try it

Say each answer before you flip the card. Then put yourself in Lister's place: carbolic acid stopped infections, but it also irritated skin. What would you have tested next?

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