Mycoplasma contamination of your cell cultures can seriously impact the reliability, reproducibility, and consistency of your experimental results. It presents a major challenge for research, as well as in the manufacturing of bioproducts.

Mycoplasma contamination is difficult to detect and therefore its presence can go undiscovered for months. In fact, it is estimated that over 10% of all cell cultures are contaminated with Mycoplasma species. This shows the importance of routine testing to make sure you are working with uncontaminated cultures. Here, we look at the challenges and best approaches for detecting, eliminating, and preventing mycoplasma contamination, including a wide range of common questions and answers on how to overcome the issue.

Where Does Mycoplasma Contamination Come From?

There are six species that account for 95% of all detected mycoplasma contaminations in continuous cell line cultures: M. orale, M. arginini, M. fermentans, M. hyorhinis, M. hominis, and A. laidlawii1. In ATCC there are about 100 Mollicute species extracted from cell culture. Typical routes of contamination are: cross-contamination from other cells (for example, via aerosols generated during pipetting), using the same media bottles, or handling more than one cell type at a time.

Other sources of contamination within your cell cultures could include direct contamination from the researcher, as well as the use of contaminated materials, such as animal sera. The high risk of contamination highlights the importance of always purchasing high-quality cell culture media  from reputable manufacturers.

How Can You Detect Mycoplasma Contamination in Your Cell Cultures?

There are several different techniques to identify if your cell cultures are contaminated with Mycoplasma. These include histochemical staining, ELISA, DNA fluorochrome staining, microbiological culture, biochemical methods, and PCR. PCR is a powerful technique to amplify specific sequences of DNA in a sample and is a sensitive and rapid method for identifying mycoplasma contamination in your cell cultures. However, there is the risk of spreading the contamination from the positive control (or any of your samples that previously tested positive for Mycoplasma) to unaffected samples during the testing process, thereby leading to false positive results. While there are many PCR detection kits commercially available, many are not able to detect all species of Mycoplasma.

Mycoplasma species produce enzymes that are not found in eukaryotes. This makes it possible to detect them by looking for the presence of these enzymes in cell cultures. One kit designed for this is the MycoAlert^TM PLUS Mycoplasma Detection Kit, which contains the luciferase enzyme as a reporter. This method works as follows: first the mycoplasma cells are lysed, releasing their enzymes. These enzymes react with the substrate from the kit, which catalyzes the conversion of ADP to ATP. Luciferase uses this ATP to create a light signal, which can then be detected using a luminometer. This is a rapid method for identifying the presence of any Mycoplasma or related Mollicute species in cell cultures.

What Options Do You Have When Your Cell Cultures are Contaminated with Mycoplasma?

Discarding your contaminated cell cultures may seem like an obvious response to mycoplasma contamination. However, if you are using a culture that you can no longer obtain, or that is particularly valuable, it could be worth trying to eliminate the contamination. Keep in mind that treatment to get rid of Mycoplasma can take weeks, if not longer.

Elimination of mycoplasma contamination can be carried out by using specially formulated reagents, such as MycoZap^TM Mycoplasma Elimination Reagent. This tool combines antibiotic and antimetabolic agents to eliminate detectable mycoplasma contamination in as little as four days. It has been optimized to clear the contamination with minimal effects to your cell culture.