Tips for practising safe cells - Part 1

Completely avoiding cell culture contamination is very difficult in busy laboratories. However, careful aseptic technique, appropriate caution and training can reduce the risk of contamination.

Aseptic technique can be used to reduce the potential of unwanted microorganisms or cell lines from being introduced into cell cultures, sterile solutions and supplies or from infecting laboratory workers.

Good aseptic technique can protect the cell line from microbial and cellular cross-contamination, prevent compromise of the cell line by misidentification and protect the value of cell lines, experiments and cell culture processes.

The following recommendations are designed to help improve aseptic techniques and to preserve the integrity of cell cultures:

• All supplies and reagents that come into contact with the cultures must be sterile.
• Wash hands before and after handling any cell culture. Handwashing stations should be readily accessible within the laboratory.
• Handle only one cell line at a time. There are intrinsic risks of misidentification or cross-contamination between cell cultures when more than one cell line is in use within the laboratory.
• Handle continuous cell lines after the handling of short-term, finite cell cultures.
• Avoid continuous long-term use of antibiotics within cell cultures. The overuse of antibiotics as prophylaxis may lead to cytotoxicity.
• Cultures should be inspected daily for signs of contamination.
• Promptly discard any contaminated cultures.

Sterile liquid transfer:

• Divide sterile solutions into small aliquots whenever possible. For instance, trypsin should be dispensed in single-use quantities of 5 to 10 mL/tube. Reagents should be prepared in sufficient amounts to only meet the requirements for the number of samples that are being processed. Solutions which are left over should be discarded rather than re-used.
• Always use separate media bottles for every cell line. This important step reduces both the possibility of cross-contamination with another cell line and limits the spread of contamination if the bottle of medium becomes contaminated.
• Avoid sharing bottles of media or other solutions with co-workers. Cross-contamination and lack of accountability arise from sharing with others.
• Do not use the same pipette with different cell lines. Never insert a pipette back into a bottle of medium after it has been used to feed a culture. This ‘double pipetting’ saves on pipettes but can easily lead to widespread contamination by other cell lines or mycoplasma.
• Do not insert the non-sterile portion of adjustable pipettors into vessels containing cells or sterile solutions; it is not worth the risk of contamination.
• Use filtered pipette tips for aseptic transfers. Using unfiltered pipette tips to transfer cells or medium can result in contamination of the pipetting device and subsequent pipette tips.
• Never mouth the pipette, even when non-hazardous substances are being transferred.
• Avoid pouring sterile liquids from one vessel into another. The drop of liquid that usually remains on the lip of the vessel can easily form a liquid bridge between the non-sterile outside and sterile inside of the vessel.
• Avoid spills or liquid bridges on the lips of dishes, bottles and flasks. They provide an easy access point for microorganisms into the vessel. Replace the caps of flasks that have wet threads or wipe dry with sterile alcohol wipes.
• Clean up any spills immediately and swab area with suitable disinfectants.

Part 2 of this article describes the impact of the laboratory environment on cell safety.