Mycoplasma Testing in Cell Culture: What Every Scientist Needs to Know
If you work with mammalian or eukaryotic cell lines, you’ve probably heard about mycoplasma contamination—and if you haven’t tested for it recently, your cells may be at risk. Mycoplasma can silently derail your experiments without visible signs, leading to unreliable data and wasted resources.
In this blog, we’ll break down:
What mycoplasma is
Why it’s so dangerous in cell culture
How to detect it using simple lab methods
And what to do if your cells test positive
What Is Mycoplasma?
Mycoplasmas are a group of tiny bacteria that lack a cell wall, making them:
Resistant to many antibiotics (like penicillin)
Small enough to pass through standard 0.22 μm filters
Invisible to the naked eye in cell culture
Unlike fungal or bacterial contamination, mycoplasma doesn’t cloud the media. This stealth allows it to persist for weeks or months without detection.
Why Is Mycoplasma a Big Problem?
Contaminated cell lines may:
Show altered gene expression and protein production
Affect cell metabolism and growth
Cause variability in transfection efficiency, signaling pathways, and drug response
Spread easily from flask to flask if undetected
In short: even low levels of mycoplasma can invalidate your experimental results.
How Do You Detect Mycoplasma?
There are several methods to test your cultures, depending on your equipment and urgency:
1. PCR-Based Detection (Most Common in Research Labs)
Detects mycoplasma-specific DNA sequences (16S rRNA genes)
Fast, sensitive, and covers multiple species
Protocol:
Collect culture supernatant or pellet
Heat-treat or extract DNA
Run PCR with universal primers
Visualize bands (~270–500 bp) on agarose gel
Pros: Sensitive, specific
Cons: Requires thermal cycler, potential for false positives if contaminated
2. Bioluminescence Assays (e.g., MycoAlert™ by Lonza)
Detects ATP-dependent enzymes unique to mycoplasma
Provides colorimetric or luminescent readout in 30 minutes
Pros: Fast, easy
Cons: Detects only viable mycoplasma; slightly less sensitive than PCR
3. DNA Staining (Hoechst or DAPI)
Use a fluorescence microscope to stain extranuclear DNA
Look for small bright dots outside the nucleus
Pros: Visual confirmation
Cons: May miss early or low-level infections; labor-intensive
4. Culture-Based Testing
Inoculate samples into a mycoplasma-specific growth medium
Incubate for up to 4 weeks and assess growth
Pros: Highly specific, detects viable mycoplasma
Cons: Slow, requires special media and lab conditions
Recommended Kits for Labs
What If Your Cells Are Contaminated?
If your culture tests positive:
Discard affected flasks (preferred in most labs)
Decontaminate equipment and incubators
Optionally treat with anti-mycoplasma antibiotics (e.g., Plasmocin®) — but this is not always 100% effective
Go back to frozen stocks or obtain fresh cells if available
Takeaway
Mycoplasma contamination is a hidden threat that can:
Alter your data
Compromise reproducibility
Waste time and funding
Make regular mycoplasma testing part of your cell culture routine—especially before publication, transfection, or downstream applications.
Other Forms of Cell Culture Contamination to Look Out For
Beyond mycoplasma, there are several other common forms of contamination in cell culture, each with its own signs and risks. Here's a breakdown:
1. Bacterial Contamination
Visible Signs: Cloudy media, color change (phenol red turns orange/yellow), rapid pH drop
Microscopic Signs: Rods or cocci moving independently
Sources: Non-sterile technique, contaminated reagents
2. Fungal Contamination
Visible Signs: Filamentous growth, floating clumps or “cottony” masses
Microscopic Signs: Hyphal networks, budding yeast cells
Sources: Airborne spores, contaminated incubators or water trays
3. Yeast Contamination
Visible Signs: Medium may appear grainy or slightly turbid
Microscopic Signs: Budding cells, larger than bacteria
Sources: Air, poor aseptic technique, human skin
4. Cross-Contamination with Other Cell Lines
Signs: Unexpected morphology, altered growth rates
Detection: STR profiling (for human lines), isoenzyme testing
Sources: Shared pipettes, mislabeled flasks, poor technique
Risk: Invalid data — one of the biggest causes of irreproducible science
5. Viral Contamination
Signs: Often none visible; can cause cell death or behavior changes
Detection: PCR, immunoassays, electron microscopy
Sources: Serum, donor tissue, infected cell lines
Risk: High — especially in clinical/preclinical research
6. Endotoxin Contamination
From lysed Gram-negative bacteria (e.g., E. coli)
Doesn’t cause visible signs but triggers immune responses in sensitive cells (e.g., PBMCs)
Detection: LAL assay
Question for You:
How often does your lab test for mycoplasma—and have you ever discovered a contaminated line?
Share your experience in the comments below or tag a colleague who needs this reminder!
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