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Mycoplasma and Sterility Technical Information
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Mycoplasma species
Mycoplasma are the smallest and simplest prokaryotes which depend on their hosts for many nutrients due to their limited biosynthetic capabilities. They have long been recognized as common contaminants of cells in continuous culture but their presence can go undetected for months and even years. As the mycoplasma competes with the cells for the nutrients in culture media, typical signs include a reduction in the rate of cell proliferation and changes in cellular responses - including gene expression. Mycoplasma have been found to exist within a number of eukaryotic organisms.
Table of Mycoplasma species found to contaminate cultured cell lines, humans and other eukaryotic species
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Detection of Mycoplasma by Biochemical Methods
A number of biochemical methods exists for detecting toxicity or Mycoplasma-specific enzymes. Many of these methods are used as the basis of several commercial kits and specifically detect viable organisms. An example is the MycoAlert® Assay (Lonza) - which exploits the activity of certain mycoplasmal enzymes. Viable mycoplasma are lysed and the enzymes react with an added substrate to catalyze the conversion of ADP to ATP. By measuring the level of ATP in a sample both before and after the addition of the substrate, a ratio can be obtained which is indicative of the presence or absence of mycoplasma. This is measured indirectly with a luminometer - recording biolumination catalysed by the reaction of the ATP and luciferase.
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Detection of Mycoplasma by DNA Staining
To directly detect Mycoplasma infection within a cell line, cell culture supernatant samples are cultivated with a Vero indicator cell line for seven days before staining with a DNA specific dye. Fluorescence, when viewed with an epifluorescent microscope, illustrates the cell nucleus, with Mycoplasma appearing as granules around the nucleus of the cell. The sensitivity of this method can vary with the user, and autofluorescence from the cell can make interpretation difficult; these issues can be addressed by enrichment of potential contaminants on an indicator cell line.
Stained Mycoplasma arginini infected cells viewed under epifluorescence
(x 1000 magnification)
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Detection of Mycoplasma by PCR
The CellBank Australia PCR detection assay identifies Mycoplasma positive samples with primers developed to Mycoplasma specific genomic DNA sequence. Although potentially very sensitive, species detection varies with the primer or probe sequence used. These methods are also unable to distinguish between an ongoing contamination and a previously treated episode, since sequence may be detected from viable and non-viable organism. The presence of contaminant mycoplasma is easily detected using agarose gel electrophoresis by verifying the bands of amplified DNA fragments from test samples against both positive and negative controls.
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Eradication of Mycoplasma
CellBank Australia strongly recommends that Mycoplasma eradication is chosen as a last resort. Antimicrobial agents used can damage the cell line and cause cell death or genomic/phenotypic changes. It is recommended that wherever possible, an alternative mycoplasma-free source of a contaminated cell line should be used.
CellBank Australia does not typically offer a Mycoplasma eradication service. But as an aid, CellBank Australia has reviewed various methods and kits and compiled them as a reference for researchers.
Mycoplasma eradication protocols
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Sterility Testing
CellBank Australia routinely tests all of cell lines and cell culture media to determine if microbial contamination is present. Samples are incubated in both:
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Tryptose soya broth (TSB) - for the detection of aerobes, facultative anaerobes and fungi
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Fluid thioglycollate medium (FTM) - for the detection of aerobic and anaerobic bacteria
Both media test methods are recommended tests by the United States Pharmacopeia (USP) and the European Pharmacopeia (EP).
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