How to Eliminate Mycoplasma? Prevention and Solutions for Mycoplasma Contamination in Cell Culture

News 28 3 月, 2025

Introduction

During cell culture, unwanted contaminants such as fungi and mold can appear unexpectedly. However, the most troublesome intruder is mycoplasma, which silently infiltrates cell cultures and causes severe damage. This article provides a comprehensive guide on what mycoplasma is, its sources, how it harms cells, and the best ways to detect and eliminate it.

What is Mycoplasma?

Mycoplasma is the smallest known prokaryotic microorganism, possessing characteristics that set it apart from both bacteria and viruses:

1. Size & Structure: It resembles bacteria but is much smaller, making it an intermediary between bacteria and viruses.
2. Simple Composition: It consists of only a cell membrane, cytoplasm, ribosomes, DNA, soluble RNA, and some internal cellular components, with ribosomes being its only organelle.
3. Lack of a Cell Wall: Mycoplasma exhibits diverse shapes, including filamentous, spherical, and flattened forms.
4. Extremely Small Size: Some species measure as little as 0.1μm, making them small enough to pass through 0.22μm filters, meaning filtration alone cannot completely remove them.

How Does Mycoplasma Contaminate Cell Cultures?

Mycoplasma contamination is invisible under a standard microscope, making it difficult to detect. However, its impact on cell health is significant, leading to:

• Altered Cell Growth: Reduced proliferation and metabolic disturbances.
• Experimental Inconsistencies: Mycoplasma can interfere with gene expression and cellular signaling pathways, leading to inaccurate results.
• Cell Death: Long-term contamination can result in apoptosis or necrosis.

Where Does Mycoplasma Come From?

There are over 20 known species of mycoplasma that contaminate cell cultures. The five most common ones (accounting for over 95% of cases) are:

• Mycoplasma orale
• Mycoplasma arginini
• Mycoplasma fermentans
• Acholeplasma laidlawii
• Mycoplasma hyorhinis

Main Sources of Mycoplasma Contamination

1. Environmental Contamination: Mycoplasma can be present in laboratory air, incubators, and biosafety cabinets.
2. Contaminated Laboratory Equipment: Pipettes, culture dishes, water baths, and other frequently used tools can harbor mycoplasma.
3. Contaminated Cell Culture Reagents: Fetal bovine serum, trypsin, media, and cryopreservation solutions can introduce mycoplasma if not properly tested.
4. Infected Primary Cells: Cells obtained from unreliable sources may already be contaminated.
5. Human Operators: Mycoplasma species such as M. orale are commonly found in the human mouth and can easily contaminate cultures through speaking, coughing, or improper aseptic techniques.

How to Prevent Mycoplasma Contamination?

Since mycoplasma contamination is difficult to detect and treat, prevention is the best strategy. Here’s how you can safeguard your cell cultures:

1. Maintain a Strict Cleaning Routine

• Regularly disinfect incubators, biosafety cabinets, pipettes, and all laboratory surfaces using 75% ethanol, UV sterilization, and autoclaving where applicable.
• Always wear sterile lab coats, gloves, and masks when handling cell cultures.

2. Use High-Quality Reagents

• Only source culture media, fetal bovine serum, and trypsin from trusted suppliers.
• Filter all reagents through a 0.1μm membrane to reduce contamination risk.

3. Follow Aseptic Techniques

• Implement standard operating procedures (SOPs) for handling cells to minimize contamination risks.
• Properly dispose of contaminated materials to prevent cross-contamination.

4. Add Mycoplasma Prevention Agents

• DNA synthesis inhibitors: Fluoroquinolone antibiotics
• Protein synthesis inhibitors: Macrolides, tetracyclines, or diterpenes
• Non-antibiotic treatments: Membrane-active peptides that specifically target mycoplasma without harming cells

How to Detect Mycoplasma in Cell Cultures?

Early detection is crucial for preventing widespread contamination. The following six methods can help identify mycoplasma in your cultures:

1. Culture-Based Detection (Gold Standard)

• Mycoplasma is grown on specialized liquid or solid media, where its presence is indicated by color changes or visible colonies.
• Pros: Highly accurate, detects most mycoplasma species.
• Cons: Time-consuming, requires several days to weeks to obtain results.

2. Fluorescence Staining (DAPI or Hoechst Stains)

• Stains mycoplasma DNA, allowing visualization under a fluorescence microscope.
• Pros: Provides clear visual confirmation.
• Cons: Dead cells may also fluoresce, leading to false positives in mild contamination cases.

3. Chemiluminescence (ATP Assay)

• Detects ATP-dependent luminescence from active mycoplasma enzymes.
• Pros: Rapid detection within 15 minutes.
• Cons: Only detects live mycoplasma, meaning dead contaminants may go unnoticed.

4. Nested PCR (Polymerase Chain Reaction)

• Uses two sets of primers to amplify mycoplasma DNA, improving specificity and sensitivity.
• Pros: High accuracy, able to detect low levels of contamination.
• Cons: Labor-intensive, requiring multiple PCR rounds.

5. Probe-Based qPCR

• Uses fluorescent probes for real-time quantification of mycoplasma DNA.
• Pros: Highly sensitive, fast results, and suitable for high-throughput screening.
• Cons: Requires a qPCR machine and strict laboratory conditions.

6. Isothermal Amplification (Colorimetric LAMP)

• A simple color-change test that amplifies mycoplasma DNA under constant temperature conditions.
• Pros: No need for complex equipment, easy visual interpretation.
• Cons: Requires multiple primers, making primer design complex.

How to Eliminate Mycoplasma from Contaminated Cells?

If your cell culture is contaminated with mycoplasma, follow these steps to eliminate it effectively:

1. Change Culture Medium

• Frequent media changes can reduce mycoplasma levels but won’t fully eliminate them.

2. Use Mycoplasma-Targeting Antibiotics

• Macrolides, tetracyclines, and quinolones can effectively clear mycoplasma.
• Warning: Overuse can lead to antibiotic resistance, requiring periodic antibiotic rotation or combination treatments.

3. Apply Non-Antibiotic Mycoplasma Removal Agents

• These agents disrupt mycoplasma membranes without harming cells.
• Pros: No antibiotic resistance, highly effective.
• Cons: More expensive than antibiotics.

4. Deep Clean the Laboratory

• If contamination occurs, dispose of affected cells, and thoroughly disinfect all equipment and surfaces.

Conclusion

Mycoplasma contamination is a persistent threat to cell cultures. While various detection and elimination methods exist, prevention is always the best approach. By maintaining strict aseptic techniques, using high-quality reagents, and regularly monitoring cultures for contamination, you can protect your cells and ensure reliable experimental results.