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Negative Control Vero cells stained with Hoechst Stain. Cells were examined using an epifluorescent microscope and a green filter



Vero cells infected with Mycoplasma hyorhinis for 5 days and stained with Hoechst stain. Cells were examined using an epifluorescent microscope and a green filter

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Invisible in cell culture but can cause serious damage!

What is Mycoplasma doing to my cell lines??

Clongen Labs
It is always a good practice to check your cell lines on a regular basis for Mycoplasma contamination. Mycoplasma contamination could be devastating to a cell culture facility and could result in heavy costs and wasted time if not detected early. At Clongen Laboratories, we offer several ways to test cell lines (or any sample source) for the presence of Mycoplasma, Ureaplasma and Acholeplasma contamination. One of our assays is a Cell-Based Assay where the test material (cell line or other) is inoculated onto a Vero cell monolayer known to be negative for the presence of Mycoplasma, incubated for a few days, fixed and stained with e Hoechst stain (DNA fluorochrome 33258). This fluorescence pattern of DNA in the cytoplasm of indicator cells indicates the infection status of the cells. Our laboratory scientists have many years of experience in evaluating microscopic fluorescence patterns and the assay is fairly sensitive (can detect 75-150 Mycoplasmas per ml). There are rare occasions where lysed cells or artifacts in the tested material can produce a false positive readout. We recommend a confirmatory assay for positive cultures other than the cell-based assay.
We also offer a highly sensitive Multiplex PCR (Polymerase Chain Reaction) assay for the detection of low copy number. The PCR assay is capable of detecting any of the 20 or more Mycoplasma and Acholeplasma species detected in cell culture.

At Clongen Labs, we offer aerobic and anaerobic cultivation of Mycoplasma on highly enriched special media used for the cultivation of Mycoplasma. Our assays are performed under GMP conditions and if requested, we offer GLP testing for Mycoplasma (21CFR 610.30) intended mainly for vaccine products. This assay detects both the cultivable and non-cultivable Mycoplasmas using the cell-culture based technique and the enriched microbiological medium to attempt to grow Mycoplasmas. We offer Mycoplasma GLP testing (9CFR113.28) for cultivable Mycoplasmas in enriched medium and the Points to Consider 1993 testing for Virus Stocks using cell-based and enriched medium growth. Protocols will be provided to clients upon request.

Mycoplasma Elimination :

Clongen Laboratories offer Mycoplamsa Elimination service that will clean up a cell line using a commercially available antibiotic cocktail (see protocol number and price at bottom of page). With this service, you will receive one PCR test at baseline for Mycoplasma presence, two PCR tests during the course of the treatment and one test five days after stopping the treatment to screen for the presence of Mycoplasma. You will receive a detailed 5-7 page report describing all work done with gel images and a certificate of quality.

Sample requirements for the most reliable results :

Cultures should be grown for a minimum of a week without antibiotics. That growth period should be longer if antibiotics such as gentamicin, kanamycin, or other potent broad-spectrum antibiotics were added to the growth medium. The presence of antibiotics could reduce the Mycoplasma copy numbers (if present) to below detection limits. The media should not be changed for at least four days prior to testing. Cultures should be submitted in 25 cm2 flasks or scraped off with a cell scraper and transferred into a 50 ml tube and the cap wrapped with parafilm to prevent leakage and cross contamination if submitting more than one cell line at once.
To select the Mycoplasma assay from our list of Biotech/Biopharm Menu and to download a submission form, please click here(Biotech/Biopharm testing)
Procedure DescriptionCat #Turnaround Time
Mycoplasma Screen by Hoechst Staining (Non-GLP)CB1141 – 2 days
Mycoplasma Screen by Hoechst Staining (GLP)CB1151 – 2 days
Mycoplasma Testing by Direct Cultivation on enriched Mycoplasma Media – Non-GLPCB1175 – 6 days
Mycoplasma Testing of Biologicals in Compliance with 21CFR 610.30CB1185 – 6 days
Mycoplasmal Testing of Biologicals in Compliance with 9CFR113.28CB1194 – 5 days
Mycoplasma Testing by Multiplex PCR (Non-GLP) ($109)1311 – 3 days
Mycoplasma Elimination from Cell lines (Price will vary depending on cell line) -
For cell lines that do not require additional handling, cost per cell line is
CB1242 – 3.5 weeks
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