Real-time qPCR Mycoplasma Contamination Detection Service

Mycoplasmas have become one of the most widespread and severe contaminants within cell culture systems in research institutions. Mycoplasma is considered the smallest free-living organism capable of self-replication. They can be as small as 0.2-0.3µm in diameter, lack a cell wall, and are known to freely pass through some filters used to remove bacteria from cell culture.

Mycoplasma can attain high densities in cell cultures (i.e. 10⁷ - 10⁸mycoplasma/ml) without ANY noticeable changes to the pH of the medium and without causing increased turbidity. Typically there is an absence of visible morphological changes. Mycoplasma contamination cannot be detected using bright-field or phase microscopy, even at high magnification. In other words, you do not know mycoplasma is contaminating the cell culture without testing.

Estimates vary but research studies performed by different groups have shown anywhere from 5-35% of all cell cultures are contaminated with mycoplasma; the real percentage may be even higher because many researchers don't know that their cultures are contaminated. If other types of bacterial contamination are a common occurrence in your lab, you most likely have mycoplasma contamination as well and just may not know it.

Due to the typically high concentrations of mycoplasma in infected cultures, mycoplasma often out-compete the host cells for essential nutrients resulting in altered growth and protein production.

Mycoplasma contamination has been shown to alter almost every cell culture property and characteristic measured including:

• Altered DNA, RNA, and protein synthesis
• Lowered ATP levels
• Altered enzyme expression and activity
• Variable growth rates and viability
• Nucleic acid synthesis which leads to aneuploidy and other chromosomal irregularities
• Cytotoxicity
• Decreased transfection efficiency and
• Culture starvation.

As  with using a misidentified cell line, using cell cultures contaminated  with mycoplasma calls into question the validity of the experiments  using that cell line.

No! Actually, mycoplasma contamination rates are much higher than average for cell lines that are routinely grown in medium containing antibiotics (72% were contaminated) versus no antibiotics in the medium (7% contaminated) (Barile, et al 1973). This is due to the mycoplasma developing partial or complete antibiotic-resistance to antibiotics such as penicillin and streptomycin. In medium containing antibiotics, other microbial contaminants disappear allowing plenty of room for antibiotic resistant mycoplasma to grow and spread through the culture undetected.

Just like human cell line authentication or mouse cell line authentication,  testing for mycoplasma should occur at a regular basis while the cell  culture is actively growing.  It is recommended that you test for  mycoplasma contamination, as applicable:

• When creating a master cell bank OR working cell bank
• Before beginning a new series of experiments
• When the cell line is not behaving as expected
• Every 2 months that the cell culture is actively growing
• Before publication of experimental data
• These  steps are crucial to the success and repeatability of the research and  to the acceptance of the research in the scientific community.