How do you do the microbiology required practical?

What does the microbiology practical investigate?

It tests how well different antiseptics or antibiotics stop bacteria growing. Paper discs soaked in each one sit on a plate covered in bacteria. Where a chemical spreads into the agar and stops growth, a clear ring forms around its disc, called the zone of inhibition. The bigger the clear zone, the better that chemical is at stopping those bacteria.

This practical is only in separate GCSE Biology. If you take Combined Science (AQA Trilogy), you do not need it. It builds on culturing microorganisms, in the cell biology topic, and relies on aseptic technique: ways of working that keep unwanted microbes out of your culture and keep the bacteria away from you.

What equipment do you need?

The core kit is a sterile agar plate with a safe culture of bacteria spread on it, and paper discs soaked in the antiseptics you are testing. Everything that touches the bacteria has to be sterile.

• A Petri dish of sterile nutrient agar
• A culture of bacteria that is safe for school use
• A sterile pipette and spreader, or an inoculating loop
• A Bunsen burner and a heatproof mat
• Paper discs, all the same size
• The antiseptics or antibiotics to test, and sterile water for the control disc
• Sterile forceps
• Sticky tape and a marker pen
• An incubator set to 25 °C
• A ruler marked in mm
• Disinfectant for the bench, and eye protection

What is the method?

In short: spread bacteria evenly over a sterile agar plate without letting other microbes in, add a paper disc soaked in each antiseptic, incubate the plate at 25 °C for about two days, then measure the clear zone around each disc.

• Wash your hands and wipe the bench with disinfectant.
• Work close to a lit Bunsen burner. Air rising from the flame carries microbes up and away from your plate.
• Lift the lid of the agar plate only slightly, and only for as long as you need to.
• Use a sterile pipette to put a few drops of the bacteria on the agar, then spread them evenly over the surface with a sterile spreader. If you use an inoculating loop instead, heat it in the flame until it glows red, and let it cool before it touches the bacteria.
• Soak each paper disc in one antiseptic for the same length of time. Soak one extra disc in sterile water: this is your control.
• Use sterile forceps to place the discs on the agar, well spaced out. Label the base of the plate so you know which disc is which.
• Tape the lid on with a few short strips, not all the way round, and turn the plate upside down.
• Incubate it at 25 °C for about 48 hours.
• Without opening the plate, measure the diameter of the clear zone around each disc.

What are the variables?

The antiseptic is what you change and the clear zone is what you measure. Everything else stays the same, so any difference between the zones comes from the antiseptics.

How do you work out and present the results?

Measure each clear zone straight across, through the middle of the disc, in mm. Halve that diameter to get the radius, then work out the area with area = πr².

Worked example: a clear zone is 16 mm across, so its radius is 8 mm. Area = π × 8² = 3.14 × 64, which is about 201 mm². A second antiseptic gives a zone 10 mm across, and π × 5² is about 79 mm². The first antiseptic stopped the bacteria growing over a much bigger area.

If you have repeats, work out the mean area for each antiseptic. Then draw a bar chart with the antiseptics along the bottom and mean area up the side. The antiseptics are categories, so the bars don't touch. If you tested one antiseptic at several concentrations, a line graph is the right choice instead.

How do you make it accurate and safe?

Spread the bacteria as evenly as you can, so every disc sits on the same even layer. If a zone is not a neat circle, measure the diameter twice at right angles and take the mean. Turning the plate upside down stops drops of condensation falling onto the agar and spreading the bacteria about. The control disc shows that the paper and water alone don't stop growth, so any clear zone is down to the antiseptic.

Here the safety rules and the aseptic rules overlap, and the specification expects you to explain each one. Petri dishes and agar are sterilised before use, and a loop is sterilised in a flame, so only the bacteria you chose can grow. The tape stops microbes from the air getting in. Taping at a few points rather than all the way round lets oxygen in, which discourages harmful bacteria that grow without oxygen. Incubating at 25 °C, well below body temperature, makes it less likely that bacteria harmful to people will grow.

Never open the plate after incubation. Look at it through the lid, then hand it back so your school can sterilise it before it is thrown away. Wash your hands when you finish.

What do exam questions ask about it?

Many questions ask you to explain one step of aseptic technique, such as why the loop goes through a flame or why the plate is kept at 25 °C and not 37 °C. The control disc comes up too: be ready to say what it shows. For an improvement, repeating with more plates and taking a mean is a safe suggestion. An odd result might come from a contaminated plate, or a disc that soaked for longer than the others. The calculation is usually an area from a diameter, and a 6-mark question may ask you to describe the whole method.

The same topic has one more bacteria calculation to be ready for. If a bacterium divides every 20 minutes, in 2 hours it divides 6 times, so one cell becomes 2⁶ = 64. Bigger numbers may need writing in standard form.

An exam-style question we wrote: "The clear zone around disc B has a diameter of 18 mm. Calculate its area. Give your answer to 3 significant figures." The radius is 9 mm, so the area is π × 9² = 254 mm² to 3 significant figures.

Common mistakes

• Putting the diameter into πr². Halve it first: r is the radius.
• Writing "25 °C keeps it safe" with no reason. Add the biology: bacteria that harm people grow best near body temperature, so a cooler incubator makes them less likely to grow.
• Forgetting the control disc, or not saying what it shows.
• Sealing the lid all the way round. A few short strips let oxygen in.
• Opening the plate to measure the zones. Measure through the lid.