Reliable and Easy Methods for Detecting Mycoplasma Infections in Cell Culture

By Juhi Rani

Most laboratories prefer not to use antibiotics in the maintenance of eukaryotic cell lines due to various reasons, including the development of antibiotic-resistant strains. This practice can inadvertently increase the risk of mycoplasma infections, which can negatively impact experimental outcomes and the quality of the research data. Therefore, it is crucial to implement reliable and convenient methods to detect mycoplasma infections in cell cultures.

Why Detecting Mycoplasma Infections Is Important

Mycoplasma infections in cell cultures can lead to severe experimental inaccuracies, altered cellular behavior, change in gene expression patterns, and reduced cellular viability. These impacts make it essential to consistently monitor and prevent mycoplasma contamination in cell cultures. Fortunately, there are several effective and easy-to-implement techniques available to detect mycoplasma infections.

Common Methods of Detecting Mycoplasma Infections in Cell Culture

1. PCR Amplification

The most reliable and widely used method for detecting mycoplasma infections is PCR amplification. This technique involves the use of specific primers designed to target mycoplasma-specific regions of the genome. Here is a step-by-step guide to carrying out PCR amplification:

Elevate the cell culture to a higher RPM to allow the eukaryotic cells to sediment, leaving behind the mycoplasma. Centrifuge the culture at a low RPM to separate the mycoplasma from the eukaryotic cells. Transfer the supernatant to a new tube and add a standard lysis buffer. Perform PCR with the extracted mycoplasma DNA using mycoplasma-specific primers. Analyze the PCR products by gel electrophoresis or any other suitable method to detect the presence of mycoplasma.

While this process may be time-consuming, it provides highly accurate results and can be performed in most laboratories with standard equipment.

2. Staining Techniques

In some cases, staining techniques can be used to detect clumps of mycoplasma in a cell culture. However, this method is limited in its effectiveness, as it relies on the ability to visualize mycoplasma clumps microscopically. It is not a reliable or definitive method for detecting mycoplasma infections, especially in early stages of infection.

Alternatives and Additional Measures

In addition to the above techniques, several other methods and preventive measures can help in detecting and avoiding mycoplasma infections in cell cultures:

Regular Monitoring: Regularly testing your cell cultures using PCR or similar methods to ensure the absence of mycoplasma contamination. Use of Mycoplasma-Free Cell Strains: Whenever possible, obtain cell strains that have been mycoplasma-tested and certified as free of contamination. Adherence to Best Practices: Adhere to protocols that minimize the risk of cross-contamination, such as using dedicated equipment for each cell culture, maintaining strict hygiene, and using disposable pipette tips.

Conclusion

Mycoplasma infections in cell cultures can be a significant issue, but reliable methods such as PCR amplification can help detect and prevent them. By integrating these techniques into your experimental workflow, you can ensure the integrity and consistency of your research outcomes.

Author: Juhi Rani