Launch practical
About 60 minutes
- Set up the bench with the 16 items of apparatus.
- Take the readings yourself, into the results table you draw first.
- Process your results and plot the graph.
- Answer exam questions on your own results, which you mark against the mark scheme.
Runs in your browser: a 3D bench with 2D close-ups of each instrument for exact readings. Nothing to install and no account.
What you measure, and how
One run takes 9 readings. You take every one yourself, the way you would in class.
- On the 3D bench (Fig. 1) you set out the apparatus and read each instrument where it stands.
- In the 2D close-up (Fig. 2) the same instrument is drawn large, from the same run, for an exact reading. It is also how the practical runs on a device that cannot show 3D.
- In your table you write what you read, to the instrument’s resolution, and the notebook checks it against the instrument.
How to read it. Held up to the light, lid taped on. Cloudy lawn; clear agar lets the light through.
What it shows
area = π r²
Each antiseptic diffuses out of its paper disc into the agar, getting weaker as it spreads. Visible bacterial growth is inhibited where the antiseptic is sufficiently concentrated, so a clear zone (a zone of inhibition) forms round the disc while a cloudy lawn grows everywhere else. Zone size depends on growth inhibition and diffusion through the agar; clear agar alone does not prove that bacteria were killed.
Recall: learn it. Halve the diameter to get the radius.
Apparatus
| Item | What it is for | Resolution |
|---|---|---|
| Sterile nutrient agar plate | The agar is food for the bacteria; the Petri dish keeps them in. | — |
| Culture of E. coli (a safe strain made for schools) in a bottle | The bacteria you grow as a lawn. | — |
| Sterile plastic pipette | Moves a few drops of culture onto the agar. | — |
| Glass spreader | Spreads the culture evenly over the agar. | — |
| Small beaker of ethanol, with a lid | The spreader is dipped in it, then flamed, to sterilise it. | — |
| Bunsen burner on a heatproof mat | Sterilises the spreader and forceps; its updraught keeps microbes away from the plate. | — |
| Filter-paper discs (6 mm) | Each one carries a liquid onto the agar. | — |
| Mouthwash, TCP, antiseptic cream and sterile water | The antiseptics to compare, and water for the control disc. | — |
| Forceps | Pick up and place the discs without touching them. | — |
| Clear sticky tape | Holds the lid on with two short strips. | — |
| Wax pencil | Labels the base of the plate. | — |
| Disinfectant spray and antibacterial hand wash | Kills microorganisms on the bench and your hands. | — |
| Discard pot of disinfectant | Used pipettes, spreader and forceps go in it. | — |
| Incubator set to 25 °C | Keeps the plate warm for 48 hours so the bacteria grow. | — |
| 30 cm ruler | Measures each clear zone’s diameter through the base. | 1 mm |
| Safety goggles | Protect your eyes from antiseptic splashes. | — |
Method
In our own words, as the Lab runs it. Your school's method sheet may differ in detail.
Which version are you doing?
Boards set this practical slightly differently. Your course setting picks one; you can change it before you start.
Why: The plate and the method are the same; what is on the discs and the exam questions follow your board.
Disinfect the bench and wash your hands
Spray your working area with disinfectant and wipe it with a paper towel. Then wash your hands with the antibacterial hand wash.
Why: The bench and your hands carry microorganisms. Disinfecting kills them, so they cannot get onto the agar and grow there.
Put on safety gogglesSafety
Put on goggles and keep them on until you have cleared away.
Why: The liquids on the discs can sting your eyes if they splash, and you are working next to a flame and ethanol.
Light the Bunsen and work beside it
Stand the Bunsen on the heatproof mat in the middle of your working area and light it. Open the air hole to a blue flame while you work, and do everything close to the flame.
Why: The flame heats the air above it, so the air rises. This updraught carries microorganisms in the air up and away from the open plate.
Label the plate
Write your name, the date and what is on each disc in a quarter of the plate with the wax pencil. Keep the writing small and near the edge.
Do: On the base, underneath the agar.
Why: You must be able to tell which disc is which after two days in the incubator.
Take a sample of the bacteria
Loosen the lid of the culture bottle. Pass its neck through the flame, take a few drops of culture with a sterile pipette, flame the neck again and put the lid back. The used pipette goes straight into the discard pot of disinfectant.
Why: Flaming the neck kills microorganisms on the rim, so only the E. coli goes onto the plate. The disinfectant kills the bacteria left on the pipette.
Open the plate
Drop the culture onto the agar. Open the plate only as much as you need to.
Do: Lift the lid at an angle, open only on the Bunsen side.
Why: Every second the agar is open to the air, microorganisms can fall onto it.
Spread the bacteria
Dip the glass spreader in the beaker of ethanol, then pass it through the flame to burn the ethanol off. Spread the culture evenly over the whole agar surface.
Do: Dip, flame, then hold it near the flame for 10 seconds to cool.
Why: An even lawn of bacteria grows over the whole plate, so every disc sits in the same lawn.
Soak the discs
Soak one filter-paper disc in each liquid you are testing and one in sterile water for the same time (10 minutes).
Do: Lift each disc out and touch it on the side of the dish so the extra drains off.
Why: Each disc should carry the same amount of liquid, so only the substance changes from disc to disc.
Flame the forceps
Pass the tips of the forceps through the flame and let them cool for a few seconds before you pick up each disc.
Why: Flaming kills microorganisms on the tips, and stops one liquid being carried to the next disc.
Place the discs
Lift the lid at an angle and put each disc on the agar with the forceps, in the quarter you labelled. Press it down gently.
Do: One in each quarter, about 2 cm from the edge.
Why: Each zone needs room to form on its own, so you can measure it.
Tape the lidSafety
Close the lid and stick it to the base with tape.
Do: Two short strips across the edge, on opposite sides.
Why: Taping stops the plate being opened by accident, so nobody is exposed to what grows.
Set up the incubator
The plate will grow in the incubator for 48 hours.
Do: Upside down (lid at the bottom), at 25 °C.
Why: Bacteria need warmth to grow quickly enough to form a lawn in two days.
Take the readings
After 48 hours at 25 °C: two diameters of the clear zone round each antiseptic disc, at right angles, in mm; then the mean diameter and the area.
Repeats: AQA’s method measures each clear zone twice, across its centre at right angles, and uses the mean, because zones are not always round. The plate itself takes two days to grow, so you cannot repeat it in the lesson: repeat plates, or the class’s results, show whether the results are repeatable.
Make safe and clear upSafety
Do not open the plate: keep the lid taped on. Put the plate in the autoclave bag for your teacher to sterilise. Put the spreader and forceps in the discard pot of disinfectant. Check the Bunsen and its gas tap are off. Spray and wipe the bench, then wash your hands.
While you take readings
Where do you measure the zones?
Keep the lid taped on. Look at the zones through the clear base of the plate.
Do: Upside down on a sheet of black card.
Why: The edge of a clear zone is where the cloudy lawn stops. It is easiest to see where the clear agar looks dark.
How many times do you measure each zone?
Put the ruler across the centre of the disc, with the zero on one edge of the clear zone, and read the other edge. The paper disc is part of the zone, so measure straight across it.
Do: Twice: one diameter, then another at right angles to it, then the mean.
Why: Clear zones are not always perfect circles, so one diameter may not be typical.
Hazards and control measures
| Hazard | What could happen | Control measure |
|---|---|---|
| Bacteria, and any other microorganisms that land on the plate and grow | Infection, if they get into your mouth, eyes or a cut | Use aseptic technique; incubate at 25 °C, not 37 °C; tape the lid on and never open the plate after incubation; the plate is sterilised in an autoclave. |
| A plate taped all the way round, so no oxygen gets in | Conditions can become anaerobic, potentially favouring unwanted anaerobic microorganisms, including some harmful to humans | Tape the lid with two short strips only, so a little air can get in. |
| Ethanol is highly flammable and is used next to a flame | The ethanol could catch fire and cause burns | Keep the beaker of ethanol away from the Bunsen with its lid nearby; dip the spreader, then pass it through the flame for a moment only. |
| A hot spreader or hot forceps straight from the flame | Burns to the fingers | Hold them by the handle and let them cool near the flame before use. |
| Antiseptics such as TCP splashed in the eyes | Sore, irritated eyes | Wear safety goggles throughout. |
Variables
- Independent: you change
- Type of antiseptic
- Dependent: you measure
- Size of the clear zone round each disc in mm
- Control: you keep the same
- The bacteria and how much culture is spread. The same culture, spread evenly over one plate.
- Size of the paper discs. All 6 mm discs from the same hole punch.
- Soaking time and volume of antiseptic on each disc. Every disc soaked for 10 minutes and drained.
- Incubation temperature and time. 25 °C for 48 hours, all on the same plate.
Results from a specimen run
| Looking at | Observation |
|---|---|
| The whole plate | A cloudy lawn of bacteria, with a clear zone round each antiseptic disc |
| Other colonies | No other colonies: the lawn is even all over |
| The water (control) disc | Bacteria grow right up to the disc: no clear zone |
| Antiseptic | Diameter 1 in mm | Diameter 2 in mm | Mean diameter in mm | Area of clear zone in mm² |
|---|---|---|---|---|
| Mouthwash | 13 | 13 | 13.0 | 133 |
| TCP | 17 | 18 | 17.5 | 241 |
| Antiseptic cream | 9 | 10 | 9.5 | 71 |
| Sterile water (control) | no zone | — | — | 0 |
Processing the data
- Area: Mouthwash
- 133 mm²
- Area: TCP
- 241 mm²
- Area: Antiseptic cream
- 70.9 mm²
- Why a bar chart
- The type of antiseptic is a categoric variable: there is nothing in between mouthwash and TCP.
- Most effective antiseptic
- TCP
- The water disc
- The paper disc on its own does not stop bacteria growing, so the zones are caused by the antiseptics.
- How a clear zone forms
- 3 of 3 right first time
| Quantity | Specimen run | Accepted |
|---|---|---|
| Mean diameter, mouthwash (class typical) | 13.0 mm | 13.1 mm |
| Mean diameter, TCP (class typical) | 17.5 mm | 19.6 mm |
| Mean diameter, antiseptic cream (class typical) | 9.50 mm | 9.70 mm |
Common mistakes and the fix
Mistakes to check in this practical, and what to do instead. In a write-up or an exam answer, the mark goes to naming the step and saying what it does to the readings.
Method: The bench looks clean
Microorganisms from the bench and your hands can reach the plate. More odd colonies grow on the lawn, and one is more likely to grow inside a clear zone and spoil a reading.
The fix: Disinfect the bench and wash your hands. The bench and your hands carry microorganisms. Disinfecting kills them, so they cannot get onto the agar and grow there.
Method: Skip them
No effect on your results, but a splash from a disc’s liquid could reach your eyes: flagged in the safety review.
The fix: Put on safety goggles. The liquids on the discs can sting your eyes if they splash, and you are working next to a flame and ethanol.
Method: Work at the side of the bench instead
Nothing carries microorganisms in the air away from the plate. More odd colonies grow, and one is more likely to grow in a clear zone.
The fix: Light the Bunsen and work beside it. The flame heats the air above it, so the air rises. This updraught carries microorganisms in the air up and away from the open plate.
The plate after 48 hours: Recorded “A cloudy lawn with clear zones, and a clear stripe across it with no bacteria”
The fix: Look again: the lawn is even all the way to the edge, with no stripe across it.
The plate after 48 hours: Recorded “A cloudy lawn with no clear zones anywhere”
The fix: Look round each antiseptic disc: there is a ring of clear agar where the bacteria did not grow.
Exam answer: Leaving out: Sterilise (flame) the equipment, e.g. pass the forceps / spreader / loop through a flame, or flame the neck of the bottle
The fix: Name a piece of equipment and how it is sterilised: pass the forceps through a flame.
Exam answer: Leaving out: Disinfect the bench / work surface (or hands); ignore clean
The fix: Say what is disinfected: spray the bench with disinfectant (“clean” is not enough).
Worked exam-style questions
Written for Study Hack in the style of exam questions, on the specimen run above. They are not taken from any exam board's papers. Try each one before you open the worked answer.
- Question 1Aseptic technique · Knowledge[2 marks]
A student prepared an agar plate of bacteria to test three antiseptics.
Give two aseptic techniques the student should use when preparing the plate.
Worked answer and how the marks are given
A full-mark answer
Pass the forceps through a Bunsen flame before using them. Lift the lid of the dish only a little, at an angle.
Mark scheme
Any 2 from:
- Sterilise (flame) the equipment, e.g. pass the forceps / spreader / loop through a flame, or flame the neck of the bottle
- Disinfect the bench / work surface (or hands); ignore clean
- Lift the lid only a little / at an angle
- Secure the lid with (adhesive) tape
- Work near a (Bunsen) flame (the updraught carries microbes away)
- Use sterilised agar / Petri dish
Where marks are usually lost
- Missing: Sterilise (flame) the equipment, e.g. pass the forceps / spreader / loop through a flame, or flame the neck of the bottle. Name a piece of equipment and how it is sterilised: pass the forceps through a flame.
- Missing: Disinfect the bench / work surface (or hands); ignore clean. Say what is disinfected: spray the bench with disinfectant (“clean” is not enough).
- Missing: Lift the lid only a little / at an angle. How is the lid opened? Only a little, at an angle.
- Question 2Area of a clear zone · Applying knowledge[3 marks]
Use your answer to Question 02.1 to calculate the area of the clear zone round the TCP disc.
Area = mm²
Worked answer and how the marks are given
A full-mark answer
r = 17.5 ÷ 2 = 8.75 mm
area = π r² = π × 8.75²
= 241 mm²Mark scheme
- radius = 17.5 ÷ 2 = 8.75 (mm) (method) Allow error carried forward.
- area = π r² (allow 3.14 for π) (method) Allow error carried forward.
- = 241 (mm²) (allow π taken as 3.14: 240; allow correct use of an incorrect mean) Allow error carried forward.
- Allow answers from 238.1 to 242.9 mm² (the marker's tolerance; correct rounding of the student's own working is also accepted).
- Allow the unit written as mm2, mm^2, square mm, square millimetres, sq mm, mm squared.
- Error carried forward: method and answer marks follow correctly from the student's own earlier value.
- A correct answer with no working scores full marks.
Where marks are usually lost
- Answer 962 mm² (gains 1 mark). You used the diameter in π r². The radius is half the diameter: r = 8.75 mm, so the area is π × 8.75² = 241 mm².
- Answer 962 mm² (gains 1 mark). You used the diameter in π r². The radius is half the diameter: r = 8.75 mm, so the area is π × 8.75² = 241 mm².
- Answer 27.5 mm² (gains 1 mark). You did not square the radius. Area = π r² = π × 8.75 × 8.75 = 241 mm².
- Question 3Planning: antiseptics and bacterial growth · Knowledge[6 marks]
A student wants to compare how well three antiseptics stop the growth of bacteria. They have an agar plate, a culture of bacteria, paper discs and the three antiseptics.
Describe a method the student could use.
Include how the student should keep the investigation safe and avoid contamination.
Worked answer and how the marks are given
A full-mark answer
1. Spray the bench with disinfectant and wash your hands.
2. Light a Bunsen burner and work close to the flame.
3. Flame the neck of the culture bottle and use a sterile pipette to put a few drops of bacteria on the agar, lifting the lid only a little at an angle.
4. Dip a glass spreader in ethanol, flame it, let it cool, then spread the bacteria evenly over the agar.
5. Soak paper discs of the same size in each antiseptic for the same time, and one in sterile water as a control.
6. Use flamed forceps to put the discs on the agar, evenly spaced apart.
7. Tape the lid on with two strips of tape and label the base.
8. Incubate upside down at 25 °C for 48 hours.
9. Measure the diameter of the clear zone round each disc twice at right angles, and calculate the mean.
10. Calculate each area using π r². The antiseptic with the largest clear zone is the most effective.Mark scheme
Indicative content:
- Disinfect the bench (and hands)
- Sterilise equipment in a flame (forceps / spreader / loop / bottle neck) or use sterile equipment
- Work near a Bunsen flame
- Lift the lid only a little / at an angle
- Spread the bacteria (culture) evenly over the agar
- Soak paper discs (same size, same time) in each antiseptic
- A control disc soaked in (sterile) water
- Place the discs on the agar (with forceps), spaced apart
- Tape the lid on (not sealed all the way round) / label the base
- Incubate at 25 °C (not 37 °C), for about 48 hours (upside down)
- Measure the diameter (or radius) of the clear zone round each disc
- Two diameters at right angles, then a mean (or repeat plates and a mean)
- Calculate the area (π r²) and compare: largest zone = most effective
Levels of response Level Marks What the answer does 3 5–6 A coherent method that would give valid results: all the important steps are there, in a sensible order. 2 3–4 A method that might not give valid results: most of the important steps are there, but some are missing or out of order. 1 1–2 A method that would not give valid results: some relevant steps are given, but they are not linked into a workable method. In Study Hack a six-mark answer is not given an automatic level: you tick the points your answer makes, then choose the level that fits it.
- No relevant content: 0 marks.
Evaluate
| Source of error | Type | Effect | Improvement |
|---|---|---|---|
| Judging where the edge of each clear zone is | Random | The lawn fades out over a millimetre or so, so each diameter can be a millimetre out either way. | Read the zones through the base against a dark background, where the clear agar looks black. |
| Zones that are not perfectly round | Random | One diameter of an oval zone can be bigger or smaller than its average width. | Measure two diameters at right angles and use the mean. |
| Discs put on dripping with antiseptic | Systematic | Extra antiseptic spreads further into the agar, so every zone is too big, by different amounts for different discs. | Soak every disc for the same time and drain off the extra before placing it. |
| A colony of another microorganism inside a clear zone | Random | A reading taken to the colony instead of the zone edge is too small. | Use aseptic technique (disinfect, work by the flame, flame the forceps, lift the lid only a little), and measure along a line that misses any colony. |
| Zones running into each other | Systematic | Where two zones merge you cannot see where one ends, so diameters measured towards a neighbour are too small. | Space the discs evenly, one in each quarter, about 2 cm from the edge. |
| Measuring from the edge of the paper disc instead of across its centre | Systematic | Every diameter is short by the width of the disc (6 mm), so every area is far too small. | Put the ruler across the centre of the disc and measure from edge to edge of the clear zone. |
| Ruler resolution (1 mm) on a small zone | Random | For the cream’s small zone, 1 mm is over 10 % of the diameter and over 20 % of the area. | Use a magnifying glass, or lay the plate on graph paper and count the squares. |
| Only one plate | Random | Plates differ (how thick the lawn is, how deep the agar is), so one plate’s zones may not be typical. | Set up three plates and calculate a mean area for each antiseptic. |
Repeatable. If you set up more plates in the same way, with the same antiseptics and bacteria, the same antiseptic gives the largest zone and the diameters agree to within a few millimetres.
Reproducible. Another group, or another school with a different batch of agar, gets the same order of antiseptics from largest zone to smallest.
Other exam boards
How this practical lines up with other GCSE boards' required or core practicals. Codes only; check your own specification for the exact activity.
| Board | Practical | Match | What is different |
|---|---|---|---|
| AQA | Required practical 2 | This page | — |
| Edexcel | 5.18B | Similar, with differences | Adds plant extracts as a test substance. |
Print it
- Blank write-up: the nine-step lab book with the correct table headings, ready to print for a real lesson.
- Specimen lab book: this page's run written up in full, with its exam answers ticked against the mark scheme.
- Printable method card: the practical on one index card. Practise with the cards in Study Hack.
- Practical workbook: a full simulated data set to process on paper, with exam questions and answers.
- Teacher and technician pack: apparatus lists, technician notes and marking points.