Contamination is the most common reason cell-culture experiments fail. It wastes time and reagents and, worse, can produce misleading results without being noticed. This guide covers the main types of contamination, how to recognise them, how to prevent them and what to do when a culture is affected.
Types of contamination
Biological contaminants
- Bacteria: the medium turns cloudy, often within a day or two, and may change colour (many media turn yellow as pH drops). Under the microscope you may see small moving particles between cells.
- Yeast: individual round or oval particles, sometimes budding; the medium becomes turbid more slowly.
- Moulds (filamentous fungi): visible filaments or fuzzy colonies, sometimes floating clumps.
- Mycoplasma: very small bacteria without a cell wall. They do not cause turbidity and are not visible with a standard microscope, yet they can change cell growth, metabolism and gene expression. They are detected only by testing.
- Cross-contamination with other cell lines: one cell line overgrows another. It is invisible without authentication and has affected published research.
Chemical contaminants include impurities in water or reagents, detergent residues and endotoxins, which can alter cell behaviour without visible signs.
How to prevent contamination
Work aseptically
- Work in a certified biosafety cabinet that is switched on in advance, cleaned before and after use, and kept free of clutter.
- Wipe items with 70% ethanol before placing them in the cabinet.
- Do not pass hands or arms over open vessels; keep caps face down or held.
- Use sterile, individually wrapped pipettes and change them between reagents and cell lines.
Organise the work
- Handle one cell line at a time, and work with clean, low-passage stocks before older or less trusted cultures.
- Label everything clearly: cell line, passage number, date and initials.
- Aliquot media and reagents so that a contaminated bottle affects only a small volume.
Maintain the environment
- Clean incubators, water pans and centrifuges on a schedule, and record it.
- Keep humidifier water clean and follow manufacturer guidance on additives.
- Limit traffic and airflow disturbance around the cabinet.
Think twice about antibiotics
Routine antibiotics can hide low-level contamination and promote resistant organisms. Many laboratories maintain cells without antibiotics so that problems become visible early, using them only for specific purposes.
How to detect contamination
- Look every day: check the colour and clarity of the medium and the morphology of the cells before working with them.
- Test for mycoplasma regularly: PCR-based kits, fluorescent DNA staining or enzymatic assays. Test new cell lines on arrival and on a routine schedule, for example every one to three months.
- Authenticate cell lines: short tandem repeat (STR) profiling confirms the identity of human cell lines and should be repeated when a line is established and at intervals.
- Keep records: a culture log with passage, observations and test results makes it possible to trace a problem to its source.
What to do when a culture is contaminated
- Isolate it. Close the vessel and move it away from other cultures.
- Discard it safely. Decontaminate according to your laboratory’s biosafety rules, typically with disinfectant or autoclaving.
- Decontaminate the workspace, including the cabinet, incubator and any shared equipment.
- Check the other cultures and reagents handled at the same time, including media and supplements.
- Return to frozen stocks from a known clean, authenticated bank.
- Record what happened and what you changed.
Trying to rescue a contaminated culture is rarely worthwhile when clean stocks are available, and it risks spreading the problem.
Build good habits early
Most contamination comes from routine handling, which is why aseptic technique is best learned by practising it repeatedly under supervision. Our hands-on Animal Cell Culture Techniques course covers aseptic work, passaging, counting, cryopreservation and contamination control, and Essential Laboratory Skills builds the foundations for any bench work.
Further reading: Geraghty RJ et al. (2014). Guidelines for the use of cell lines in biomedical research. British Journal of Cancer 111:1021–1046.
Written by the Vivofold team for research and education. Found an error? Tell us and we will correct it.