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Aseptic technique

Aseptic technique - steps to follow

Aseptic technique is one of the fundamental principles of microbiology and is crucial to prevent cross-contamination of the cultures and media and importantly to maintain our own health and safety.

Purity plating or streaking was first developed by Loeffler and Gaffky in Robert Koch’s laboratory in the 1880s. It works on the principle that you inoculate the plate at first with high inoculum, and each time you streak it further you gradually reduce the numbers of bacteria until you reach the point you are manipulating a single bacterium, which then grows to form a single colony.

This is crucial to allow the picking of a single (clonal population) but also to identify if a mixed population is present (contamination). Both are crucial for antimicrobial susceptibility testing.

The following is a list of steps to prevent contamination of your media and yourselves. You will probably be able to suggest a few more.

  • Reduce and prevent draughts and unexpected airflow in the area you are working (e.g. no open windows).
  • Keep working surfaces cleaned with a suitable disinfectant and ensure you clean up any spills quickly.
  • Prepare your working area beforehand making sure you have all necessary reagents and equipment easily to hand.
  • Carry out procedures efficiently but without hurrying.
  • When opening tubes or plates limit the amount of time they are open to contamination as much as possible. If you must rest a lid on the bench, make sure it is facing upwards so that the rim does not touch the surface.
  • Allow liquids to settle after shaking to avoid aerosol formation.
  • Take care with sterile equipment you will use to inoculate bacteriological media – for example loops, pipettes, etc. If you touch something (e.g. another surface, clothing, your own hand) other than the intended media – discard the object or re-sterilize it.
  • Make sure to work with only sterile equipment and reagents and visually inspect media for potential contamination.
  • Waste containers should be covered when not in use, and emptied regularly.
  • Store bacterial growth plates appropriately and do not leave them out on the lab bench when not in use. Plates should be stacked no more than 10 high in the incubator (inoculated side face down), or placed in a sealed autoclave bag when ready for discard.
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Bacterial Genomes: Antimicrobial Resistance in Bacterial Pathogens

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