Cell Structure » Required Practical: Antibiotics and Bacterial Growth

The aim of the practical

Some chemicals stop bacteria growing. Antibiotics are drugs that kill bacteria inside the body. Antiseptics are chemicals put on skin and wounds to kill bacteria. In this practical you investigate how well different antibiotics or antiseptics work, or how well different concentrations of one antiseptic work.

You do it by growing a lawn of bacteria on an agar plate and adding the chemicals to the plate. Where the chemical works, the bacteria cannot grow and a clear area appears.

Key terms:

• Antibiotic: a drug that kills bacteria inside the body.
• Antiseptic: a chemical applied to skin or wounds to kill bacteria.
• Zone of inhibition: the clear area around a disc where bacteria cannot grow.
• Hypothesis: a testable prediction, based on science, about what will happen and why.

The method

Use the same aseptic technique you met in Culturing Microorganisms. Then follow these steps:

1. Use a sterile spreader to spread a liquid culture of bacteria evenly over a sterile agar plate. This makes a lawn of bacteria.
2. Soak small paper discs in the antibiotics or antiseptics you are testing. Use a different disc for each one.
3. Soak one more disc in sterile water. This is the control disc.
4. Use sterile forceps to place the discs on the agar, spaced well apart. Press each one down gently.
5. Label the base of the dish (not the lid) with a marker pen. Tape the lid on and turn the dish upside down.
6. Incubate the dish at 25°C for about 48 hours.
7. Look at the plate. Measure the clear zone around each disc.

The control disc should have no clear zone. This shows that the water and the paper do not stop bacteria growing, so any clear zone around another disc is caused by the chemical.

Reading the results

A diameter is not the best way to compare results because the zone is a circle and its size depends on the radius squared. Doubling the diameter makes the area four times as big. So we calculate the area.

Calculating the area of a clear zone

The area of a circle is πr², where r is the radius. The radius is half the diameter. Use π = 3.14 unless told otherwise.

Hypotheses, variables and accuracy

A good hypothesis says what you expect and why. For example: A higher concentration of antiseptic will give a bigger zone of inhibition, because more of the chemical will be present to kill the bacteria.

To make the test fair, only one thing changes and everything else stays the same:

• Independent variable (what you change): the type of antibiotic, or the concentration of antiseptic.
• Dependent variable (what you measure): the area of the clear zone.
• Control variables (what you keep the same): the species of bacteria, the amount of bacteria spread, the type and depth of agar, the size of the discs, the volume of chemical on each disc, the incubation temperature and the incubation time.

To be accurate, measure the diameter carefully to the nearest millimetre. Measure each zone twice, in different directions, and use the mean. Repeat the experiment and calculate a mean area for each chemical, which makes the results more reliable.

Choosing the best antibiotic or concentration

The best antibiotic is the one with the largest zone of inhibition, because it stopped the most bacteria growing. In the same way, the most effective concentration of an antiseptic is the one that gives the largest zone.

Safety and ethics

Exam-style question

A student tested three antibiotics on a lawn of bacteria. She placed a disc soaked in each antibiotic on the agar, plus one disc soaked in sterile water. After incubation the diameters of the clear zones were: antibiotic X 10 mm, antibiotic Y 16 mm, antibiotic Z 0 mm.

(a) Why did she use a disc soaked in sterile water? [1 mark]
(b) Which antibiotic was the most effective? Give a reason. [2 marks]
(c) Calculate the area of the clear zone for antibiotic Y. Use π = 3.14. [2 marks]