Core practical 5: Antiseptics, antibiotics and plant extracts

Aim

To find out which antiseptic, antibiotic or plant extract is most effective at stopping bacteria growing, by measuring the clear zone around discs on a bacterial lawn.

Background

Bacteria can be grown in the laboratory on nutrient agar jelly in a petri dish. The agar and dish are sterilised first (in an autoclave) so that only the bacteria you want to study grow. Aseptic technique stops other microorganisms getting in and stops the bacteria you are growing escaping.

Antibiotics are medicines that kill bacteria or stop them growing, without harming the host's cells. Antiseptics are chemicals used on living tissue, such as skin, to kill bacteria. Some plants make chemicals that also stop bacteria growing, so plant extracts can be tested in the same way.

If a substance stops bacteria growing, the bacteria close to it die or cannot divide. This leaves a clear zone (an inhibition zone) in the lawn around the disc. The bigger the clear zone, the more effective the substance. The clear zone is roughly circular, so its area is found using πr², where r is the radius.

Hypothesis

The antibiotic will produce the largest clear zone, because it is designed to kill bacteria, and the water disc will produce none, because water has no effect on bacterial growth.

Variables

Equipment

• Sterile nutrient agar plates (petri dishes)
• Broth culture of a harmless bacterium (for example Escherichia coli K12 or Micrococcus luteus) from a reputable supplier
• Sterile pipette and sterile spreader
• Sterile filter paper discs (about 6 mm across)
• Sterile forceps
• Test substances: an antibiotic solution, an antiseptic solution, one or two plant extracts (for example crushed garlic or mint leaves in a little water) and sterile distilled water
• Disinfectant, adhesive tape and a marker pen
• Incubator or warm cupboard set at 25 °C
• Transparent ruler with a millimetre scale
• Eye protection (disposable gloves if your teacher asks)

Risk assessment

Method

1. Wash your hands and wipe the bench with disinfectant. Put on eye protection. If your teacher tells you to, work close to a lit Bunsen burner, whose rising hot air carries microorganisms away from the plate.
2. Turn a sterile agar plate over and use a marker pen to divide the base into five sections. Write a code for each substance on the base, near the edge. Do not write on the lid.
3. Lift the lid just enough to work. Using a sterile pipette, transfer a small volume (about 0.1–0.2 cm³, as your teacher instructs) of the bacterial broth onto the agar.
4. Spread the broth evenly over the whole surface with a sterile spreader to make a bacterial lawn. Close the lid straight away.
5. Soak a sterile paper disc in each test substance. Use a separate disc for each one and let the extra liquid drip off.
6. Lift the lid slightly and use sterile forceps to place each disc in its labelled section, well spaced from the others and from the edge. Press each disc gently onto the agar.
7. Place a disc soaked in sterile distilled water in the fifth section as a control.
8. Fasten the lid with two short strips of tape. Do not seal it all the way round, so that oxygen can get in.
9. Turn the dish upside down and incubate it at 25 °C for 48 hours.
10. After incubation, do not open the dish. Look at it from underneath. Measure the diameter of each clear zone, including the disc, to the nearest millimetre. Measure twice at right angles and calculate the mean.
11. Halve each mean diameter to find the radius and calculate the area of each clear zone using area = πr².
12. Record your results in the table. Return the dish to your teacher to be sterilised, clean the bench with disinfectant and wash your hands.

Results

Fill this table in as you go. Print the PDF for a copy to write on.

Drawing the graph

Draw a bar chart. Put the substance on the x-axis (categoric data, so bars with gaps between them) and the area of the clear zone (mm²) on the y-axis. Label both axes and use a sensible scale.