Detecting and Preventing Cell Culture Contamination

Biological contamination Bacteria: • Common examples include Escherichia coli, Bacillus cereus, Enterococcus malodoratus and Staphylococcus epidermis. • Easily introduced by poor aseptic technique or contaminated water baths. • Often cause visual changes in the media, such as increased turbidity and color change due to pH shifts. Mycoplasma: • Common contaminating species include Mycoplasma fermentans, Mycoplasma orale and Mycoplasma arginine. • Difficult to detect as it doesn’t cause any visible or morphological symptoms. • Introduced by operator cross‑contamination or cell culture supplements. Cell line crosscontamination: • Often caused by mislabeling and use of shared reagents. • Can result in an overgrowth of cells with unexpected characteristics. • Problematic cell lines have been estimated to have been used in around 16.1% of published papers. Fungi: • Typically caused by molds, such as Aspergillus or Penicillium, or yeasts, such as Candida species. • Easily introduced by poor aseptic technique and airborne transmission. • Often results in cloudy media or floating clumps. Viruses: • Often silent contaminants. • Difficult to detect using routine microscopy. • Can originate in the cell line itself or from animal serum. Chemical contamination Endotoxins: • Outer membrane components of Gram-negative bacteria. • Introduced from cell culture media and supplements. • Can affect cell viability and response. Free radicals: • Reactive species introduced through oxidative damage or poor reagent storage. • Can stress or kill cells. Metal ions: • Leached from containers or equipment. • Can disrupt cell signaling and enzymatic activity. Disinfectant residues: • Introduced from poorly rinsed equipment or surfaces. • Can be toxic to cells. Common sources of contamination Anyone working in the lab, along with the reagents and equipment they use, can unintentionally introduce microbes, unwanted cells or chemical contaminants into cell cultures. Contaminated reagents/media Unsterile lab equipment Unsuitable environment such as poor airflow Cultures should be routinely monitored for deviations in media appearance, cell growth, viability and morphology.3 When contamination is suspected, a range of detection methods are available for further testing. Not all contaminants are visible, so routine testing is advised. Detecting cell culture contamination Method Used to detect Microscopy Bacteria and fungi PCR Mycoplasma, viruses ELISA Endotoxins STR profiling Cell line authentication Visual inspection Bacteria and fungi How to prevent cell culture contamination Due to the negative impact of contamination, reducing the chances of it happening should be a priority for all labs. Several steps can be taken to minimize contamination risk and prevent it from escalating into a major problem when it does occur.4 / Practice good aseptic technique. Use suitable PPE, such as gloves, masks and lab coats. Regularly disinfect surfaces with 70% ethanol. Don’t eat or drink in the lab. / Use sterile media and reagents. Don’t share media between cell lines. / Quarantine new cell lines until tested. / Routinely test for Mycoplasma and cell identity. / Use good labeling practice. Standardize records and be careful with acronyms. / Discard suspect stocks promptly. / Limit time cultures spend outside controlled environments. / Maintain equipment regularly to ensure sterility and performance. / Use antibiotics strategically, not routinely. Overuse can mask contamination. / Build a cell repository. Store early-passage, authenticated stocks in liquid nitrogen. / Promote a culture of shared responsibility. Provide training and SOPs to all lab members. / Consider lab design. Optimize equipment placement and restrict access. Good Cell and Tissue Culture Practice (GCCP) 2.0 provides detailed guidance for reproducible, contamination-free culture.5 Contamination cannot be fully eliminated, but awareness, vigilance and good practice greatly reduce risk. By investing time and effort to protect cells, labs can ensure the reliability, reproducibility and credibility of their data. References: 1. Mirjalili A, Parmoor E, Moradi Bidhendi S, Sarkari B. Microbial contamination of cell cultures: A 2 years study. Biologicals. 2005;33(2):81–85. doi: 10.1016/j. biologicals.2005.01.004 2. Weiskirchen S, Schröder SK, Buhl EM, Weiskirchen R. A beginner’s guide to cell culture: practical advice for preventing needless problems. Cells. 2023;12(5):682. doi: 10.3390/cells12050682 3. Verma A. How to detect and avoid cell culture contamination. Technology Networks. http://www.technologynetworks.com/tn/how-to-guides/how-todetect- and-avoid-cell-culture-contamination-372612. Updated August 16, 2023. Accessed August 27, 2025. 4. Boussommier-Calleja A. Preventing and avoiding cell culture contamination. Technology Networks. http://www.technologynetworks.com/tn/how-toguides/ preventing-and-avoiding-cell-culture-contamination-299231. Updated October 29, 2024. Accessed August 27, 2025. 5. Pamies D, Leist M, Coecke S, et al. Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0). ALTEX. 2022;39:30–70. doi: 10.14573/ altex.2111011 SPONSORED BY: Cell culture plays a vital role in biological and biomedical research, providing scientists with tools to study cellular processes, model diseases and develop biotherapeutics. However, the reliability of these models depends on maintaining contamination-free conditions. Even small lapses can waste time and resources, compromise data and damage credibility. This infographic explores common sources and types of cell culture contamination and outlines best practices for preventing and detecting contamination in the lab. Detecting and Preventing Cell Culture Contamination Written by Anna MacDonald | Designed by Janette Lee-Latour Why cell culture contamination matters Contamination can affect several cell characteristics, including growth, metabolism, migration and morphology. This can lead to1: Lost time Unreliable data Costly setbacks Health hazards The effects of cell culture contamination can range from the inconvenience of discarded plates or flasks to significant impacts on reproducibility, wasted funding and reputational damage. Types of contamination Once isolated from tissue, cells in culture are vulnerable to two main categories of contamination: biological and chemical.2 Human error and poor aseptic technique