How should I prepare samples for mycoplasma testing?
1) Cryopreserved cell cultures should be cultured for circa 2 weeks prior to testing to ensure a sufficient mycoplasma titer. Thus, we recommend submission of samples from growing cultures to minimize chargeable costs for shipment and cell culture.
2) For the same reason, cell lines must be cultured without antibiotics for at least two weeks prior to submission.
3) We recommend submission of samples taken on Monday's (conditioned medium from a three days culture or older) and immediate shipment. Do not send the samples later than Wednesday to ensure same-week delivery.
4) Work in DSMZ laboratories is only permitted for cell cultures categorized up to and including risk group 2.
Suspension cell cultures:
For suspension cell lines, stand culture flasks vertically allowing cells to settle for about 30 minutes prior to removal of 2 ml of the supernatant and transfer to two sterile tubes.
Adherent cell cultures:
Grow the cultures almost to confluence and remove 2 ml supernatant. Transfer the supernatant to two sterile reaction tubes.
These procedures should yield optimum numbers of cells to detect mycoplasma adhering to the eukaryotic cells to facilitate detection of adherent mycoplasma.
How is the mycoplasma PCR assay performed?
We first isolate the cellular particles present in 1 ml culture supernatant by centrifugation, and wash with PBS. Sample DNA is separated and purified using Wizard DNA Clean-Up system (Promega) and aliquots used as template in PCR reactions with primers for 16S rDNA regions of mycoplasma species predominantly found in cell cultures. In the event of contamination, a 504 - 519 bp DNA fragment (depending on the mycoplasma species) is visible in the ethidium bromide-stained agarose gel. As internal control, we amplify a 975 bp DNA fragment added in a limiting dilution in a parallel PCR reaction to confirm a successful PCR amplification. Positive, negative, and water controls are also included.
For comparison of the PCR method with other mycoplasma detection assays, please refer to the following publication:
Uphoff CC, Drexler HG: Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell Dev Biol Anim 38: 79-85 (2002).
How sensitive is the PCR assay?
The determination of the sensitivity of a mycoplasma detection assay is problematical because an objective reference is necessary to quantitate mycoplasmas. The counting of colony forming units (cfu) is inexact because mycoplasmas often grow as clumps and one colony might be seeded from multiple mycoplasmas. Thus, we have cultivated pure cultures of mycoplasmas, isolated and quantitated the DNA, and performed PCR reactions with a dilution series of the DNA.
The following calculation is an approximation to determine the sensitivity:
- Amount of DNA after column preparation: 300 ng in 20 μl
- The genome size is assumed to be 1000 kb. 300 ng DNA corresponds to ca. 3 x 10E8 mycoplasmas (1 ng DNA / 10E6 mycoplasmas).
- 1 μl of a 1:100.000 dilution of the original DNA solution may be detected by the PCR reaction --> 300 ng / 20 μl : 100,000 = 150 fg/μl
- 150 fg correspond to a DNA amount of about 150 mycoplasma genomes (1 fg / mycoplasma genome)
How long does the elimination procedure take?
Depending on the antibiotic used, the eradication procedure takes at least four to five weeks.
How is mycoplasma elimination performed at the DSMZ?
We apply different antibiotics to eliminate the mycoplasmas. After treatment, the cell cultures are further cultured for at least two weeks without antibiotics. Two independent detection assays are then performed (PCR and microbiological culture). When the cultures are free from mycoplasma, the cells are expanded and five ampoules are frozen as stocks. Four ampoules are sent to the client and one stored for about 6 months in case of recurrence contamination. For further details on the elimination procedure and the antibiotics applied, please refer to the following publication:
Uphoff CC, Drexler HG: Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell Dev Biol Anim 38: 86-89 (2002).