Recognise bacterial, fungal, mycoplasma and cross-contamination in cell culture and respond correctly.
Why it matters
Contamination is the most common way cell culture goes wrong. Some contaminants are obvious overnight; others, such as mycoplasma or a different cell line, can sit unnoticed for months and invalidate every result produced from those cells. In biomanufacturing a contamination event can mean losing a batch worth a great deal of money and investigating the whole facility. A technician who recognises contamination early, and responds by the procedure rather than by instinct, protects the whole lab.
The main types
| Contaminant | What you see | Notes |
|---|---|---|
| Bacteria | Medium turns cloudy and often yellow within a day or two; tiny moving dots between cells under the microscope | Fast growing; sometimes a film on the surface |
| Yeast | Cloudy medium; round or oval particles, sometimes budding, often in chains | pH may change slowly at first |
| Mould (fungi) | Thin filaments (hyphae), later fuzzy clumps visible by eye | Spores spread through air and incubators |
| Mycoplasma | Usually nothing visible: no cloudiness, no obvious pH change | Very small bacteria without a cell wall; alter cell metabolism and gene expression; not killed by penicillin |
| Viruses | Usually nothing visible | Important in biomanufacturing; tested with specific assays |
| Cross-contamination | Cells look and behave like "your" line but are not | Another cell line has taken over; HeLa is the classic culprit |
Detecting the invisible ones
- Mycoplasma is tested routinely (for example monthly, and always for new lines entering the lab) using PCR, enzyme-based luminescence kits or DNA staining. Bought or shared lines go into quarantine until they test clean.
- Cell line identity is confirmed by STR profiling (short tandem repeat analysis) for human lines, compared against reference databases. The International Cell Line Authentication Committee (ICLAC) keeps a register of known misidentified lines: https://iclac.org/databases/cross-contaminations/ (Checked on 2026-10-23.)
- In GMP manufacturing, cell banks and production batches are tested for sterility, mycoplasma and adventitious viruses before release.
Prevention
- Aseptic technique in a certified biosafety cabinet, every time.
- One cell line at a time in the cabinet, with cleaning between lines, so lines cannot mix.
- Separate, labelled media bottles for each line, never shared.
- Clean incubators and water pans on a schedule, and keep the lab tidy.
- Work from a tested master and working cell bank, and keep passage numbers low.
- Minimise routine antibiotics, which can mask low-level contamination.
Responding to contamination
When you suspect contamination, follow your lab's procedure. Typically:
- Do not open the vessel in the cabinet with other cultures, and do not try to "rescue" it unless the culture is irreplaceable and your supervisor approves a specific procedure.
- Tell your supervisor and record what you saw, with photographs if possible.
- Decontaminate and discard the culture as your institution's biosafety procedure requires (for example by disinfectant treatment or autoclaving).
- Check everything that was shared: other cultures from the same incubator, media and reagent bottles used with it, and the cabinet.
- Clean the cabinet and incubator as required.
- Investigate the cause (technique, a reagent lot, incubator, a new cell line) and record corrective actions, using the deviation and CAPA approach from the lab data course.
Worked example. Monday you see cloudy, yellow medium in one of three flasks of the same line, with moving dots under the microscope. The other two flasks look normal. Bacterial contamination of one flask is likely. You report it, discard the flask as required, check the other two flasks and the shared medium bottle, and clean the cabinet. Because all three were fed from the same medium bottle on Friday, you take a sample of that bottle for testing and quarantine it until the result is back.
Safety and training
Contaminated cultures, and human or animal cell lines in general, are biohazards. Handling, disinfection and waste rules depend on your institution and biosafety level, and you learn them through institution training under supervision. This course never asks you to handle cultures at home.
Common mistakes
- Assuming no cloudiness means no contamination (mycoplasma and cross-contamination are invisible).
- Trying to treat a contaminated flask with extra antibiotics and carrying on.
- Opening the contaminated flask in the shared cabinet.
- Skipping quarantine for a new line from a collaborator.
- Not recording the event, so a pattern across weeks is missed.
Quick recap
- Bacteria, yeast and mould are usually visible; mycoplasma, viruses and cross-contamination usually are not.
- Test mycoplasma routinely and authenticate lines (STR); check the ICLAC register.
- Prevent with aseptic technique, one line at a time, separate media and tested cell banks.
- Respond by the procedure: contain, report, discard safely, check shared items, clean, investigate, document.
Try it
The Do tab has a quiz with contamination scenarios.