At best, cell culture contamination can be a minor inconvenience, resulting in the loss of a few plates or flasks. At worst, it can represent a major setback, casting doubt on the validity of past or current work. This article outlines the main types of cell culture contaminants, and their common sources, and offers practical strategies for controlling cell culture contamination in the lab.
Cell culture contaminants can take many forms. Biological contaminants, such as bacteria, molds, and yeasts, are typically easy to detect as their presence in cell culture media often results in pH shifts (commonly seen as media color changes), turbidity, and cell damage or death. Other biological contaminants, including viruses, protozoa, insects, mycoplasmas, and other cell lines, are more difficult to identify. For example, mycoplasmas can reach very high densities in cell cultures (107 to 109 colony forming units/mL) without any visible signs of contamination, due to their small size and their lack of a cell wall.
While many researchers are quick to blame biological contaminants for changes in cell growth or morphology, chemical contamination may also be responsible. Chemical contaminants include metal ions and endotoxins in media and culture supplements; impurities in gases used in CO2 incubators; and free radicals generated in media through exposure to fluorescent light. In small amounts, most chemical contaminants are present at low, non-toxic levels. However, in combination, they may cause stress responses or even lead to culture loss.
One of the main causes of biological contamination is the unintentional use of non-sterile supplies, media, or solutions during routine cell culture procedures. Such products may be contaminated due to improper sterilization or storage or may become contaminated during use. Biological contamination may also result from airborne particles and aerosols, which are frequently generated during routine lab activities, including pipetting, aspiration, and centrifugation. Further sources include microorganisms entering culture vessels due to improper sealing or handling, as well as accidental cross-contamination with other cultured cells.
There are many steps that researchers can take to minimize the risk of cell culture contamination. Here are our five practical strategies:
• Use good aseptic technique
Good aseptic technique includes working in a certified biosafety cabinet, wearing a clean lab coat and gloves, and utilizing sterile materials. It is also important to use properly sealed culture vessels (e.g., vented cap flasks) and filtered pipet tips when possible, minimize clutter in the biosafety cabinet, work with only one cell line at a time, and aliquot reagents into smaller volumes to reduce repeated handling.
• Reduce opportunities for accidental contamination
Avoid mix-ups and cross-contamination by clearly labeling solutions and other supplies, carefully labeling cell lines, and utilizing standardized record-keeping forms to document and track cell culture activities. The use of written protocols and formulation sheets when preparing media and solutions—listing the reagents used, lot numbers, weights, and other critical information—is also recommended.
• Keep the laboratory clean
Reducing the amount of airborne particulates and aerosols in the laboratory will help lower contamination risk. Cleaning should include routinely wiping floors and work surfaces; periodically disinfecting incubators, biosafety cabinets, and water baths; and regularly vacuuming the cooling coils on refrigerators and freezers. Waste containers should be emptied daily.
• Routinely monitor for contamination
The key to developing a realistic contamination monitoring program is to keep it as simple as possible while ensuring that it can get the job done. Bacteria, fungi, and yeasts can be detected using standard microbiological testing methods. Mycoplasma contamination can be identified directly in cultures (e.g., PCR-based testing) or via indirect tests that measure specific mycoplasma characteristics (e.g., enzymatic assay). Methods for confirming cellular identity include karyotyping, DNA fingerprinting, and isoenzyme analysis using electrophoresis. Detecting chemical contaminants is more difficult, but careful documentation and consistency in materials and processes can help identify potential sources.
• Use frozen cell repositories strategically
Cell repositories enable researchers to convert continuously maintained cultures into a series of short-term working stocks. This simplifies the amount of routine monitoring required and limits potential problems from cryptic contaminants. However, a cell repository is only a reliable resource if the cultures it contains have been properly tested, well documented, and stored under appropriate conditions.
Although cell culture contamination cannot be totally avoided, it can be managed to reduce how often it occurs and limit the seriousness of its consequences. Corning Life Sciences offers an extensive range of products that can help to combat contamination, including vented cap flasks, filtered pipet tips, and sterile tubes. To learn more, visit corning.com/contamination