Mycoplasma in Cell Culture: What You Need to Know

Mycoplasmas are parasitic bacteria that can infect cell cultures and cause a variety of issues such as slowed cell growth, altered gene expression, and even cell death. Regular mycoplasma testing is essential for preserving the quality of cell cultures and achieving precise results. In this article, we will discuss methods of mycoplasma detection, preventative measures, and steps for removing Mycoplasma from a cell culture.

What Mycoplasma species are contaminating cell cultures?

There are more than 190 known species of mycoplasma. In cell culture only 20 distinct species of have been discovered. These 20 species are responsible for the large majority of mycoplasma contamination in cell culture, with eight of them being the most prevalent species and causing up to 95% of all cell culture contaminations. M. arginini (bovine), M. fermentans (human), M. hominis (human), M. hyorhinis (porcine), M. orale (human), M. pirum (human), M. salivarium (human), and Acholeplasma laidlawii (bovine) account for approximately 95% of all mycoplasma contamination cases in cell culture.

Methods of Mycoplasma Detection

Mycoplasma contamination in cell culture can be detected by a number of methods, each of which has its own advantages and disadvantages. Here we present the most common methods for Mycoplasma detection:

• PCR-based techniques are extremely sensitive able to detect even small amounts of mycoplasma DNA in a sample.
• Culture-based methods involve cultivating the suspected mycoplasma-contaminated cells in a particular culture medium and identifying contamination based on distinctive growth and morphology.
• ELISA and other antibody-based techniques use specific antibodies to detect mycoplasma antigens in a sample.

It is recommended to use multiple methods to improve detection accuracy as available kits and methods can vary in their sensitivity. Each method has his pitfalls with PCR-based methods being highly sensitive but unable to distinguish between live or dead Mycoplasmas, culture-based methods being useful for identifying the specific mycoplasma species but being very time-consuming, and antibody-based methods being good for identifying low amounts of Mycoplasma contamination but being not as sensitive as PCR-based methods.

Prevention of Mycoplasma Contamination

Preventing Mycoplasma contamination in cell culture is essential for ensuring the quality and integrity of cell cultures and the reliability of scientific findings. Recommended measures for preventing mycoplasma contamination are:

• Work sterile and employ antiseptic techniques when working with cell cultures
• Use mycoplasma-free cell lines and reagents
• Test regularly for mycoplasma and combine multiple detection methods
• Decontaminate the laboratory and its equipment on a regular basis
• Avoid antibiotics in cell culture because they can mask mycoplasma contamination
• Reduce the use of extensively passaged cell cultures, as they are more likely to be contaminated
• Develop a procedure for quarantining new and untested cell lines

Note: Be aware of risk factors for Mycoplasma contamination such as high humidity, low air flow, and highly dense cultures, and take measures to reduce these risks in the laboratory.

Insect cultures and Mycoplasma: Mycoplasma growth is temperature dependent, and they grow quickest when the incubation temperature is increased from 28°C to between 35 and 37°C. Keep this in mind when working with insect cell culture, as researchers may wish to alter the incubation temperature to inhibit mycoplasma growth.

Decontamination and Recovery of Contaminated Cell Cultures

How is Mycoplasma removed from cells?

Generally, autoclaving is regarded as the most efficient method for eliminating mycoplasma contamination in cell culture but it also kills the cell cultures.

When a clean culture cannot be obtained, alternative methods for removing mycoplasma and recovering as many viable cells as possible must be used.

The use of antibiotics designed to specifically target mycoplasmas is one method for decontaminating mycoplasma-contaminated cell cultures. Unfortunately Mycoplasmas are immune to common cell-culture antibiotics like penicillins and streptomycins, necessitating the use of anti-mycoplasma antibiotics.

The following three classes of antibiotics are effective against mycoplasmas at relatively low concentrations:

1. Tetracyclines
2. Macrolides
3. Quinolones

The tetracyclines and macrolides inhibit protein synthesis by interfering with the ribosome translation process. Alternatively, quinolones inhibit the DNA replication of mycoplasma, effectively halting its growth. The choice of antibiotic depends on the species of mycoplasma contaminating the cell culture and its susceptibility to the antibiotic in question. Additionally, it is essential to consider the potential effect of the antibiotic on the cultured cells.

Once mycoplasma contamination has been eradicated, the cells can be passed and retested for mycoplasmas after a few days in culture to confirm that decontamination was successful. However, keep in mind that recovered cell cultures may not be identical to those prior to contamination.

Can mycoplasma pass through less than 1 micron filter size?

Mycoplasmas are small enough (typically around 0.2 micrometers in size) to easily pass through filters with pores smaller than 1 micron. Traditional sterilization techniques such as filtration of culture media may not be effective at removing mycoplasmas.

Final words

Mycoplasma contamination in cell culture is a common issue that can result in cell death or inaccurate research findings. Preventative measures against mycoplasma contamination, and regular mycoplasma testing, can aid in preventing or detecting a contamination early and reduce the possibility of inaccurate results. If contamination is confirmed, researchers can try to decontaminate the affected cell cultures and recover as many viable cells as possible.

However, it is essential to keep in mind that recovered cells may not be identical to those that existed prior to contamination, and to keep this in mind when interpreting results. If possible, obtain clean cultures and work with these.