How to Eliminate Mycoplasma From Cell Cultures?
It is well known that a significant percentage of cell cultures are contaminated with mycoplasma. The extent of this contamination problem has been surveyed many times over the last three decades and found to vary from 10% to 70% in mammalian cultures. It is thus safe to say that the chances of cultures in your lab being contaminated with mycoplasma now or in the future are moderate to high.
What is Mycoplasma Contamination?
Mycoplasma is a genus of bacteria that is characterized by the lack of a rigid cell wall. It is known as the smallest (0.3-0.8 µm), self-replicating organism on this planet. Further, they can shrink to diameters below 0.45 µm due to their flexible cell membrane, allowing them to pass through the typical antibacterial filters. These microscopic organisms are renowned for their ability to evade detection, making them a constant threat to the reliability and validity of experimental results. Mycoplasma contamination can have far-reaching consequences, affecting everything from cellular metabolism to gene expression, and can even lead to the misidentification of cell lines.
Effects of Mycoplasma Contamination on Cell Cultures
Mycoplasma are extremely simple organisms and, due to their parasitic nature, rely on eukaryotic host cells for metabolism needs. In the early stages, Mycoplasma will attach to the host cell and eventually fuse with its membrane. After this point, the Mycoplasma will replicate until they outnumber the host cell by 1000-fold. Mycoplasma contamination can have severe consequences on cell cultures, affecting their growth, morphology, and genetic stability.
- Growth inhibition. Contaminated cultures often exhibit slowed growth rates due to competition for nutrients. Mycoplasma can consume essential nutrients and release metabolic byproducts that inhibit cell proliferation.
- Morphological changes. Mycoplasma can induce morphological alterations in host cells. For example, contaminated cells may exhibit abnormal shapes, detachment from the culture substrate, or cytoplasmic vacuolation.
- Genetic and metabolic disruptions. Mycoplasma can interfere with host cell DNA, RNA, and protein synthesis, leading to genetic instability and altered metabolic profiles. These changes can compromise the reproducibility and reliability of experimental data.
- Impact on biopharmaceutical production. In the context of biopharmaceutical production, mycoplasma contamination can lead to inconsistent product yields, reduced efficacy, and potential safety risks.
What Causes Mycoplasma Contaminations?
Mycoplasma contamination can arise from various sources, necessitating vigilant monitoring and preventive measures.
- Contaminated reagents. Reagents such as serum, media, and trypsin are common sources of mycoplasma contamination. Even commercially available reagents can harbor mycoplasma if not properly screened.
- Cross-contamination. Cross-contamination between cell cultures can occur through shared equipment, personnel, or aerosolized particles. Inadequate aseptic techniques and poor laboratory practices often exacerbate this issue.
- Laboratory environment. Environmental sources, including contaminated air, water, and surfaces, can introduce mycoplasma into cell cultures. Regular monitoring and rigorous cleaning protocols are essential to mitigate this risk.
How to Detect Mycoplasma Contamination?
The lack of a cell wall and how Mycoplasma attach themselves to their host cells makes them invisible to the naked eye, even with microscopy. Also, Mycoplasma do not cause turbidity in the growth medium. Only by observing your cells very carefully, you may notice changes in proliferation, morphology, or transfection efficiency - yet at this point, you would most likely discard your culture. As of today, there are many ways to detect Mycoplasma contamination, and each of them may have its particular advantages and disadvantages.
| Methods | Introduction | Advantages |
| Polymerase Chain Reaction (PCR) | PCR is a highly sensitive and specific method for detecting mycoplasma DNA. It allows for the rapid identification of mycoplasma species, even at low contamination levels. | High sensitivity and specificity; Rapid results; Ability to detect multiple mycoplasma species |
| Enzyme-Linked Immunosorbent Assay (ELISA) | ELISA detects mycoplasma antigens using specific antibodies. This method is useful for screening large numbers of samples quickly. | High throughput; Specific antigen detection; Quantitative results |
| Direct and Indirect Staining | Staining methods, such as Hoechst staining, can visualize mycoplasma under a fluorescence microscope. These methods are often used as confirmatory tests. | Visual confirmation; Simple and cost-effective |
How to Prevent Mycoplasma Contamination?
Although it can be tricky to identify and eliminate a Mycoplasma contamination, there are some simple practices you can follow, to prevent it - most of which are good lab practices anyway!
- Good laboratory practices. Implementing stringent aseptic techniques and maintaining rigorous laboratory protocols are fundamental to preventing mycoplasma contamination.
- Aseptic techniques. Regular and thorough hand washing or use of hand sanitizers before handling cell cultures. Using sterilized pipettes, culture vessels, and other lab tools to prevent introducing contaminants. Maintaining a clean working environment, including regular disinfection of work surfaces and equipment.
- Use of antibiotics. Do not indiscriminately use antibiotics to prevent contamination, as Mycoplasma are resistant to the standard antibiotics (e.g., penicillin, streptomycin). This will merely act like a mask: bacteria that cause turbidity are eliminated, while Mycoplasma survives without detection.
Creative Bioarray Relevant Recommendations
| Cat. No. | Product Name |
| MD-BC | Test™ Mycoplasma Biochemical Detection kit |
| MD-PR | Test™ Mycoplasma PCR Detection kit |
| MD-RT | Test™ Mycoplasma RT-qPCR Detection kit |
| ME-AG | Cure™ Mycoplasma Elimination Solution |
