Mycoplasma contamination is the silent killer of cell biology experiments. Unlike bacteria or fungi, mycoplasmas don’t cloud media or produce visible colonies. They quietly alter gene expression, growth rates, metabolism, and immune responses — often for months before anyone notices.
Why mycoplasmas are different
Mycoplasmas are the smallest self-replicating prokaryotes. They lack a cell wall, which means antibiotics like penicillin and streptomycin don’t affect them. They pass through 0.22 µm filters routinely used to sterilize media. And they can reach densities of 10⁸ organisms per mL with no visible turbidity.
Sources of contamination
- Other contaminated cell lines in the same incubator
- Bovine serum (still the most common historical source)
- Operator-derived contamination from oral or skin flora
- Reagents introduced during experiments
How to detect mycoplasma
PCR-based detection
The most common modern approach. Primers target conserved 16S rRNA regions. Sensitive (10–100 organisms per reaction), fast (under a day), well-suited to routine screening. Many commercial kits available.
DNA staining (Hoechst or DAPI)
Mycoplasma DNA appears as small punctate fluorescence around cell nuclei and on the cell surface. Cheap and direct, but requires experience to interpret and is less sensitive than PCR.
Culture-based detection
Considered the gold standard but slow (28 days) and unable to detect non-cultivable strains. Now mainly used for regulatory compliance.
Biochemical assays
Detect mycoplasma-specific enzymes. Fast and sensitive but more expensive than PCR.
Prevention is everything
- Quarantine new cell lines for at least one passage and test before introducing them to your main incubator.
- Test routinely — once a month is reasonable for active lines; before banking is mandatory.
- Avoid working with multiple lines simultaneously in the same hood without thorough decontamination between.
- Use validated FBS lots, ideally heat-inactivated or gamma-irradiated.
- Don’t share media bottles across users.
- Clean hoods, incubators, and water baths on a documented schedule.
If you find contamination
The safest action is to discard the line and thaw a clean stock. Treatment with anti-mycoplasma antibiotics (Plasmocin, ciprofloxacin, BM-Cyclin) can work but is imperfect — strains can become resistant, and treated lines often retain altered phenotypes. If the line is irreplaceable, treat with cycling antibiotics for 2–3 weeks, then retest at least three times over a month before declaring it clean.
The cost of ignoring it
Estimates vary, but 5–35% of cell lines in active use are believed to be contaminated. Mycoplasma alters cytokine secretion, viral infection rates, drug responses, and gene expression — making any data from a contaminated line fundamentally suspect. Many high-impact retractions trace back to contamination that wasn’t tested for.
Mycoplasma testing is the cheapest insurance in cell biology. Build it into your routine and your data will be cleaner, more reproducible, and more publishable.
