Mycoplasma Detection and Elimination in Cell Culture

• The Invisible Threat to Cell Lines and Cultures
• Common Sources of Mycoplasma and How to Control Them
• Mycoplasma Detection Methods
• Mycoplasma Elimination Solutions
• Materials

THE INVISIBLE Contamination THREAT TO CELL LINES AND CULTURES

The maintenance of contamination-free cultures is fundamental to cell-based research. Among the most pervasive contaminations in eukaryotic and stem cell cultures is mycoplasma. Members of this genus of bacteria are typically sub-micron in size and lacking a cell wall. This makes it difficult or impossible to detect by microscopy and resistant to many antibiotic treatments.

Although mycoplasma contamination does not usually cause cell death, it can cause a variety of effects on cultured cells. These include an altered metabolism, slowed growth rates, and chromosomal aberrations. Such changes may not be immediately noticeable but can ultimately affect the reliability of the culture data. Contaminated cell lines can be so compromised that they behave as a completely different line.

In short, mycoplasma contamination compromises the validity of affected cell lines for providing meaningful data for life science research.

COMMON SOURCES of Mycoplasma IN THE LAB AND HOW TO CONTROL THEM

Sources of mycoplasma contamination in the laboratory are very challenging to control with certainty. As several species are found on human skin, they can be introduced to cultures through poor aseptic technique.

These bacteria may also be introduced via contaminated supplements, such as fetal bovine serum, or by transmission from other contaminated cell cultures. This cross contamination is a very common way to introduce mycoplasma contamination in the lab. Because mycoplasma strains are small (0.2-0.8 µm), they often escape standard sterile media filtration methods, and can also quickly spread to contaminate other areas of the lab through aerosols and particulates generated during culture handling.

Strict adherence to good laboratory practices such as conscientious aseptic technique is key. Routine screening of cultures is also highly recommended for the successful control of contamination. For more information about other sources of contamination and ways to prevent contamination, read our article on cell contamination troubleshooting.

Decontamination sprays and wipes designed for laboratory equipment and surfaces can provide additional protection against contamination, while enhancing the accuracy of PCR screening results.

MYCOPLASMA DETECTION METHODS

The products below represent a complete toolkit for mycoplasma detection. Researchers may wish to use a second assay to confirm a positive mycoplasma contamination result. Multitest kits can also ensure that testing is conducted at a sufficient frequency to protect vital cultures. Three effective detection methods are:

• Mycoplasma culture: bacterial culture media optimized for mycoplasma are inoculated with culture test samples and grown on a mycoplasma agar plate. This is the most direct test for mycoplasma contamination, but it is also takes the longest time. Cultures should be allowed to incubate for at least two weeks before evaluating, and results from mycoplasma culture can take up to a month to obtain.
• DNA staining method: This simple method relies on staining of bacteria nuclei with Hoechst or DAPI, which is visible by fluorescence microscopy. While rapid, this method is not definitive for contamination with mycoplasma.
• PCR (polymerase chain reaction): which will amplify the bacterial DNA if it is present in culture samples. PCR-based mycoplasma detection has become a trusted method for routine cell culture maintenance, and is the method employed by companies that offer mycoplasma detection services, as well as the way cell repositories protect their own stocks. Well-designed PCR primers are important to eliminate false positives and ensure that valuable cultures aren't destroyed if they aren't contaminated. This test can also be performed quickly, making it helpful in a potential outbreak situation.

PCR tests are extremely sensitive and can provide rapid results, allowing researchers to respond quickly to isolate and eliminate mycoplasma contamination when it’s detected. LookOut^® Mycoplasma PCR Detection Kits are exceptionally sensitive, with a detection limit of as little as 2 genomes per μL of sample. Our newest one-step PCR kit is designed to reduce pipetting to a minimum, as the PCR mix includes all reagents required for the reaction: primers, nucleotides, polymerase and the internal amplification control, provided in a ready-to-use lyophilized reaction mix. View our method for Optimized PCR-based Detection of Mycoplasma.

DNA contamination can influence the results of a mycoplasma PCR test. Elimination of contaminating DNA from the PCR environment is therefore important to ensure that the PCR tests are accurate. DNA elimination spray (L8917) and wipes (L9060-50) remove nucleic acid contaminants from the PCR environment, leading to more accurate PCR results.

MYCOPLASMA ELIMINATION SolutionS

Once mycoplasma is detected, cell culturists can either eliminate the contamination from the cultures or start over. Often, scientists will want to eliminate the contamination because the cell lines are important to the project or expensive.

Our mycoplasma elimination kits are a convenient, efficient choice for ridding cell cultures of contamination.

MATERIALS FOR DETECTION, PREVENTION, AND ELIMINATION OF MYCOPLASMA CONTAMINATION