Cell culture contamination is often not detected easily, until experimental variability becomes too large to ignore. One of the most persistent threats is mycoplasma species. These are small, cell-wall-deficient bacteria that bypass normal filtration mechanisms. They are capable of modifying:
- Cellular metabolism
- Gene expression
- Growth kinetics
It is important to know the source of contamination. It enables you to have specific controls in place as opposed to reactive remedies. Below, we explain some major risk areas.
Contact with infected cell lines
Some laboratories move cell stocks without a quality-assurance record. Poor internal handling procedures can compromise samples.
You can mitigate this risk by:
- Isolating all newly obtained cell lines.
- Mycoplasma testing before they are introduced into common culture areas.
- Performing authentication and contamination screening before expansion or cryopreservation.
There must be a high level of separation between quarantined and validated cultures. This ensures that infected lines do not compromise the rest of the laboratory inventory.
Poor aseptic technique
Mycoplasma species may be introduced by laboratory staff either by:
- Respiratory droplets
- Contact with infected surfaces
- Poor practices in the biosafety cabinet
- Inadequate changes of gloves
- Congested work areas.
You can reduce this risk by:
- Providing formal training for aseptic techniques
- Periodic evaluation of competence
- Having standard workflows
- Sanitizing biosafety cabinets before and after use
- Using tested agents effective against Mycoplasma.
- Limiting talking when handling open culture.
Frequent mycoplasma testing of high-risk cultures is one more insurance. Especially where there are many operators or a high rate of experiments in the laboratory.
Contaminated reagents and biological materials
Unless there are adequate sourcing and manufacturing controls, mycoplasma may be carried by:
- Serum
- Trypsin
- Media supplements
- Other biologically derived reagents.
Most commercial suppliers usually have quality systems. But there are always reports of contamination incidents.
Here is how to mitigate this risk:
- Your source reagents vendor should provide analysis certificates. They should have tested negative for Mycoplasma.
- Store reagents under recommended conditions upon receipt.
- Do not subject them to repeated warming cycles.
- Aliquot high-use reagents where possible to reduce recurrent exposure.
- Install lot tracking. This makes it easy to identify contamination and throw the batches away.
Shared Incubators and equipment
Indirect contamination can also happen in labs through:
- Incubators
- Water baths
- Centrifuges
- Microscopes.
Shared CO 2 incubators are especially troublesome. Contamination may occur through aerosolized particles or contact between flasks.
To minimize such exposure:
- Assign separate incubators for quarantined vs validated cell lines where possible.
- Introduce periodic decontamination procedures. Use high-temperature sterilization.
- Clean water pans regularly.
Combine these practices with periodic mycoplasma testing of representative cultures. This can aid in the early detection of equipment-related transmission.
Cross-handling multiple cell lines
Lastly, some people manage two or more cultures in the same biosafety cabinet. This heightens the risk of cross-contact, particularly where media bottles, pipettes, or waste containers are moved across lines.
Instead:
- Implement a one-cell-line-at-a-time policy in the cabinet.
- Use specialized media and pipettes on each line during active work.
- Label all materials clearly.
- Physically separate culture vessels.
This, plus routine mycoplasma testing, will detect cross-transfer events before they can affect large datasets or batches of production.