Q & A Mycoplasma Detection Will the ATCC® Universal Mycoplasma Detection Kit be compromised if the sample contains too many cells? Ideally, for both adherent and suspension cells, the cell range should be between 104 and 106 cells. The use of cells greater than this volume could potentially inhibit the PCR. Can the kit be used in clinical diagnostics? The Universal Mycoplasma Detection Kit is intended for laboratory research only, not for clinical use. In our lab, we use a commercially available, PCR-based mycoplasma detection kit. The problem which I currently face is that the positive control is not amplifying. Also, our internal controls have amplification problem sometimes as well. How efficient is ATCC® Universal Mycoplasma Detection PCR kit? The ATCC® Universal Mycoplasma Detection Kit is highly sensitive and efficient. It can detect target DNA as low as 2.5 fg and does not cross-react with related bacterial or mammalian genomic DNA. The positive control, which is DNA from Mycoplasma arginini, contains a total of 50 pg (50 µl of 1pg/µl) and has no problem with amplification. In the ATCC® Universal Mycoplasma Detection Kit do you use cell media or cell lysates for detection of mycoplasma? The kit is sensitive enough to detect mycoplasma in just cell media. However, certain mycoplasma directly adhere to cells. This could negatively affect detection. It is therefore highly recommended that cell lysates be used. Is it necessary to use internal controls such as GAPDH or b-ACTIN when using the mycoplasma detection kit? The ATCC® Universal Mycoplasma Detection Kit comes with its own positive control and no additional controls are needed. Is the kit approved by the FDA? The ATCC® Universal Mycoplasma Detection Kit has not been approved by the FDA for biopharmaceutical or clinical use. Can I only use PCR-based method to detect mycoplasma contamination? As a service, ATCC offers a Direct Culture Mycoplasma testing service that is approved by the FDA. If this level of scrutiny is required, as with biopharmaceutical products, this service can be utilized. For research purposes, the ATCC® Universal Mycoplasma Detection Kit is designed and proven to detect mycoplasma without any additional testing required. Do you have any detailed protocols for the antibiotic treatment of mycoplasma positive cells? We recommend discarding mycoplasma contaminated cells since they are considered a source of contamination in the lab. If the cells are really valuable and rare, there are many chemical agents that can be used to treat them to eliminate mycoplasma infection; examples are BM-cyclin and plasmocin. Just follow manufacturer instructions. More helpful information - https://www.hindawi.com/journals/bmri/2012/267678/ If a cell line is cultured for ten passages only, how often should a cell line (CHO, for example) be tested for mycoplasma contamination? According to best practices, a mycoplasma test should be done when on the original vial if the cells are received from an unreliable source and before a master stock is prepared. If the cells are being isolated from tissues generated after primary, it may be necessary to use antibiotics in the primary culture, but they should be removed as soon as possible and the cells tested for mycoplasma contamination. The test should be repeated when the master stock is expanded to a working stock and the final working stock should be tested as well. There is no limit to the number of passages after which the cells should be tested, but it is advisable to screen cultures regularly if they are in continuous culture. What are the easiest methods and protocols used to detect mycoplasma contamination in cancer cell cultures? The Universal Mycoplasma Detection Kit is one of the quickest methods to detect mycoplasma in cell culture. The entire procedure only takes approximately three hours. Also, as alternate, ATCC offers a PCR-based mycoplasma detection service. Can you comment on using routine anti-mycobacteria antibiotics in media as a preventative measure? As a general rule, it is discouraged to use antibiotics in cell culture media for most cases. Antibiotics/antimycotics can alter the physiology and health of cells in culture. However, for certain reasons, it may be necessary. You can find additional information about the use of antibiotics and antimycotics in our free-to-download cell culture guides. Could insect cells such SF9 be contaminated by mycoplasma? All cells in culture can be contaminated by mycoplasma. Even if the stock cells are mycoplasma free, contamination can occur due to inadequate cell culture techniques.