����ý

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

Aseptic technique

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.

Preparation of the work area

1. Clear the work space of all non-essential items.
2. Clean the desk with disinfectant.
   • Reason - this kills all unwanted bacteria and so decreases the chance of the agar plate becoming contaminated.

Preparing the agar plates for growth of a colony of bacteria

• Glass petri dishes and agar gel must be sterilised before use by using an autoclave, or pre-sterilised plastic petri dishes can be bought.
  • Reason - this will kill any unwanted bacteria that are present in the solution or on the petri dishes.
• Pour the agar into the sterile petri dishes and allow to set fully.
  • Reason – this provides the selected bacterium with all the nutrients needed for them to grow.

Plating the bacteria

1. The following should be done beside a blue Bunsen flame.
   • Reason - to create an updraft to stop the agar media getting contaminated with unwanted bacteria from the air.
2. Swirl (do not shake) the bacterial suspension to make sure that the bacterial culture is well mixed.
   • Reason - to make sure that the bacteria aren't all at the bottom of the container
3. Sterilise the inoculating loop, by heating it in the Bunsen burner flame. Leave it to cool. Alternatively, sterilise it by placing it in pure alcohol for a few seconds.
   • Reason - kills any unwanted bacteria that are present on the loop.
4. Remove the lid from the bacterial bottle and put the mouth of the bottle in the Bunsen flame.
   • Reason - to kill off any unwanted bacteria that could be on the bacterial bottle.
5. 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.
6. Replace the lid of the petri dish as soon as possible and secure with tape. Allow the plate to dry then label the half of the petri dish containing the media (do not label the top). Invert the plate and store it upside down.
   • Reason - The lid 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 this identifies the growing bacterium. If the lid is separated from the petri dish for some reason, the label will stay with the part that has the bacteria on it, so it can be identified.
7. Incubate at a maximum temperature of 25°C in schools and colleges.
   • Reason - this reduces the chance of growing harmful pathogens, which would grow at 37°C in a human body. Hospital laboratories would incubate plates at 37°C (body temperature) to allow quick growth and identification.

Clearing up following the activity

1. All contaminated materials need to be disposed of either in autoclave bags (for disposable materials that need to be sterilised, eg spreaders/Petri dishes) or pots (for items that are to be washed, sterilised and then reused).
2. It is essential that all work surfaces need to be thoroughly disinfected at the end of the activity. Ensure that hands are washed with soap and water at the end of the activity.