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Investigate the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zones of inhibition

The effectiveness of antibiotics or antiseptics can be tested experimentally using uncontaminated agar plates.

How to culture microorganisms

This method is an example of aseptic technique.

This allows the selected bacteria to be grown under laboratory conditions, and it requires skill and experience. Additional contaminating bacteria will complicate the experiment and possibly confuse the results. Aseptic technique is vital when the effectiveness of antibacterial substances is being tested.

Preparing the agar plates of a colony of bacteria

1. Glass Petri dishes and agar gel must be sterilised before use in an autoclave that uses high pressure to heat steam to 121°C to sterilise equipment and growth media, or pre-sterilised plastic petri dishes can be bought.
   • Reason - this will kill any bacteria and their spores that are present in the solution or on the petri dishes.
2. Pour the sterile agar plates and allow to set fully.
   • Reason - this provides the selected bacterium with all the nutrients needed to grow.
3. Sterilise the inoculating loop, by heating it in the Bunsen burner flame.
   • Reason - kills any bacteria that are present on the loop.
4. Dip the inoculation loop into the microorganism solution and make streaks on the surface of the agar plate.
   • Reason - this allows the bacteria to spread out and to grow in individual colonies on the agar plate. A lawn of bacteria can be produced by using a sterile spreader to evenly spread the bacteria across the whole of the plate.
5. Replace the lid as soon as possible, secure with tape. Label and invert the plate, and store upside down.
   • Reason - This stops additional unwanted bacteria in the air contaminating the plate. Do not fully seal the lid, as this will stop oxygen reaching the bacterium, and this may encourage harmful anaerobic bacteria to grow. Labels are important, as they identify the growing bacterium.
6. Incubate at a maximum temperature of 25°C in schools and colleges.
   • Reason - this reduces the chance of growing harmful pathogens. Hospital laboratories would incubate plates at 37°C (body temperature) to allow quick growth and identification.

Testing the effectiveness of different antimicrobials

Adding antibiotic or antiseptic soaked patches to pre-prepared agar plates.

By adding filter paper soaked in a variety of anti-microbial solutions to the pre-prepared agar plate, the effect of the solutions can be tested experimentally. A clear area (zone of inhibition) indicates that the bacteria have been killed by the solution or have not been able to reproduce.

1. Soak small (5 mm) filter paper disks in a variety of solutions, or different concentrations of the same solution.
   • Reason - the effectiveness of the solutions at killing the bacteria can be tested.
2. Add the discs to the surface of a bacterial lawn, prepared using aseptic techniques.
3. Seal the Petri dishes, and label the solutions used on each of the discs.
4. Incubate the Petri dishes and allow sufficient time for bacterial to grow.
5. Examine patterns of bacterial growth around each of the paper discs.
6. Calculate the clear area around the soaked filter paper disks. A control disk must also be included.
   • Reason - size of zone indicates the effect of the substance tested on the growth of the specific bacterium.