Core practical
Investigate the effect of antiseptics, antibiotics or plant extracts on microbial cultures
The effectiveness of antibioticSubstance that controls the spread of bacteria in the body by killing them or stopping them reproducing. or antisepticA substance that kills or stops the growth of germs which cause disease. can be tested experimentally using agar plateA Petri dish that contains agar gel and usually some nutrients. Agar plates are used to culture (grow) bacteria and fungi in the lab. covered with a lawn of known bacteria.
Method A - Preparing the agar plates of a colony of bacteria
- Glass Petri dishes and agar gelA jelly-like substance that is derived from a type of seaweed and used in the lab as a medium on which to grow bacteria and fungi. must be sterilised in an autoclaveA strong heated container used to sterilise equipment. before use or pre-sterilised plastic Petri dishes can be bought.
- Reason – this will kill any bacteriaSingle-celled microorganisms, some of which are pathogenic in humans, animals and plants. Singular is bacterium. that are present in the solution or on the Petri dishes.
- Pour the sterile agar plates and allow to fully set.
- Reason – this provides the selected bacterium with all the nutrients needed to grow.
- Use a sterile pipette to add a few drops of the microorganism solution to the agar. Close the lid of the agar plate and place the pipette in disinfectant.
- Reason – this solution contains the bacteria that will grow on the agar.
- Unwrap a sterile spreader or sterilise a spreader in ethanol. Use the spreader to spread the micro organismsMicroscopic living things such as archaea, bacteria and some species of eukaryotes. solution, across the entire surface of the agar plate.
- Reason – this allows a lawn of bacteria to be produced across the whole of the plate. 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 anaerobicWithout oxygen. bacteria to grow. Labels are important, as this identifies the growing bacterium.
- Incubate at a maximum temperature of 25°C in schools and colleges.
- Reason – this reduces the chance of growing harmful pathogenMicroorganism that causes disease.. Hospital laboratories would incubate plates at 37°C (body temperature) to allow quick growth and identification.
This method is an example of aseptic techniquesName given to the laboratory procedures carried out to prevent the contamination of pure cultures of microorganisms..
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.
Method B - 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 (method A), the effective of the solutions can be tested experimentally. A clear area (zone of inhibitionThe area around an antibiotic or antiseptic on agar where bacterial growth is not visible. The size of the zone depends on how effective the antibiotic or antiseptic is.) indicates that the bacteria have been killed.
- Soak filter paper disks in a variety of solutions, use either different concentrations of the same solution, or a variety of different solutions.
- Reason - the effectiveness of the solutions at killing the bacteria can be tested.
- Measure the clear area around the soaked filter paper disks. A control disk must be also included.
- Reason - size of zone indicates the effect of the substance tested on the growth of the specific bacterium.