5.12 Required Practicals: Communicable Diseases

A free GCSE Biology lesson for AQA (8461) from OBA School: required practical: investigating the effect of antiseptics or antibiotics on bacterial growth using agar plates and clear zones. Watch the video, read the explanation and worked examples, then try the revision questions. You can work through the full lesson, with every video, practice quizzes and printable worksheets with mark schemes, in our free online course.

What you will learn

• Plan and carry out the antiseptic (or antibiotic) investigation using aseptic technique
• Identify the independent, dependent and control variables, and explain the control disc
• Calculate the mean diameter and the area of a clear zone using π r²
• Evaluate results, identify anomalies and suggest improvements

Key words

• Clear zone: The area around a disc where bacteria have been killed or could not grow (zone of inhibition).
• Lawn: An even layer of bacteria spread over the whole surface of the agar.
• Control: A disc soaked in sterile water. It shows that any clear zone is caused by the antiseptic, not the disc or handling.
• Anomaly: A result that does not fit the pattern of the others. It is left out when calculating the mean.
• Repeatable: If you repeat the experiment with the same method and equipment, you get similar results.

Aim, equipment and method

Aim: to find out which antiseptic is most effective at killing a type of bacterium (or how the concentration of one antiseptic affects bacterial growth).

Equipment: sterile agar plate; broth culture of a safe bacterium (e.g. Micrococcus luteus or a safe strain of E. coli); sterile pipette or spreader; filter-paper discs; antiseptics; sterile water; sterile forceps; Bunsen burner; disinfectant; tape; marker pen; incubator; ruler.

Method

1. Wipe the bench with disinfectant and wash your hands. Light the Bunsen burner to create rising air.

2. Label the base of the agar plate: your name, date, and a letter for each disc position.

3. Lift the lid slightly. Use a sterile pipette and spreader to make an even lawn of bacteria over the agar. Replace the lid.

4. Use sterile (flamed and cooled) forceps to dip a paper disc in antiseptic P. Shake off excess and place it on the agar. Repeat for the other antiseptics.

5. Place a control disc soaked in sterile water on the plate.

6. Tape the lid on with two or four short strips (not sealed). Turn the plate upside down.

7. Incubate at 25 °C for about 48 hours.

8. Without opening the plate, measure the diameter of each clear zone through the base. Repeat the whole experiment at least twice more.

Variables and risk assessment

• Independent; In this investigation Type of antiseptic (or its concentration)
• Dependent; In this investigation Area of the clear zone (from its diameter)
• Control; In this investigation Type of bacterium and volume of culture; size and type of paper disc; time discs are soaked; volume and depth of agar; incubation temperature and time

• Bacteria; Risk A pathogen may grow and infect you; Control measure Aseptic technique; 25 °C maximum; never open plates after incubating; wash hands; autoclave plates before disposal
• Bunsen flame; Risk Burns; ethanol-based antiseptics may catch fire; Control measure Keep antiseptics away from the flame; tie hair back
• Antiseptics; Risk Irritate skin or eyes; Control measure Wear eye protection; wash off spills

Processing the results

For each disc, measure two diameters at right angles and take the mean. Then find the mean of the repeats, leaving out anomalies. Halve the mean diameter for the radius and use area = π r².

• Antiseptic P; Plate 1 / mm 16; Plate 2 / mm 17; Plate 3 / mm 15; Mean diameter / mm 16.0; Area / mm² 201.1
• Antiseptic Q; Plate 1 / mm 24; Plate 2 / mm 23; Plate 3 / mm 25; Mean diameter / mm 24.0; Area / mm² 452.4
• Antiseptic R; Plate 1 / mm 11; Plate 2 / mm 12; Plate 3 / mm 10; Mean diameter / mm 11.0; Area / mm² 95.0
• Sterile water; Plate 1 / mm 6; Plate 2 / mm 6; Plate 3 / mm 6; Mean diameter / mm 6.0; Area / mm² 28.3

Worked example: antiseptic P

Mean diameter = (16 + 17 + 15) ÷ 3 = 16.0 mm, so r = 8.0 mm.
Area = π × 8.0² = 201.1 mm² (1 d.p.).
The water disc is 6 mm across: this is just the paper disc, so water did not kill the bacteria.

Answer: Q is the most effective antiseptic (largest area, 452.4 mm²)

Exam tip: The area of the clear zone is a better measure than the diameter, because it shows the real amount of agar where no bacteria grew. Doubling the diameter makes the area four times bigger.

Errors and improvements

• Clear zones are not perfect circles; Improvement Measure two diameters at right angles and use the mean; or count squares on graph paper
• Edges of zones are fuzzy, hard to measure to 1 mm; Improvement Use a magnifying lens or photograph the plate next to a scale
• Discs soak up different volumes; Improvement Use a micropipette to add the same volume to each disc
• Zones overlap if discs are too close; Improvement Space discs evenly; use fewer discs per plate
• Contamination (colonies of different colour or shape); Improvement Better aseptic technique; discard the plate

Antiseptics spread out through the agar by diffusion. A more effective (or more concentrated) antiseptic kills bacteria further from the disc, giving a bigger clear zone. The same method can be used to compare antibiotics, using discs already soaked in each antibiotic.

Common mistakes

• Using the diameter instead of the radius in π r².
• Including an anomalous result when calculating the mean.
• Opening the plate to measure the clear zones. Measure through the base with the lid taped on.
• Forgetting the control disc, so you cannot show the clear zone is caused by the antiseptic.
• Saying the plate is incubated at 37 °C "so bacteria grow faster". In schools the maximum is 25 °C for safety.

Revision questions

Try these, then check your answers below each one.

Question 1. A student compares three antiseptics using paper discs on an agar plate covered in bacteria. What is the independent variable? (Choose from: The type of antiseptic, The area of the clear zone, The incubation temperature, The type of bacteria.)

Answer: The type of antiseptic

Working: The student chooses which antiseptic goes on each disc, so the type of antiseptic is the independent variable.

Question 2. In the same investigation, what is the dependent variable? (Choose from: The size of the paper disc, The area of the clear zone, The volume of antiseptic, The type of agar.)

Answer: The area of the clear zone

Working: The student measures the clear zone, so its area (or diameter) is the dependent variable.

Question 3. True or false? A disc soaked in sterile water is included as a control.

Answer: True

Working: True. The water disc shows that any clear zone is caused by the antiseptic, not by the paper disc or handling.

Related lessons

• Previous lesson: 5.9 Human Defence Responses
• Next lesson: 6.1 Vaccination
• In this chapter: 5.1 Health and Disease
• In this chapter: 5.2 Pathogens and Disease
• In this chapter: 5.3 Growing Bacteria in the Lab

Continue in the full course

This lesson is section 5.12 (ARPBH0512) of our free GCSE Biology course for AQA. In the full course you get every video for this section, a self-marking practice quiz, a printable worksheet with a mark scheme, chapter tests, and your progress saved. Go to the full GCSE Biology course.