Mycoplasma Testing in Cell Culture
Quick Facts
| Feature | Information |
|---|---|
| Purpose | Detection of mycoplasma contamination in cell cultures |
| Organism | Wall-less bacteria (Class Mollicutes) |
| Detection Frequency | Monthly (recommended) |
| Common Detection Method | PCR |
| Biosafety Impact | Essential Quality Control |
| Applicable To | Cell lines, primary cells, stem cells, organoids, cell banks |
Overview
Mycoplasma contamination is one of the most common and serious quality control issues in cell culture laboratories. Unlike bacterial or fungal contamination, mycoplasma infections are usually invisible, do not cause media turbidity, and may remain undetected for extended periods.
Because contamination can profoundly alter cell physiology and experimental outcomes, routine mycoplasma testing is considered an essential component of good cell culture practice.
What are Mycoplasmas?
Mycoplasmas are extremely small bacteria belonging to the class Mollicutes.
Unlike most bacteria, they:
- Lack a cell wall
- Grow slowly
- Are resistant to many common antibiotics
- Are generally invisible under routine light microscopy
| Feature | Description |
|---|---|
| Size | 0.1-0.8 μm |
| Cell Wall | Absent |
| Growth Rate | Slow |
| Microscopic Visibility | Usually not visible |
| Antibiotic Resistance | Resistant to many cell wall-targeting antibiotics |
Why Mycoplasma Testing Matters
Mycoplasma contamination can alter numerous cellular processes, including:
- Cell proliferation
- Apoptosis
- Differentiation
- Cellular metabolism
- Signal transduction
- Immune responses
- Gene expression
- Protein expression
These changes can lead to:
- False biological phenotypes
- Poor experimental reproducibility
- Invalid research conclusions
Common Sources of Contamination
Typical sources include:
- Cross-contaminated cell lines
- Laboratory personnel
- Contaminated sera or reagents
- Poor aseptic technique
- Shared culture media and pipettes
Why Detection is Difficult
Unlike bacterial contamination, mycoplasma infection usually does not produce obvious visual changes.
Typical characteristics include:
- Clear culture medium
- Normal pH indicator color
- Continued cell growth
- No visible turbidity
Possible indirect signs include:
- Reduced proliferation
- Increased cell death
- Morphological abnormalities
- Experimental variability
- Unexpected differentiation failure
When Should Cells Be Tested?
Routine screening is recommended throughout the cell culture workflow.
| Time Point | Recommendation |
|---|---|
| Routine culture | Every 4 weeks |
| Before cryopreservation | Required |
| After thawing | Recommended |
| Before distribution | Required |
| Before major experiments | Recommended |
Examples of critical experiments include:
- RNA-seq
- Proteomics
- Drug screening
- Stem cell differentiation
- Genome editing
Detection Methods
| Method | Sensitivity | Speed | Typical Use |
|---|---|---|---|
| PCR | High | Fast | Routine testing |
| qPCR | Very High | Fast | Clinical and GMP laboratories |
| DNA Fluorescent Staining | Moderate | Fast | Rapid screening |
| Culture-Based Detection | Very High | Slow | Confirmatory testing |
| Luminescence Assays | High | Very Fast | Routine laboratory screening |
PCR remains the most widely adopted method because it combines high sensitivity with relatively rapid turnaround.
Recommended Testing Strategy
For most research laboratories:
Routine Screening
↓
PCR or Luminescence Assay
↓
If Positive
↓
Confirm by PCR (if required)
↓
Appropriate corrective action
Interpreting Results
Negative Result
- No detectable mycoplasma contamination
- Continue routine monitoring
Positive Result
- Culture considered contaminated
- Immediate corrective action required
- Prevent spread to other cultures
Prevention Strategies
Good laboratory practices remain the most effective prevention strategy.
Recommended practices include:
- Routine monthly testing
- Quarantine newly received cell lines
- Separate validated and unverified cultures
- Use dedicated reagents when possible
- Maintain strict aseptic technique
- Perform regular surface disinfection
Importance for Stem Cell Research
Routine testing is particularly important for:
- Human iPSCs
- Embryonic stem cells
- Organoids
- Neural stem cells
- Gene-edited cell lines
Contamination may alter:
- Pluripotency
- Differentiation efficiency
- Gene expression
- Genome editing outcomes
- Omics datasets
Untested cultures should never be used for:
- Cell banking
- CRISPR editing
- Clone generation
- Clinical or translational studies
Laboratory Quality Control
A comprehensive quality control program should include:
- Monthly mycoplasma screening
- Testing before cryopreservation
- Testing after thawing
- Testing before cell distribution
- Testing before publication-quality experiments
Common Problems
| Problem | Possible Cause | Recommended Solution |
|---|---|---|
| Repeated positive results | Cross-contamination | Improve segregation and replace contaminated stocks |
| False positives | Sample contamination | Repeat testing with confirmatory assay |
| False negatives | Low organism load | Repeat testing using PCR-based methods |
Best Practices
- Test cultures every month
- Verify cultures before cryopreservation
- Test all newly acquired cell lines before routine use
- Confirm cultures are mycoplasma-negative before publication
- Never establish Master Cell Banks from untested cultures
Related SOPs
- SOP: PCR-Based Mycoplasma Detection
- SOP: MycoAlert® Luminescence Assay
- SOP: DAPI/Hoechst Mycoplasma Screening
- SOP: Mycoplasma Decontamination
- SOP: Cell Line Quarantine
References
- Uphoff CC, Drexler HG. Detection of Mycoplasma Contamination in Cell Cultures. Current Protocols in Molecular Biology (2014).
- Young L et al. Detection and Control of Mycoplasma Contamination in Cell Cultures. Cytotechnology (2010).
- International Cell Line Authentication Committee (ICLAC) Recommendations.
- Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications.