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Guide to Mycoplasma-Free Cultures: Mycoplasma Testing Protocol

CLYTE research team
Nov 14
8 min read

In the world of cell culture, the most dangerous threat is the one you cannot see. Mycoplasma contamination is the silent sabotage of research—an insidious, common, and often undetected problem. Unlike bacterial or fungal contamination, Mycoplasma does not cause turbidity, a pH shift, or visible cloudiness. Instead, it quietly hijacks your cell culture.

You might also be interested in this article about the cost of contamination!

Ask Sophie about Mycoplasma testing in cell culture!

Because these tiny bacteria lack a cell wall, they are resistant to common antibiotics like penicillin and streptomycin. They pass through standard 0.2-micron filters and can spread rapidly through aerosols or shared reagents. Once present, Mycoplasma contamination systematically invalidates research by:

Altering cell metabolism and nutrient competition.
Modifying gene expression and signal transduction.
Inducing chromosomal aberrations.
Reducing transfection efficiency.
Slowing cell proliferation.

It is estimated that 15-35% of all continuous cell cultures are contaminated. Therefore, routine testing is not optional; it is a fundamental pillar of Good Cell Culture Practice (GCCP) and research integrity. This article provides a comprehensive protocol for the detection, prevention, and elimination of Mycoplasma, based on leading industry and academic standards.

A Guide to Mycoplasma Detection Methods

There is no single "perfect" test. The most robust quality control (QC) protocols often combine a rapid, highly sensitive method (like PCR) with a viability-based method (like microbiological culture).

Method 1: PCR-Based Detection (The Rapid Standard)

Polymerase Chain Reaction (PCR) is the most widely used method for routine Mycoplasma detection. It is exceptionally fast, sensitive, and reliable.

How it Works: The test uses specific primers to amplify a highly conserved region of the Mycoplasma genome, typically the 16S rRNA gene. This allows for the detection of a broad range of Mycoplasma species, including the six species that account for 95% of all contaminations.
- Culture Preparation (Recommended): For maximum sensitivity, grow the cell line for 2-3 passages without antibiotics before testing. This prevents suppression of the contamination, which could lead to a false negative.
- Grow Cells: Culture your cells until they reach 80-90% confluency. This is when Mycoplasma titers are generally highest. Avoid using over-confluent or old cultures.
- Collect Sample: Aseptically transfer 1-2 mL of the cell culture supernatant (the liquid medium, cell-free) into a sterile microcentrifuge tube.
- Heat Inactivation (Optional): Many protocols recommend heating the supernatant sample at 95°C for 5-10 minutes. This lyses any remaining cells, releases Mycoplasma DNA, and inactivates DNA-degrading enzymes (DNases).
- Run Test: Use the prepared supernatant (or lysate) as the template for your PCR reaction, following your specific kit's instructions.
- Validate with Controls: Every PCR run must include:
  - Negative Control: A sample of sterile, fresh culture medium.
  - Positive Control: A sample containing the known Mycoplasma DNA standard (usually provided in the kit).

Pros:
- Rapid: Results can be obtained in just a few hours.
- Highly Sensitive: Can detect very low levels of contamination, often down to a few genomes per microliter.
- Specific: Greatly reduces the chance of false positives compared to other methods.
Cons: PCR can detect DNA from both living and dead Mycoplasma. A positive result may linger after successful antibiotic treatment, though this "dead" DNA will dilute out with subsequent passages.

Method 2: DNA Staining (The Visual Check)

This indirect method uses fluorescent DNA-binding dyes, such as DAPI (for fixed cells) or Hoechst 33258 (for live or fixed cells), to visualize contamination.

How it Works: Cells are grown on a coverslip and stained. The researcher then uses a fluorescence microscope to observe the sample. The cell nuclei will fluoresce brightly. If Mycoplasma is present, it will appear as a pattern of small, distinct fluorescent dots in the cytoplasm or in the extracellular spaces around the cells. This is often described as a "starry sky" appearance.
- Prepare Sample: Seed and grow your cells on sterile glass coverslips (or a chamber slide) until they reach a sub-confluent density.
- Stain Cells:
  - For Fixed Cells (DAPI or Hoechst): Fix the cells (e.g., with a methanol-based fixative) and then apply the DNA-binding dye solution (DAPI or Hoechst 33258) according to the manufacturer's protocol.
  - For Live Cells (Hoechst 33258 only): Apply the live-cell compatible Hoechst stain directly to the culture media and incubate for the recommended time.
- Wash and Mount: Gently wash the cells to remove excess dye and mount the coverslip onto a microscope slide with mounting medium.
- Observe: Use a fluorescence microscope with the appropriate filter set (for blue fluorescence).
- Interpret Results:
  - Negative: You will see only the large, bright, and clearly defined nuclei of your cells. The surrounding cytoplasm and background will be dark.
  - Positive: In addition to the cell nuclei, you will see small, distinct fluorescent dots or filaments scattered in the cytoplasm or in the spaces between cells. This is often described as a "starry sky" pattern and represents the Mycoplasma DNA.

Pros:
- Relatively fast (results in one day).
- Inexpensive and uses standard lab equipment (fluorescence microscope).
- Can visually confirm the presence of an organism.
Cons:
- Less Sensitive: Requires a high level of contamination (e.g., >10⁵ cfu/mL) to be clearly visible.
- Subjective: Interpretation requires a trained eye and can be ambiguous.

Method 3: Microbiological Culture (The Gold Standard)

This direct culture method is the traditional "gold standard" and is still required by many regulatory bodies (e.g., for vaccine production).

How it Works: A cell-free sample of the culture supernatant is inoculated into a specialized, nutrient-rich broth. It is then subcultured onto agar plates and incubated for up to 28 days.
- Inoculate Broth: Collect a cell-free sample of your culture supernatant. Aseptically inoculate this sample into a specialized, highly nutritious Mycoplasma broth.
- Incubate (Broth): Incubate the broth under the recommended conditions (e.g., specific temperature and CO2) for several days to allow the Mycoplasma to grow.
- Subculture to Agar: After the initial broth incubation, take a small volume from the broth and plate it onto specialized Mycoplasma agar plates.
- Incubate (Agar): Incubate the agar plates for up to 28 days, checking them periodically under a microscope.
- Interpret Results: A positive result is confirmed by the visual identification of classic "fried egg" shaped Mycoplasma colonies on the agar plate.

Pros:
- Detects Viable Organisms: This is the only method that definitively proves an active, living infection.
- Highest Sensitivity: Can detect as few as 1-10 colony-forming units (CFUs) per mL.
- Allows for species identification.
Cons:
- Extremely Slow: Results take 3-4 weeks, making it impractical for routine lab QC.
- Difficult: Requires specialized media and expertise. Some fastidious (picky) Mycoplasma strains may not grow in culture.

A Standard Operating Protocol for Routine Testing

An effective Mycoplasma control plan relies on a consistent testing schedule and proper sample collection.

When to Test? A Routine Schedule

On Arrival: Test all new cell lines immediately upon arrival. Keep the new line in quarantine, away from your main stocks, until it is confirmed to be negative.
Before Cryopreservation: Always test a culture before freezing it down to create master and working cell banks.
Routinely: Test all active, in-use cultures every 1-3 months.
If Suspicious: Test immediately if you observe any unexplained changes in cell morphology, growth rate, or experimental results.

Sample Collection & Preparation (for PCR)

Culture Preparation: For highest sensitivity, grow cells for 2-3 passages without antibiotics (if possible), as some antibiotics can suppress—but not eliminate—Mycoplasma, leading to false negatives.
Reach Confluency: Grow cells to 80-90% confluency. This is when Mycoplasma titers are typically at their highest. Do not use cultures that are 100% confluent or have been sitting for several days, as PCR inhibitors may accumulate.
Collect Sample: Aseptically collect 1-2 mL of the cell culture supernatant (the liquid medium) and transfer it to a sterile microcentrifuge tube. This cell-free supernatant is the primary sample.
Heat Inactivation (Optional but Recommended): Many protocols recommend heating the supernatant sample at 95°C for 5-10 minutes. This lyses any cells, inactivates DNases, and releases Mycoplasma DNA.
Run Controls: Every test run must include:
- Negative Control: A sample of sterile, fresh culture medium.
- Positive Control: A sample containing a known, non-infectious Mycoplasma DNA standard (included in most commercial kits).

Handling a Positive Result: Elimination Protocol

Receiving a positive Mycoplasma test result can be alarming, but a clear action plan is essential.

The Best Option: Discard The safest, cheapest, and most recommended solution is to discard the contaminated culture immediately. Also, discard any media or reagents (e.g., aliquots of FBS, Trypsin) that were used exclusively with that culture. Autoclave all associated flasks and waste.
Decontaminate the Lab Thoroughly clean and decontaminate the biosafety cabinet, incubators, water baths, and any pipettors used with the infected culture. Mycoplasma can persist on dry surfaces.
Treat (Only if Irreplaceable) If the cell line is invaluable and cannot be replaced, you can attempt elimination. This is a last resort.
- Quarantine the culture in a dedicated incubator.
- Treat the culture with a specialized anti-Mycoplasma antibiotic (e.g., Plasmocin, BM-Cyclin, or Mycoplasma Removal Agent) for 2-3 weeks, following the manufacturer's protocol. These are often quinolone or tetracycline derivatives.
- After treatment, culture the cells for another 2-3 weeks without any antibiotics.
- Re-Test: You must re-test the culture (preferably using both PCR and the gold-standard culture method) to confirm that the infection has been truly eliminated and is not just suppressed.

Conclusion: Protect Your Science

Mycoplasma contamination is a preventable disaster. By integrating a routine testing schedule, practicing strict aseptic technique, and quarantining new cell lines, you can protect the integrity of your lab. A culture that is not tested is a culture that cannot be trusted. Make this protocol a non-negotiable part of your lab's quality control to ensure your results are valid, reproducible, and reliable.

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Frequently Asked Questions (FAQ) About Mycoplasma Testing

What is the best way to test for Mycoplasma?

There is no single "best" method; the best approach depends on your needs.

For Routine Lab QC: PCR-based testing is often considered the best choice. It offers an excellent balance of speed (results in hours), high sensitivity, and reliability.
For Regulatory Compliance (cGMP): The microbiological culture method is the required "gold standard" and is the best (and only) method to definitively prove a viable infection.
Best Practice: The most robust quality control strategy often involves using two different methods, such as rapid PCR for routine screening and the culture method for final validation (e.g., of cell banks).

What is the protocol for Mycoplasma culture?

The microbiological culture protocol involves growing the organism on specialized media. The steps are:

Inoculate Broth: A cell-free sample (supernatant) from your cell culture is added to a specialized, nutrient-rich Mycoplasma broth.
Incubate Broth: The broth is incubated for several days to enrich the sample and allow any Mycoplasma to multiply.
Plate on Agar: A small amount of the incubated broth is then transferred (subcultured) onto a specialized Mycoplasma agar plate.
Incubate Agar: The agar plate is incubated for up to 28 days and checked periodically under a microscope.
Identify: A positive result is confirmed by finding the characteristic "fried egg" shaped colonies on the agar.

What is the gold standard for Mycoplasma testing?

The microbiological culture method (Method 3 in this article) is the gold standard. It is considered this because it is the only test that can definitively prove the presence of viable, living Mycoplasma organisms. While other tests like PCR are very sensitive to DNA, they cannot distinguish between DNA from a live organism and leftover DNA from a dead one.

Does Mycoplasma show on culture?

Yes, but with two very important distinctions:

In Your Cell Culture Flask: No. This is why it's a "silent" contaminant. Mycoplasma does not cause visible cloudiness (turbidity) or pH changes in your standard cell culture medium, unlike typical bacterial or fungal contamination.
On a Specialized Lab Culture: Yes. Mycoplasma will grow when intentionally cultured on the specialized broth and agar media described in the "gold standard" protocol. On this agar, they form unique, microscopic "fried egg" colonies that confirm their presence.