How to Detect Mycoplasma Contamination in Cell Culture

The presence of mycoplasma represents one of the most common and impactful compromises to cell culture integrity. Since these contaminants do not create visible cloudiness, specialized methods are required for accurate identification. At ExCell Bio, we support a multi-faceted approach to this problem, where the correct method of mycoplasma detection protects research validity and production continuity. This article outlines the primary techniques employed for effective mycoplasma detection in a laboratory setting.

Molecular Methods: PCR-Based Assays

Nucleic acid amplification techniques, particularly Polymerase Chain Reaction (PCR), are now a standard for mycoplasma detection. This method targets specific, conserved DNA sequences unique to mycoplasmas. The primary advantage of a PCR-based mycoplasma test is its high sensitivity and rapid turnaround, often providing results within a few hours. It can detect very low levels of contamination and is effective for a wide range of mycoplasma species. However, it requires careful design to avoid cross-reactivity with cellular DNA and does not distinguish between viable and non-viable organisms, a factor considered during data interpretation.

Traditional Microbiological Culture

The cultural method, while slower, remains a definitive and highly sensitive approach for a mycoplasma test. This technique involves inoculating a sample into specialized broth and agar media that support mycoplasma growth. A positive result is confirmed by a characteristic "fried egg" colony morphology on solid agar after an incubation period of up to 28 days. Its strength lies in its ability to detect a broad spectrum of species and confirm the presence of viable, dividing organisms. Many pharmacopoeial guidelines still require or recommend this method for final product release testing due to its proven reliability.

Direct Staining and Indirect Indicators

Alternative methods include direct DNA staining with fluorescent dyes like Hoechst 33258, which binds to all DNA. When viewed under a fluorescence microscope, mycoplasmas appear as extranuclear speckles on the surface of infected cells. This method is useful as a quick screen but requires experience to interpret accurately and is less sensitive than PCR or culture. Indirect methods, such as monitoring for shifts in cellular metabolism or growth rates, can signal a potential problem but are not specific. These indicators should prompt a formal mycoplasma detection investigation using one of the primary, definitive methods listed above.

Reliable mycoplasma detection relies on selecting an appropriate, validated method aligned with the stage of work and regulatory needs. A robust quality program often incorporates more than one technique; for instance, using PCR for frequent, rapid screening and cultural methods for final verification. Establishing a scheduled mycoplasma test protocol is a critical defense, providing the necessary assurance that cell cultures are free from this covert source of experimental variability and product risk.