Mycoplasma Testing

Tissue cell cultures can become contaminated by mycoplasma species without you knowing, which can be a serious problem. Mycoplasma can cause alterations in cell growth and metabolism, or induce morphological changes or chromosomal abnormalities. Potential sources of contamination are laboratory personnel, contaminated products used for cell culture, or exposure to contaminated cultures.

The FAF offers mycoplasma testing using a PCR based MycoDtect kit from Greiner Bio-One. A fluorescent primer pair is used to amplify the conserved 16S – 23S intergenic spacer region of ribosomal RNA. Amplified fragments are hybridized to a MycoDtect DNA-array for detection of all mycoplasma species using a universal probe while simultaneously identifying nine of the most common mycoplasma species with species-specific probes. Species identification can help to identify and treat the source of contamination.

Screening for mycoplasma contamination is recommended on a regular basis, and before cell banking and cryopreservation.


Johns Hopkins customers please place your order through our secure online ordering system, Genesifter. Genesifter is now behind the JHMI firewall. This means it is ONLY accessible within the Hopkins environment. If you are off campus you will need to access the server via VPN, just like your email and many other JHU secure services.  The order form can be found under FAF Order Forms: Mycoplasma Testing or Cell Line Authentication and Mycoplasma Testing for combined service.

External non-Johns Hopkins customers please use this downloadable Excel order form for placing requests. Our online ordering system, Genesifter, is no longer available to external customers due to security risks. We hope to have a solution in the future. If you have a Genesifter account with us and need access to your old data files please contact Laura Kasch at Sorry for the inconvenience.

Sample Preparation

Samples should be submitted from growing culture or as frozen ampoules. We accept cells, cell culture supernatant or frozen stocks. If submitting samples for mycoplasma testing and cell line authentication follow the below directions under Instructions for Growing Culture and Cell Pellets.

Instructions for Growing Cultures: We recommend passage of cells in antibiotic-free media twice. Do not change the medium or dilute with fresh medium during the last 72 hours before testing. Cells should be 50-80% confluent. Submission of cells at higher confluency may inhibit the PCR assay due to the accumulation of PCR inhibitors. Samples grown in antibiotics or given fresh medium prior to testing may have artificially low mycoplasma levels if present or may give a false negative result.

Cell Pellets: 10E5 to 10E6 cells
Adherent cells: Use a cell scraper to dissociated cells from the substrate. Do not treat with trypsin or other enzymatic agents for dissociation. Collect 5 to 7 ml of medium in a T25 flask or 8 to 10 ml in a T75 flask. Centrifuge to pellet cells, then remove supernatant.

Suspension culture: Count cells. Remove suspension culture to a 15ml conical tube.

Collect 5-10ml of supernatant medium of adherent cells or suspension cultures and submit in a 15ml conical tube. Paraffin the top if shipping.

Frozen Stock: Submit ampoule.

Turnaround time:

Samples submitted for mycoplasma testing are run once a week on Wednesday, with results emailed Friday of the same week or following Monday. However, to keep services costs down we try to fill the processing arrays so turnaround may be longer. Johns Hopkins customers should drop off samples in Blalock 1004 by 4PM on Tuesday for inclusion in that week’s processing schedule. External customers should ship samples to the lab to be received by 11AM Wednesday for inclusion in that week’s processing schedule. Samples received after 11AM Wednesday through Friday of any given week will be held and processed the following week.

JH Customers: Drop samples off in Blalock 1004 with the GeneSifter order number, Monday – Friday from 9 AM – 4:00 PM.

External Customers: Send the samples with the GeneSifter order number to:

Laura Kasch
GRCF, Fragment Analysis Facility
Johns Hopkins University
725 N. Wolfe Street, PCTB G20
Baltimore, MD 21205
Phone (443) 287-7948

Samples should be sent for delivery Monday through Friday only.


Mycoplasma testing only: $70/sample

Mycoplasma AND Cell Line Authentication (STR profiling): A cell pellet is needed for combined service, extracted DNA is not suitable.

  • GenePrint 10: $145/sample (Standard Service)
  • GenePrint 10: $180/sample (Standard Service plus Profile Search)
  • PowerPlex 18D: $179/sample (Standard Service)
  • PowerPlex 18D: $214/sample (Standard Service plus Profile Search)

John Hopkins investigators: IO number or credit card (VISA or MasterCard)
External U.S. customers: P.O. or credit card (VISA or MasterCard)
External foreign customers: P.O., check or credit card (VISA or MasterCard)

International customers using a credit card will have an additional $25 processing fee added in addition to the administrative fee.

Procedure and sensitivity

DNA is extracted from the cell culture supernatant using a DNEasy Blood & Tissue kit automated with a QIAcube (Qiagen). A MycoDtect internal control is added prior to isolation to monitor the DNA extraction process and template use for the amplification. Using a highly conserved Cy5 fluorescent-labeled primer pair the DNA is polymerase chain amplified (PCR) for the 16S-23S rRNA intergenic spacer region of any mycoplasma species present. A PCR control within the PCR MasterMix allows for validation of the PCR. The labeled products are then hybridized to complementary sequences on the MycoDtect chip. Each mycoplasma species, the universal probe, and the internal and PCR controls are each detected by five measuring points on the chip. Non-specifically bound probes are removed by washing. The bound labeled probes are detected by stimulation with monochromatic light and analyzed using CheckReport software. The MycoDtect assay will detect both viable and non-viable mycoplasma, it will not distinguish between the two.

Greiner Bio-One has tested MycoDtect with the following Mollicutes species: A. axanthum, A. laidlawii, A. modicum, A. morum, A. oculi, A. vituli, M. alkalescens, M. arginini, M.arthritidis, M. bovigenitalium, M. bovirhinis, M. bovis, M. bovoculi, M. buccale, M. californicum, M.canadense, M. canis, M. eqhirhinis, M. faucium, M. fermentans, M. flocculare, M. gallinaceum, M.gallinarum, M. gallisepticum, M. genitalium, M. glycophilium, M. hominis, M. hyopharyngis, M.hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M.salivarium, M. synoviae, S. citri, S. kunkelii, U. diversum, U. urealyticum.

In addition, MycoDtect was tested by Greiner Bio-One with a broad range of closely related bacteria. None of the tested bacteria showed a signal with the specific probes, but some hybridized to the universal probe. For a detailed list, please contact Laura Kasch.

FAF services may only be used for research purposes. Any data or samples generated by the facility may not be used for human diagnostic or therapeutic use.