Unfortunately, it is challenging to quickly and reliably detect mycoplasma contamination in cell cultures. One reason is that mycoplasma contamination does not typically trigger the turbidity changes that are common with other types of bacterial or fungal contamination. In addition, the bacteria are too small to observe using optical microscopy without the need for specific labeling.

However, cell cultures can show subtle changes as a result of mycoplasma contamination, meaning careful analysis can provide some insight. For example, as the Mycoplasma will compete with your cells for nutrients in the culture medium, one of the first visible signs of contamination is a reduced rate of cell proliferation. Other indications of contamination include cell aggregation, morphological changes, and poor transfection efficiencies (which can be easier to spot when you are working with cells that used to show high transfection efficiencies in the past).

The only way to detect Mycoplasma species is to explicitly test for them. 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 PCR¹. Recently, a research group even managed to show the presence of Mycoplasma species by light microscopy using oblique illumination, which renders the mycoplasma cells visible as white foci due to light scattering².

The best assays are highly sensitive and specific, but can also be performed quickly. Based on these criteria, we discuss the most commonly used assays below.

Using PCR to Detect Mycoplasma Contamination

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.

Culture-based Methods for Mycoplasma Detection

There are several standard culture methods available for the detection of Mycoplasma. These are:

1) The Broth and Agar Cultivation Procedure

This approach involves growing the contaminant in broth, plating the cells on nutrient agar plates, and then identifying if the contamination is due to Mycoplasma from the colonies formed. This method is highly specific and sensitive.

2) The Indicator Cell Culture Method

In this method, the bacterial contaminant is grown in a ‘test’ cell culture – a culture which is known to support high levels of mycoplasma proliferation. To identify if the contaminant is likely to be Mycoplasma, the bacterial DNA is fluorescently stained³. While highly sensitive, this technique has limited specificity for mycoplasma DNA.

3) The Sensor-cell Approach

It is possible to use specially engineered cells to detect the presence of Mycoplasma in cell cultures. These ‘sensor cells’ detect mycoplasma cells through their toll-like receptor (TLR) 2. In the presence of Mycoplasma species, this receptor is activated and triggers a signaling cascade, with the outcome being an observable color change. This is a highly sensitive method but has low specificity in comparison to other methods.

Manufactured and licensed biological products produced via cell substrates (e.g. viral vaccines, monoclonal antibodies, and similar products) need to be tested by methods (1) and (2) above to ensure the absence of mycoplasma contamination – this is a requirement for regulatory mycoplasma testing. A positive control with live mycoplasma bacteria and/or contaminated cells is also necessary in these assays. When the use of a positive control might compromise the sterility of a laboratory, it may be preferable to outsource the mycoplasma testing.

Biochemical Tests to Detect Mycoplasma Contamination

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 Mycoplasma species in cell cultures.

Using Fluorescence Microscopy to Detect Mycoplasma Contamination

It is also possible to detect mycoplasma DNA by fluorescence microscopy by using a non-specific DNA stain⁴. This stain is added to the culture medium and the presence of mycoplasma DNA can be confirmed by the appearance of bright foci/small clusters amongst the cells, when observed using a fluorescence microscope.

References:

1) Drexler HG, Uphof CC (2002). Cytotechnology 39: 75–902) Boslett et al. (2014). BioMed Research International, article ID 5321053) Young et al. (2010). Nature Protocols 5:5

2) Boslett et al. (2014). BioMed Research International, article ID 532105

3) Young et al. (2010). Nature Protocols 5:5

4) Rottem et al. (2012). Book chapter 3, book: Biomedical Tissue Culture, ISBN 978–953–51–0788–0