Most cell culture contaminations caused by bacteria, yeasts and fungi are usually rapidly detected during routine cell cultivation. However, slow-growing bacteria, e.g. some mycoplasma and mycobacteria species, are frequently found in cell cultures and may be chronic and not evident during routine cell culture monitoring. Thus, sensitive methods are required to detect such contaminations.

Mycoplasma contaminations are particularly widespread among all types of human and animal cell cultures. Figure 1 depicts a mycoplasma contaminated eukaryotic cell. According to our experience, ca. 20% of cell cultures received from other laboratories are contaminated with mycoplasmas. These microorganisms are usually introduced by direct contact with other infected cultures, but other sources of contamination cannot be excluded (e.g. laboratory personnel or serum). For this reason, we quarantine all incoming cell cultures until their mycoplasma status is known. In addition, DSMZ laboratory cell cultures are routinely tested for mycoplasmas. All cell lines distributed by the DSMZ are tested mycoplasma negative.

Cell lines are initially tested by at least two different assays: the classical broth-agar microbiological culture method (Figure 2) and by a polymerase chain reaction (PCR) assay. Many cell lines have been also tested by a third or even fourth assay such as the DNA fluorochrome staining, DNA-RNA liquid hybridization assay (Gen Probe), fluorescent in situ hybridization (FISH), or a luminometrical method (MycoAlert). For further detailed information on the above-mentioned detection assays, please consult our publications (1, 2, 3, 4) and the section on mycoplasma contamination on our cell culture technology information site.

On detection, mycoplasma positive cell lines are normally discarded and replaced by mycoplasma free culture. When the latter are unavailable, mycoplasmas are eradicated with various antibiotics. The most effictive antibiotics for mycoplasmas belong to the fluoroquinolones (ciprofloxacin, enrofloxacin, sparfloxacin, and MRA – Mycoplasma Removal Agent), the tetracyclins (Minocyclin), and the macrolides (Tiamulin). Treatment times cover one to three weeks depending on the antibiotic used, and the cells are held without antibiotics for at least two weeks post-treatment before testing. Only cell lines subsequently testing negative may be distributed. The comparative effectiveness of various ant-mycoplasma agents used at the DSMZ is shown in Figure 3. Protocols and detailed information have been published (5, 6).

Other occult microbiological contaminations found in exceptional cases have turned out to be caused by bacteria of the genus Mycobacteria (Mycobacterium avium-Mycobacterium intracellulare complex, M. chelonae). These contaminations can be detected by the microbiological broth-agar culture method or by PCR using prokaryotic consensus primers. Usually, these bacteria are resistant against a multitude of antibiotics and antibiograms may be required to find effective antibiotics.

References:

  1.  Hopert A, Uphoff CC, Wirth M, Hauser H, Drexler HG: Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines. J Immunol Methods 164: 91-100 (1993). PubMed ID 8360512
  2. 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). PubMed ID 11928999
  3. Uphoff CC, Drexler HG: Detection of Mycoplasma Contaminations. in: Basic Cell Culture Protocols, Third Edition (eds Helgason CD, Miller CL), Humana Press, Totowa, NJ (2005), pp. 13-24. PubMed ID 15361652
  4. Uphoff CC, Drexler HG: Mycoplasma contamination of cell cultures. in: The Encyclopedia of Industrial Biotechnology, Bioprocess, Bioseparation and Cell Technology, Vol. 5 (ed Flickinger MC), Wiley, New York (2010), pp. 3611-3630
  5. Uphoff CC, Drexler HG: Eradication of Mycoplasma Contaminations. in: Basic Cell Culture Protocols, Third Edition (eds Helgason CD, Miller CL), Humana Press, Totowa, NJ (2005), pp. 25-34. PubMed ID 15361653
  6. Uphoff CC, Drexler HG:Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.In Vitro Cell Dev Biol Anim 38: 86-89 (2002). PubMed ID 11929000