Mycoplasma Contamination - Detection and Elimination

Mycoplasma Contamination - Detection and Elimination_Mycoplasma Bacteria_Parker Bioscience FiltrationWhen compared to bacterial contamination, Mycoplasma contamination isn't immediately obvious.

The outcome of any contamination event is the same — the disposal of the contaminated media and decontamination of equipment used in the process — but the methods of detection and the timelines differ enormously. 

You would know if a culture was contaminated with bacteria within 24 hours: the contamination would be obvious — as would the need to immediately remove and dispose of the culture. 


Mycoplasma's secret weapon

Mycoplasma contamination, however, presents a different scenario. A culture could have a Mycoplasma infection at as much as 10,000 Mycoplasma per mammalian cell, with no detectable effect.

Mycoplasma stays under the radar and co-exists with a cell line, with no immediately visible side effects. There is no change in the media appearance, or in the pH or DO demand. What will happen is that the Mycoplasma will compete for nutrients at a low level, resulting in potentially slower cell growth and lower yields. 

As well as depleting the nutrient supply, the Mycoplasma's metabolic activity will result in the release of toxic and/or cytolytic metabolites which will have a negative effect on the cells.

The results? Production levels could be lower than expected and R&D decisions could be made based on compromised data, due to an undetected Mycoplasma infection affecting cell growth and productivity. 


Mycoplasma Contamination - Detection and Elimination_Preventing Mycoplasma Contamination White Paper_Parker Bioscience Filtration


If you'd like to learn more about Mycoplasma contamination and how to tackle it read our white paper: Preventing Mycoplasma Contamination





How the wider supply chain is affected

A Mycoplasma contamination event can have serious consequences for patient safety as it can disrupt supply of critical drug products. It will result in unplanned downtime, leading to disruption in the supply chain causing reputational damage and financial consequences. 

From an operational perspective, Mycoplasma contamination will lead to an investigation and remedial works, all followed by the cleaning in place (CIP) / sterilizing in place (SIP) of fixed assets. If a facility is using single-use systems, CIP and SIP won't be needed, however, the entire site will still need to be decontaminated. 


So what options are available for testing?

Direct culture on agar

This is the classic method for detecting a Mycoplasma contamination and is the regulatory test to show clearance for clinical use. 

While this test is effective and sensitive, it is the most difficult and time-consuming method - it takes 28 days for results to be obtained. This method also requires live Mycoplasma cultures to be maintained to act as positive controls. However, bearing in mind how easily Mycoplasma can spread, this may not be something that is advisable to have in the same building as a cell culture lab. As a result, this test tends to be outsourced — which further extends the time taken for a result to be attained. 


PCR is a sensitive and analytical testing method. It is relatively quick and effective in detecting contaminations which can be difficult to spot. However, it can't distinguish between viable and non-viable organisms. In addition, to avoid giving false-positive results, the testing laboratory must implement strict measures to ensure containment to prevent cross-contamination and environmental contamination. 

DNA fluorochrome staining

This is a simple and fast method that stains DNA using a fluorescent dye which can then be viewed under a fluorescence microscope. It requires positive and negative controls and while in normal circumstances, the requirement for positive controls would count against a method, these controls are commercially available. And as the Mycoplasma have already been fixed onto slides, they present no contamination risk to the lab. 

ELISA testing

Enzyme-linked immunosorbent assay (ELISA) is a technique used to detect infectious agents in a sample. This form of testing can yield quick results, however, it may not be able to detect uncommon sources of contamination. 

But which is the best practice?

Best practice (and that required by the FDA) is a combination of the direct culture method (given its high sensitivity) and DNA Fluorochrome testing, which will detect low-level fastidious Mycoplasma contaminations.


So how can Mycoplasma be eliminated?

Once identified, you must eliminate any Mycoplasma infected material, solutions or equipment. An autoclave cycle will have a 100 percent success rate. But if a cell line is unique and you cannot afford to lose it, you have no option but to try to recover the cell line. 

You could turn to antibiotics — but that should be a last resort and only used if a highly valuable or unique cell line can't be thrown away. The process lasts for a number of weeks and antibiotics may also be detrimental to the cells, resulting in the loss of the culture you are trying to preserve.

Once treatment with antibiotics is complete, thorough screening is required to ensure that contamination has been eliminated and not just suppressed.

Tetracyclines, macrolides and fluoroquinolones can be used, but their use should be limited to avoid resistance.

All equipment should be decontaminated either by SIP or autoclave cycles. Disinfectants that target Mycoplasma are also available. If it is not possible to apply these methods after suspected contamination, any equipment needs to be removed from the facility and replaced. 

To avoid cross-contamination events, media and reagents should only be used for one cell line and never stored in the laminar flow hood. Any media and reagents that have been near a contaminated culture should be disposed of immediately.

Implementing a system to isolate cell lines, reagents and equipment — combined with good housekeeping procedures — should help to prevent contamination from spreading. 


Prevention is always better than cure

The key is knowing the source of the cells, using media from reputable sources and following a good aseptic technique.

When looking at a mitigating risk from media and components that are heat labile, a particularly successful method is filtration using a 0.1 micron rate membrane such as Parker Bisocience Filtration's PROPOR MR. 

Incorporating a highly retentive 0.1 micron PES membrane, PROPOR MR filters provide fast and effective removal of Mycoplasma from cell culture media


Mycoplasma Contamination - Detection and Elimination_Preventing Mycoplasma Contamination White Paper_Parker Bioscience FiltrationIf you'd like to learn more about Mycoplasma contamination and how to tackle it read our white paper: Preventing Mycoplasma Contamination


Mycoplasma Contamination Detection and Elimination - Tackling the Source_Guy Matthews Author_Parker Bioscience Filtration

This post was contributed by Guy Matthews, division marketing manager, Parker Bioscience Filtration, United Kingdom.


Parker Bioscience Filtration specializes in automating and controlling single-use bioprocesses. By integrating sensory and automation technology into a process, a manufacturer can control the fluid more effectively, ensuring the quality of the final product. Visit to find out more.



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