Preventing, Detecting, and Addressing Cell Culture Contamination

Cell culture contamination can be devastating for a laboratory—causing the loss of valuable time, effort, and reagents, not to mention potentially irreplaceable cell lines. Of further concern is undetected contamination; producing potentially erroneous results, it risks damaging your laboratory’s reputation. Biological contaminants such as bacteria, molds, yeasts, viruses, and mycoplasma as well as cross-contamination by other cell lines form a major part of this problem, and while contamination can never be completely eliminated, proactive measures can help you prevent, detect, and handle cell culture contamination—limiting occurrences and their impact.

Prevention

As Alison Armstrong, Senior Director and Global Head of Field Development Services at MilliporeSigma, explains, “There are two known avenues for potential introduction of contamination: personnel and environment.” To limit the risk of contamination from these sources, good aseptic technique, sterile products, and a clean working environment should be used at all times. Methods to ensure such stringent practices are maintained should also be established—regular cleaning routines, equipment maintenance, and staff training are critical to success in reducing cell culture contamination.

In addition to good working practices, a proactive contamination monitoring program should also be established. All contamination, however minor, should be recorded, and each occurrence should be reviewed for possible causes and severity. Understanding the frequency and cause of contamination within your laboratory is essential to prevent further outbreaks in the future.

To prevent cross-contamination, “Cell lines should only be purchased from trusted sources that employ best practices regarding regular screening and sterile filtration,” explains Markus Markowich, Technical Support Specialist at Corning Life Sciences. Any cell lines that enter the laboratory, “should be quarantined until contamination screening has been completed. Once the cells are cleared, it is advisable to screen them regularly for contaminants at least every three to four months.”

And what about antibiotics? While many labs routinely use antibiotics in cell culture, their overuse can result in resistant strains and poor technique. As Fang Tian, Principal Scientist at ATCC, makes clear, “Rather than relying on antibiotics, using cell culture best practices and proper aseptic technique can effectively help prevent contamination.”

Detection

If contamination happens, quick detection is vital to controlling and limiting its impact. Different biological contaminants can be detected in different ways. “Contaminant organisms such as bacteria multiply very quickly and may secrete organic substances,” says Markowich, meaning they can be detected within a few days. A drop in pH, a cloudiness or visual turbidity of the media, or a yellow color change in phenol-red containing medium may be noticed, while bacteria themselves can appear as tiny moving objects under a light microscope. As Matthew Dallas, Senior Manager for R&D in Cell Biology at Thermo Fisher Scientific, explains “Cell culture scientists should keep a close eye on their cultures and look for anything out of the ordinary, as these could be early signs that the culture has been contaminated.” A Gram’s stain test can then confirm their presence.

By contrast, yeast and mold contamination show very little change in culture pH until the contamination is at an advanced stage. Early detection therefore relies on visual inspection; under light microscopy yeast appear as ovoid or spherical particles, while mycelia usually appear as thin, wispy filaments or denser clusters of spores. Such a method is ineffective for detecting viruses however. Because of their size, and because they do not usually adversely affect their host cell, viruses are very difficult to detect. Viral detection relies on electron microscopy, immunostaining, ELISA assays, or PCR.

Mycoplasma detection is equally challenging and often shows no visible signs until a chronic infection is established. This means it “can go undiscovered for months until it seriously affects the cells,” explains Markowich, and for this reason “regular screening of the cell lines is strongly advised.” Infection can be tested by two basic methods. Direct microbial culture is the most sensitive method, but is challenging and time-consuming, and should ideally be conducted by an outside testing facility. Indirect methods, although less effective, give quicker results and can be conducted in-house. These include PCR-based kits, DNA fluorochrome staining, ELISA, or specialized test kits.

As Tian summarizes, “The best way to detect contamination is to have a protocol for routine testing of cell cultures before they exhibit symptoms.” This can make outbreaks easier to handle.

Handling contamination

Despite all good intentions and proactive measures, contamination still occurs. And once it does, knowing how to deal with it quickly and efficiently is key.

If contamination is detected, the best practice is always to discard the sample and start again. Autoclaving is guaranteed to eliminate the infection, so all possible contaminated cultures and reagents should be autoclaved, and the incubator thoroughly cleaned. Other cultures sharing the incubator should be closely monitored over the following days and weeks for any signs of infection.

If a cell line is truly irreplaceable, “Prophylactic use of an antibiotic can prove useful for short durations,” explains Tian. Although penicillin and streptomycin are commonly used, resistant mycoplasma infections require the use of tetracyclines, macrolides, and quinolones. Care should be taken, however, as high doses of antimicrobial compounds can affect cell health and viability. So, while treating valuable cultures may be a risk worth taking; it has no guaranteed success and can lead to repeated contamination or problems with cell health later.

Summary

Contamination is a constant risk in every cell culture laboratory. And while the threat of contamination will always remain, good working practices and proactive screening are essential to reduce the likelihood and severity of outbreaks—protecting your research and reputation. As Matthew Dallas explains, while “Technologies that can prevent microbes from surviving on surfaces may one day make their way into the laboratory environment, there is no replacement for good aseptic technique.”