GUIDE

Mycoplasma are small free-living organisms, considered to be among the simplest of bacteria. Due do their lack of a cell wall, they can be resistant to many common antibiotics. Mycoplasma contamination can come from infected cells from another lab, contaminated cell medium reagents, and/or laboratory personnel infected with strains like M. orale or M. fermentans.

These organisms may spread, making experiments with contaminated cells potentially useless by changing how the cells react, which can lead to an extensive waste of time and resources. Regular testing of cell lines for mycoplasma contamination is recommended to protect your cells and research when receiving lines from another laboratory, after 10 passages, after preparation for a cell bank, and/or when in doubt.

• Good aseptic techniques
• Cleaning surfaces regularly
• Proper PPE (lab coat, protective eyewear, long pants, and closed toe shoes)
• Routine testing for potential contamination
• Keeping media and ideally incubators separate for each cell line

• PCR-based testing
• Staining
• Agar and broth culture

The PCR method (Polymerase Chain Reaction) is a lab technique used to make multiple copies of DNA, which is highly sensitive at identifying many species of bacteria and provides rapid results.

ABS Bio’s sensitive and specific PCR-based method tests your cells or media samples for multiple strains of mycoplasma. We use an Agilent 2100 Bioanalyzer for the analysis of amplified PCR fragments from any mycoplasma presents, resulting in precise, fast, and cost-effective mycoplasma testing.

First, prepare the samples using standard laboratory procedures. If necessary, you may need to also prepare a diluted positive/negative control.

Dependent on the machine or kit you are using, each will come with its own set of instructions. Some companies choose to forego this tedious process and outsource it to another lab, such as ABS, which takes care of all of this and provides the information by the assigned timeline given.

• Destroy contaminated cells and start over
• Treat with trypsin, S-bromouracil, antisera, etc. (may help)
• Antibiotics can help, but you’ll need to know which myco strains you are fighting
  • Using a mix of various antibiotics may offer best results

Cleaning up a cell line contaminated with mycoplasma is difficult and can be a waste of resources and not generally recommended. However, if the cell line is unique or hard to acquire, it would be best to treat it, as you may not be able to acquire that particular group or cells/cell line in time for your project.

Plasmocin®, a broad-spectrum mycoplasma and related cell wall-less bacteria removal reagent, is effective against common Mycoplasma strains, both extracellular and intracellular. Different procedures will need to be followed for adherent and suspension cultures.

If you choose to clean the mycoplasma from a cell line, removing it away from other cell lines (ideally in an environment such as a dedicated Quarantine laboratory space) will prevent the infection of other cell cultures that might cause a potentially catastrophic chain reaction.

After a Mycoplasma test comes back negative with normal media, the cells are deemed Mycoplasma negative and the job for Mycoplasma Cleanup can be continued if there are vials to freeze. Testing should be repeated over a several week period because some

Mycoplasma strains grow slowly and may not be immediately detectable.      In all cases, mycoplasma test results should be recorded for recordkeeping purposes.

Generally, this procedure is lengthy and can waste precious research time for any scientist or laboratory. Outsourcing this project to ABS could save you the time, money, and resources. Our laboratory personnel make it a priority to get your cells where they need to be, so you can focus on more important tasks.

Request a consultation at ABS to talk to one of our highly skilled and knowledgeable sales team who will speak with you about your projects and needs. Together, we will come up with a personalized plan of action to help you reach your goals with us.