Mycoplasma Contamination of Cel Cultures: Detection and Eradication
Cell culture is a vital component of biological and medical research. It is used in numerous applications including the investigation of cell physiology, the testing of chemical compounds on specific cel types, the synthesis of valuable biologicals, and the creation of genetically-modified animals. Contamination of cell cultures can profoundly affect research results by decreasing cell growth, altering phenotypic and metabolic characteristics, inhibiting differentiation, and cell death. Mycoplasma sp. are the most common contaminants detected in cell cultures (Table 1). % Positive Infectious agent
Lactic dehydrogenase elevating virus (LDEV) 1.1 Parvoviruses
Lymphocytic choriomeningitis virus (LCMV)
Table 1. Prevalence of cel culture contaminants detected in 19,666 samples submitted to RADIL in 2009.
Mycoplasmas belong to the class Mollicutes, which includes over 100 species of Mycoplasma and the very closely related genus, Acholeplasma(1). The most prevalent Mycoplasma and Acholeplasma contaminants of cultured cells are Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hominis and Acholeplasma laidlawi (2). Infections of cell cultures with Mycoplasma pulmonis, the Mycoplasma species most commonly found in rodents, can occur, but are extremely rare. Detection
Among bacterial infections, Mycoplasma contamination poses a particular challenge because it does not conspicuously overgrow the cell culture, can only be detected using special assays, and is resistant to many commonly used antibiotics. Thus, contaminations may be insidious al owing continued undetected propagation of Mycoplasma, which is then spread to other cultures within the laboratory, and may be further disseminated by interchange of cell lines among laboratories. It is important to
employ rigorous testing to identify Mycoplasma infected cell lines. Many methods have been used to detect Mycoplasma sp. including staining techniques, electron microscopy, biochemical and immunological tests, and some hybridization assays (3). These methods can be complicated and time consuming, may only detect a few Mycoplasma species, have a low sensitivity, be difficult to interpret, or require specialized equipment. Recently, PCR assays have been developed to detect the presence of Mycoplasma sp. IDEXX RADIL’s Mycoplasma assay is highly sensitive, detecting as little as 1-10 organisms, and employs two distinct PCR assays which amplify different regions of the genome. Positive results with both primers provide a high level of confidence that all lines are infected with Mycoplasama. Diagnostic testing results are available within 5 business days after receipt of samples at IDEXX RADIL’s secure website. Additional y, an email is sent to the submitter as soon as results are available.
Samples may be submitted as pellets of cells in growth medium or phosphate buffered saline or vials of frozen cells with cryoprotectant. The IDEXX RADIL’s assay detects all species of Mycoplasma known to infect cell cultures and includes Acholeplasma laidlawi and Spiroplasma mirum, two other members of the Mollicutes class that also can contaminate cultured cells (Table 2). Table 2. Bacteria detected by RADIL’s PCR assay. Eradication Mycoplasma contamination of cell cultures adversely affects research. Once detected, it is important to eradicate the infection to prevent further spread of the contaminant. Many of the antibiotics commonly used in cell culture are ineffective against Mycoplasma, but several anti-Mycoplasma antibiotics have been developed that are available commercially. The infection can be completely eliminated by autoclaving the infected cultures and replacing them with fresh stocks, however, that is not possible if the entire cell line has been contaminated or if it is an irreplaceable culture. There are four general categories of antibiotics that can be used to eliminate contamination (Table 3). Reagent (category) Mode of Action Effect on bacteria Treatment period Table 3. Antibiotics used in the eradication of Mycoplasma contamination (4).
It is recommended that cells be passaged an additional 3-5 times after treatment in the absence of antibiotics and then retested for Mycoplasma to ensure that the infection has cleared. Note: The prophylactic use of anti-Mycoplasma antibiotics is not recommended because it involves more manipulation of the culture which could compromise sterile technique and the bacteria could develop resistance to the antibiotic. References
1. Author unknown. “Mycoplasma: The Insidious Invader of Cell Cultures”. InvivoGen Insight.
InvivoGen, November/December 2005. Web. 9 May 2011.
2. Drexler HG, Uphoff CC. “Mycoplasma contamination of cell cultures: Incidence, sources, effects,
detection, elimination, prevention”. Cytotechnology 39(2002): 75-90.
3. Drexler HG, Uphoff CC. “Contamination of cell cultures, Mycoplasma.” Encyclopedia of Cel Technolog. Eds. E Spier, B Griffiths, AH Scragg. Wiley, New York, 2000. 609-627.
4. Uphoff CC, Meyer C, Drexler HG. “Elimination of mycoplasma from leukemia-lymphoma cell
lines using antibiotics”. Leukemia 16(2002):284-288.
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