Aseptic technique video protocol

Aseptic technique prevents contamination of cell cultures and reagents by microorganisms. Watch our aseptic technique video protocol that shows you how to sterilize work areas and use appropriate sterile handling techniques, personal protective equipment and good hygiene (2:34 minutes).

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​Sterile work areas

• Cell culture should be carried out in a laminar flow safety cabinet located in a room that is specific to cell culture.
• The interior of safety cabinets should be wiped with Trigene disinfectant followed by 70% ethanol before and after use. Ultraviolet (UV) light can also be used to sterilize safety cabinets between uses.
• Areas of work should be uncluttered and contain only the items required for the current procedure.
  
  

Sterile handling techniques

• Spray gloved hands with 70% ethanol before commencing work in the safety cabinet.
• When bringing equipment or reagents into safety cabinets, spray them beforehand with 70% ethanol.
• Never pour cell culture media or other reagents. Instead use sterile glass or disposable plastic pipettes to manipulate liquids and use each one only once.
• Many cell culture reagents are certified sterile from the manufacturer; however, they can become contaminated during handling. Sterilize reagents either by filtration (0.2 µm filters) or autoclaving if necessary.
• Never leave bottles of reagents, flasks or dishes open in the hood longer than necessary. Only open vessels immediately before use and if a lid has to be placed on the surface of the safety cabinet, ensure the opening is facing down. Replace lids as soon as possible.
• Never open multiple flasks, dishes or bottles at the same time, this may result in cross contamination.
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Personal protective equipment and good hygiene

• Always wash hands thoroughly before and after working with cell cultures.
• Wear a lab coat, gloves and safety glasses to protect yourself from any hazardous materials and to prevent contamination of the cell cultures from microbes present on skin and clothes.
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View our cytopresevation of mammalian cell lines protocol.

Sources of contamination and how to address them​

The most common source is through contamination of cultures with microorganisms such as bacteria, fungi and yeasts, which can usually be observed under the microscope.

Another important contaminant is mycoplasma, which is resistant to traditional cell culture sterilisation techniques, isn’t visible and requires specific routine testing to detect. Once a mycoplasma contamination has been detected the only way to ensure a mycoplasma free environment is through hydrogen peroxide fumigation, also known as “bombing”, of the lab. All cell lines from Abcam are supplied mycoplasma-free, so this is something you should not need to worry about.

Video transcript

Aseptic technique prevents contamination of cell culture and reagents from microorganisms in the environment. This video will outline the procedures necessary to work in a sterile manner within a cell culture hood.

Before commencing work ensure that you have washed your hands and are wearing the correct personal protective equipment, this includes gloves, a lab coat, and safety glasses. Thoroughly spray the inside of the cell culture hood with disinfectant and wipe clean with tissue.

Dispose of used tissue in the appropriate waste bin. Next, spray the inside of the hood with 70% ethanol and wipe clean with tissue. The hood is now ready to use. Take the cell culture media and supplements from the fridge and place in a 37^oC incubator to warm. To prepare the media aseptically spray each item with ethanol. Aim to work quickly and efficiently to minimize the risk of contamination and place each bottle in easy reach. Add each supplement to the bottle of media using a new sterile tip or pipette. Only open bottles immediately before use and if the lid is put down ensure the opening is facing downwards. Never sweep your hands and sleeves over the top of an opened bottle as this could cause contamination. To minimize the risk of this happening replace the lid as soon as you are finished with the bottle.

Label the media bottle with your initials the date and details of the contents.