mycoplasma.md

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Checking for Mycoplasma Contaminations

Mycoplasma is a bacteria that can infect cell cultures and is one of the most common contaminants in cell culturing.

Characteristics of mycoplasma:

• they are the smallest self-replicationg organism known (0.3-0.8μm in diameter)
• they lack a cell wall
• they are fastidious in their growth requirements

Mycoplasma have the ability to alter their host culture's cell function, growth, metabolism, morphology, attachment, membranes, virus propagation and yield, interferon induction and yield, cause chromosomal aberrations and damage, and cytopathic effect including plaque formation.

These 3 simple characterists, combined with their ability to alter virtually every cellular function and parameter, make mycoplasma the most serious, widespread, and devastating culture contaminants.

It is therefore crucial to regularly check for mycoplasma contamination.

Sources of mycoplasma contamination

The number one source of mycoplasma contamination is other infected cell lines. Cell lines cultured in for example the same hood can easily become infected if there is a contamination in a single one.

The second common source is the cell culturist; good training especially in aseptic techniques combined with the strategic use of a tested cell repository will greatly reduce the opportunities for contamination via this route. Talking and sneezing can generate significant amounts of aerosols that have been shown to contain mycoplasma. Also diry lab coats can be a source of contamination when a dust-laden sleeve is put into a laminar flow hood and dust particles fall into cultures.

Contaminated sera and other filtration products have, in the past, been a major source of contamination, but nowadays they only play a minor role in mycoplasma contaminations.

Mycoplasma detection

There are two bacis testing methods for mycoplasma:

1. Direct culture in media

Direct culture is the most effective and sensitive method for detecting mycoplasma, but it is also the most difficult and time-consuming.

2. Indirect tests that measure specific characteristics of mycoplasma

There are a wide range of indirect test methods available for mycoplasma detection, including PCR-based kits, DNA fluorochome staining, autoradiography, ELISA, immunofluorescence, and specific biochemical assays. These tests are faster than direct culture and are often commercially available as kits.

It is important to know what aspect of contamination the test is designed to detect, how well the test performs, its specificity (i.e., what strains of mycoplasma it detects and any likely causes of false positive or false negative reactions) and for detectable contamination, what level of sensitivity is achievable under the prescribed sampling and test conditions. Selection of test methods should be based on evaluation of the potential specificity and sensitivity of detection and the likelihood of inhibition of a positive result.

Mycoplasma Control Kits

If you are not sure what test methods or kits to use, please do not hesitate to ask your collegues at LCSB.

Here are some examples of kits used by LCSB researchers:

• MycoAlert Mycoplasma Detection Kit Westburg
• MycoAlert Control Set Westburg
• Mycoplasma Detection Kit InvivoGen
• Lookout Mycoplasma PCR Detection KIT Sigma Aldrich
• Universal Mycoplasma Detection kit ATCC

Control Intervalls

The longer the intervalls between tests, the more research has to be put into questions and the more work has to be repeated to be sure about the results.

The LCSB, therefore, recommands to test cell cultures for mycoplasma contaminations at time of arrival and aftewards at monthly intervalls. You do not only prevent your own research results to be invalidated, but help your colleagues' cell cultures to remain free of mycoplasma contaminations.