Titered Mycoplasma Reference Strains Panel

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Prevalence of Mycoplasma Contamination

Mycoplasmas are frequent contaminants of cell cultures and bioprocessing fluids. Numerous studies have been published that establish mycoplasma contamination of continuous cell cultures in the range of 15-35%, with primary cell cultures exhibiting a minimum 1% contamination rate1, 2. There are over 190 species of mycoplasma, but only 20 distinct species of human, bovine and porcine origin have been identified in cell culture3, 4. Of those twenty, eight species account for approximately 95% of all mycoplasma contamination in cell culture, including M . arginini (bovine), M. fermentans (human), M. hominis (human), M. hyorhinis (porcine), M. orale (human), M. pirum (human), M. salivarium (human), and Acholeplasma laidlawii (bovine).

Importance of Routine Testing

Mycoplasma can affect the phenotypic and functional characteristics of cells in vitro, including morphology, protein expression, and virus production. Implementation of early detection methods that are rapid and sensitive is an important step towards preventing the deleterious and costly effects of mycoplasma on research and development projects.

Assay Development & Validation

ATCC mycoplasma reference standards with low genome copy (GC) to colony forming unit (CFU) ratios are ideal for use in the development and validation of PCR-based methods of detection. The Titered Mycoplasma Reference Strains Panel (ATCC® No. MP-7™) represents a unique collection of species that are:

  • Commonly associated with cell culture contamination
  • Essential for comparing the sensitivity of nucleic acid-based testing against conventional methods
  • Useful for evaluating the entire isolation and detection process

The panel is composed of 10 species of mollicutes isolated from both clinical and environmental sources. Each titered sample provides 0.5 mL mycoplasma in suspension that has been:

  • Evaluated for genome copy number*
  • Quantified by colony forming units (CFU)**
  • Rigorously characterized and authenticated by ATCC ISO 9001 certified laboratories
  • Optimized to yield high-viability upon thaw

Items from the ATCC Titered Mycoplasma Reference Stains Panel can be ordered individually with bulk discounts available. Genomic DNA from each strain is also available upon request.

ATCC Genuine Cultures® included in the Titered Mycoplasma Reference Strains Panel (ATCC® No. MP-7™)
ATCC® No. Organism Designation Source
27545-TTR™ Mycoplasma hominis Human - blood
15531-TTR™ Mycoplasma pneumoniae FH strain of Eaton Agent Human - atypical pneumonia
23206-TTR™ Acholeplasma laidlawii PG8 Sewage
23064-TTR™ Mycoplasma salivarium Saliva
25204-TTR™ Mycoplasma synoviae WVU 1853 Hock joint of chicken
19989-TTR™ Mycoplasma fermentans PG18 Ulcerative balanitis
23838-TTR™ Mycoplasma arginini G230 Mouse brain experimentally infected with scrapies
19610-TTR™ Mycoplasma gallisepticum Suspension of tracheal and air sac tissues of chickens with chronic respiratory disease
17981-TTR™ Mycoplasma hyorinis BTS-7 Nasal cavity of pig
23714-TTR™ Mycoplasma orale CH 19299 Human - oropharynx of child
Sources of Mycoplasma Contamination

Despite having limited metabolic pathways and no cell wall, mycoplasmas are resilient. Mycoplasmas are dispersed by aerosol droplets or particles generated while pipetting and handling media and different cell types simultaneously. Possible sources of mycoplasma contamination are1:

  • Infected incoming cells (cross-contamination)
  • Cell culture media, sera or trypsin
  • Laboratory personnel

The primary source of mycoplasma contamination is cross contamination from infected cell cultures. Laboratory personnel are also a key source of contamination, as human mycoplasmas (M. orale, M. fermentans and M. salivarium) are major species detected in cell cultures. Laboratory equipment, benches and flow hoods have also been identified as sources of contamination, along with cell culture reagents such as media and sera.

Effect of Mycoplasma in Cell Cultures

Mycoplasma contamination can cause a wide variety of adverse effects on the function and activities of cells in culture, leading to misinterpretation of results and compromising the validity of data generated for research and/or development projects. Consequences of mycoplasma contamination include1, 4, 6:

  • Inhibition of cell metabolism
  • Induction of chromosomal abnormalities
  • Disruption of DNA and RNA synthesis
  • Changes in virus and antibody production
  • Interference with growth rate of cells
  • Depletion of arginine
  • Change in pH
  • Activation of B cells
  • Altered gene expression in cells
Antibiotics and Mycoplasma

Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack a cell wall. Streptomycin inhibits about half the mycoplasma strains but is ineffective against many others. Gentamycin is generally ineffective at the concentrations routinely used in cell culture. In fact, mycoplasmas are generally resistant to most antibiotic mixtures commonly used in cell culture1.

References
  1. Drexler, H.G. and Uphoff, C.C. Mycoplasma contamination of cell cultures: incidence, sources, effect, detection, elimination, prevention. Cytotechnology 39:75-90 (2002). PMID: 19003295
  2. McGarrity G. J., Steiner, T. and Vanaman, V. Detection of Mycoplasma Infection of Cell Cultures by DNA Fluorochrome Staining. In Methods in Mycoplasmology, Vol. 2, J. G. Tully and S. Razin, eds. (Academic Press, New York, 1983) pp. 183-190.
  3. Barile, M. F., Hopps, H. E., Grabowski, M. W., Riggs, D. B. and Del Giudice, R. A. The Identification and Sources of Mycoplasmas Isolated from Contaminated Cell Cultures. Ann. NY Acad. Sci. 225:251- 264 (1973).
  4. Rottem, Shlomo. Interaction of Mycoplasmas With Host Cells. Physiol Rev 83: 417–432 (2003).
  5. Freed, J. J. and Schatz, S. A. Chromosome aberrations in cultured cells deprived of single essential amino acids. Exp. Cell Res. 55:393-409 (1969). PMID: 12663864
  6. McGarrity G. J., Vanaman, V. and Sarama, J. Cytogenetic Effects of Mycoplasmal Infection of Cell Cultures: A Review. In Vitro 20 (1):1-18. (1984). PMID: 6199287

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*The genome copy number calculation is determined using genome-size reported for the strain or the species and the concentration of genomic DNA determined by PicoGreen© from three separate extractions; the values provided for each distribution lot are an average of these three results. Using an alternative method of gDNA quantification may yield different results.

**CFU are quantified by absorbance or plate counts, depending on the ability of each strain to be cultured on solid media. Use of media or culture conditions other than those recommended by ATCC may yield different results.