Mycoplasma Detection

Tissue cell cultures can be contaminated by mycoplasma species without you knowing and can be a serious problem. Mycoplasmas are self-replicating bacteria that lack a cell wall and range in diameter from 0.15 to 0.3 uM. Mycoplasmas 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 Core mycoplasma test uses PCR amplification of the conserved 16S - 23S intergenic spacer region of ribosomal RNA with a highly conserved fluorescent primer pair. The amplified fragments are then hybridized to a MycoDtect DNA-array (Greiner,Bio-One). A universal mycoplasma DNA-probe tests for the presence of ALL mycoplasma species, while species-specific probes detect nine of the most common mycoplasmas. Species identification can help to identify and treat the source of contamination.

Testing for Mycoplasma contamination is recommended on a regular basis, and before cell banking and cryopreservation.

Ordering, Sample Preparation and Submitting Samples

Ordering instructions and a request form for Mycoplasma testing are available through the below links.

Mycoplasma Testing Ordering Instructions-JHU
Mycoplasma Testing Request Form-JHU

Grow cells in antibiotic-free media for 7 days. Do not change the medium or dilute with fresh medium during the last 24-72 hours before testing. Cells should be 50-80% confluent. 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.

Remove all but approximately 5 to 7 ml of medium in a T25 flask or 8 to 10 ml in a T75 flask. Use a cell scraper to dissociated cells from the substrate. Pipet the cell suspension into a 15 ml concial tube.

Bring sample(s) to Blalock 1005 Monday through Friday between 9 AM and 4 PM with the Customer Info page of the request form.

What you will receive

Mycoplasma results are presented in a Word document. Included with the results are a table indicating in the samples are negative or positive for mycoplasma and, if positive, which of the nine species identified with the kit were positive.

Turn around time

Samples for Mycoplasma testing are run once a week on Wednesday, with results emailed usually on Friday. Samples received after 4 PM on Tuesdays will be held until the following week for processing. Any failed samples from a Wednesday run will be re-run the following Wednesday with all results being emailed that Friday.

Pricing

Mycoplasma testing only
  • 1-10 Samples......................$80/sample
  • Groups of 11 Samples........$50/sample
  • Samples 12 -21...................$50/sample for the first 11, $80.00/sample for #12-21
Mycoplasma and STR Profiling Analysis (kit dependent):
  • PowerPlex 1.2 System:
    • 1-10 Samples......................$135/sample
    • Groups of 11 Samples........$100/sample
    • Samples12 -21....................$100/sample for the first 11, $135/sample for #12-21
  • StemElite:
    • 1-10 Samples......................$155/sample
    • Groups of 11 Samples........$120/sample
    • Samples12 -21....................$120/sample for the first 11, $155/sample for #12-21
  • Identifiler:
    • 1-10 Samples......................$185/sample
    • Groups of 11 Samples........$150/sample
    • Samples12 -21....................$150/sample for the first 11, $185/sample for #12-21

Please see our website for more information about STR profiling of cell lines

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. The DNA is polymerase chain amplified (PCR) for the 16S-23S rRNA intergenic spacer region of any mycoplasma species present using a highly conserved Cy5 fluorescent labeled primer pair. A PCR control within the PCR MasterMix allows for validation of the PCR. The labeled products are hybridized to complementary sequences on the MycoDtect chip. Each mycoplasma species, the universal probe, and the internal and PCR controls are 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 limit of detection determined by the manufacturer with the universal probe for the nine specific species identified is 1 colony forming unit (CFU) except M. fermentas, which is 10 CFU. MycoDtect testing by the manufacturer found the universal probe to detects 40 mollicute species.

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.