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Preparation of Conditioned Media for Mycoplasma Detection

Mycoplasma Details

• Tests are scheduled once a week on Wednesdays and samples should be delivered to the TCSR by Tuesday evening.
• Test results can be obtained the same day.
• Mycoplasma tests should be performed on all cells arriving in the LCC from outside laboratories and as a routine check on existing stocks.
• All cell lines used in TCSR shared labs must be demonstrated to be mycoplasma negative.
• The TCSR provides incubators and hoods free of charge for quarantining new cell lines prior to testing.

Sample Preparation

1. Cell lines must be cultured for at least two passages prior to testing.
2. Cell lines must be cultured in the absence of antibiotics and selection agents during this time. (such as Pen-Strep, G418, gentamycin, geneticin, fungizone, puromycin, or zeocin) (Mycoplasma is reduced but not eliminated in the presence of routine tissue culture antibiotics, ie, you will get a false negative!)
3. Media should be collected after it has been in contact with cells for three days.
4. Transfer approximately 10 ml of the media into a centrifuge tube.
5. Bring the sample and a completed request form to TCSR.
6. Please store samples at 4oC until you bring them to the TCSR.
7. Don't forget to label your sample, yours is not the only one we will have!

Background on Mycoplasma
or
Why You Need To Test Your Cells!

Mycoplasma testing has been provided by the Shared Resource as a service to LCC investigators since April 1990. When present in a culture mycoplasma can induce chromosomal abnormalities and alter the antigenicity of cell membranes. They can affect cellular metabolism by competing for nutrients and influence cell fusion procedures. Therefore, since so much of the research of the LCC is based upon tissue culture, the detection of mycoplasma infection is considered a quality control measure of the utmost importance. The assay performed is based upon nucleic acid hybridization of a DNA probe with a chemiluminescent label which is complementary to the ribosomal RNA of the genera Mycoplasma and Acholeplasma . This probe detects all species that commonly infect laboratory cultures. The following species have been found to be reactive in the test: A. axanthum, A. granularum, A. oculi, M. arthritidis, M. fermentans, M. hominis, M. muris, M. pirum, M. salivarium, S. citri, A. laidlawi, A. morum, M. arginini, M. capricolum, M. genitalium, M. hyorhinis, M. orale, M. pneumoniae, S. apis, S. floricola.