Although mycoplasma is the smallest known prokaryotic organism with a tiny size range of 0.15 to 0.35 µm, it can quickly become a huge problem in cell culture labs!
Mycoplasma was first identified by researchers working with HeLA cell cultures in 1956. Even though more than 60 years have passed since then, it is estimated that a staggering 15-35 % of the cultures in research labs around the world today are contaminated by mycoplasma (1). This is partially because mycoplasma can easily go undetected for several reasons; it doesn’t cause the turbidity or cloudiness that is usually seen with bacterial or yeast contamination, and it doesn’t alter the colour or smell of the culture medium. Furthermore, it doesn’t produce many metabolic byproducts and it is not visible under standard compound microscopes.
More than 120 species of mycoplasma have been described to date. However, only a handful of these cause 95 % of the infections seen in lab cultures: Mycoplasma arginini, M. fermentans, M. hominis, M. hominis, M. pirum, M.hyorhinis, M. orale and Acholeplasma laidlawii.
What Does Mycoplasma Do to Your Cells?
Mycoplasma can interfere with cell cultures in many ways, ultimately impacting experimental data:
- It decreases cell growth by competing for nutrients and biosynthetic precursors, thus interrupting nucleic acid, amino acid, and ATP synthesis (1,2)
- It may cause mutations and introduce chromosomal modifications (1,3)
- Its presence can impact gene expression, including genes for receptors, growth factors and cell signalling pathways, in particular affecting immune cells through perturbation of the NF-kB pathway (4,5)
Beyond direct effects on cell culture and data quality, mycoplasma contamination may also impact a lab economically over time because of effects on transfection efficiency, recombinant virus production, and other cell culture-based workflows that rely on healthy cells.
Sources of Mycoplasma Contamination
Here are three main sources of mycoplasma contamination and tips to help you minimise the risk!
- The biggest source of mycoplasma is human hands. Therefore, you should always consider laboratory personnel as a serious contamination risk and take appropriate precautions:
- Always use protective equipment such as gloves and lab coats
- Follow sterile working techniques
- Cross-contamination from infected to healthy cultures via droplets is another important source of mycoplasma contamination. You can minimise this risk by:
- Working with one cell line at a time
- Keeping open lids and cultures to a minimum
- Not using a laminar flow cabinet to store reagents or other materials.
- A spike in the presence of bovine mycoplasma species in lab cultures in the 60s and 70s eventually revealed culture reagents, growth media and bovine serum as major sources of mycoplasma contamination. Nowadays, serum is generally controlled for mycoplasma in order to prevent culture contamination but bear in mind that mycoplasma-free serum is not a guarantee. So, it’s good practice to monitor for mycoplasma periodically using some of the detection methods described below.
Detecting the Silent Enemy
- Direct detection – culture
Here, mycoplasma is cultivated on agar plates or in selective broth. Although this is a very sensitive test and is considered to be the gold standard, it is time-consuming. It takes approximately 2-4 weeks to obtain results and these should be interpreted by specially trained personnel.
- Indirect detection – DNA staining
This method uses Hoechst DNA staining and imaging with fluorescence microscopy. It is very easy to perform and it is cost-effective. However, since Hoechst stains all nucleic acids, it can be hard to differentiate bacterial DNA from host DNA.
- Biochemical detection
Biochemical detection depends on determination of metabolic or enzymatic activities that occur in mycoplasma but not in mammalian cells. For example, one popular approach is to detect the mycoplasma-mediated conversion of ADP into ATP as mycoplasma interacts with a substrate in a luciferase detection assay.
This is an easy and fast method that works by detecting the presence of mycoplasma-specific 16s rRNA sequences. It is highly specific and allows species-level determination, but it does require a PCR optimisation step. Nordic BioSite has a range of PCR detection kits for mycoplasma, e.g., Mycoplasma TaqMan PCR Kit and TransDetect PCR Mycoplasma Detection Kit.
Better Safe Than Sorry
Even though mycoplasma can go unnoticed, its presence can be detrimental to cells, and nowadays some scientific journals request evidence for mycoplasma-free cultures as a requisite for accepting peer-reviewed manuscripts. It is therefore worthwhile implementing a mycoplasma detection protocol for regular quality control testing. In the case of a positive result, it’s best to simply discard all contaminated cultures as well as other cultures incubated in the same area (if they are easily replaceable) and sterilise the working area.
Because mycoplasma lacks cell walls, they are not susceptible to the commonly used antibiotics in cell culture, e.g., penicillin and other cell-wall targeting antibiotics. In cases where the contaminated cell lines are not easily replaceable, one can try to use specialised antibiotics, e.g., TransSafeTM Mycoplasma Elimination Reagent, which is a combination of a macrolide antibiotic and a tetracycline derivative.
Need Help With a Mycoplasma Problem?
If you suspect mycoplasma in your cultures or if you would like help with implementing a mycoplasma detection protocol, please don’t hesitate to get in touch with us at firstname.lastname@example.org.
- Drexler, H. G., & Uphoff, C. C. (2002). Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39(2), 75–90.
- Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell journal, 13(4), 203–212.
- Ji Y, Karbaschi M, Cooke M. S. (2019). Mycoplasma infection of cultured cells induces oxidative stress and attenuates cellular base excision repair activity. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 845(403054).
- Hoff, F. W., et al. (2018). Mycoplasma contamination of leukemic cell lines alters protein expression determined by reverse phase protein arrays. Cytotechnology, 70(6), 1529–1535.
- Logunov D.Y., et al. (2008). Mycoplasma infection suppresses p53, activates NF-kappaB and cooperates with oncogenic Ras in rodent fibroblast transformation. Oncogene. 27(33): 4521–4531.