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Prepare a 0.8 mM trypan blue solution in phosphate-buffered saline (PBS). Mix cells and trypan blue solution in a 1:1 ratio. Inspect and count dead and alive cells under a light microscope in a haemocytometer. Dead cells will stain blue due to trypan blue uptake, while live cells appear transparent. Cells should not be exposed to trypan blue solution for more than 20 min. Prolonged incubation will increase cell death and reduce viability.
A variety of fl uorescent dyes can also be used to monitor cell viability, typical in the combination with fl ow cytometry. Propidium iodide and 7-actinomycin D are commonly used for this purpose.
Assessing cell viability at the population level has become very popular. These assays are typically carried out by using different tetrazolium compounds. Among the most popular are MTT ((3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), MTS, XTT and WST-1. Among these MTT is positively charged and readily penetrates viable cells, while the others are negatively charged and are excluded from viable cells. The MTT assay is perhaps the most popular to assess cell viability and proliferation in a population of cells. It was the fi rst assay to be developed for high trough put screening in a 96-well format. Viable cells convert MTT into a purple colored formazan product. When cells die, they lose ability to convert MTT into formazan. The mechanism behind this process is not well under- stood, although many publications suggest that the MTT assay refl ects changes in mitochondrial activity. Commercial kits containing MTT and a solubilization reagent can be obtained from several vendors like Sigma Aldrich, Promega and Millipore, among others.
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belong to only six species of bovine, swine and human origin. Bovine and swine mycoplasmas typically derive from contaminated sera or other animal-derived products used in the cell culture laboratory. Currently, many infections are trans- fered from already infected cultures within the same laboratory due to inadequate aseptic and cell culture techniques or from equipment that has been contaminated through prior handling of infected cells. However, laboratory personnel also repre- sent an important source of mycoplasma infections, and mycoplasma species from humans, mainly of oral origin, are responsible for 40–80 % of mycoplasma infec- tions in cell cultures. In total it is estimated that 15–35 % of all cultures based on continuous cell lines are mycoplasma infected (Barile et al. 1973 ), while contami- nated cultures based on primary cells are seldom found.
Their fl exibility in shape due to the lack of cell wall, allows most mycoplasmas to pass through 0.2 μm fi lters traditionally used for sterilization of media and medium ingredients. For the same reason they are diffi cult to observe in cell cultures. Moreover, despite the richness of standard culture media, mycoplasmas grow slowly, a fact that contributes to the problems with detecting them, visually or by change in turbidity. Although we tend to consider mycoplasmas mainly as a problem in cell culture work, it should be remembered that the mycoplasma family also includes a number of human pathogens.
How will mycoplasma infection affect the cultivated cells? Different mycoplasma species will introduce different problems, but in general, mycoplasma infections tend to infl uence a number of cellular processes leading to decreased growth rate due to inhibition of protein-, DNA- and RNA synthesis. Additionally, they introduce changes in gene expression that can be measured as down-regulation of cytokine and growth factor secretion, expression of receptors, intracellular signaling molecules, ion channels etc.
How to avoid contamination by mycoplasmas? A few major guidelines should be followed: (1) Strict aseptic techniques are an absolute requirement. Minimize talking, and do not practice mouth pipetting when working with cell cultures. Never pour medium between bottles and fl asks, and avoid crowding in the laminar fl ow hood. Unnecessary equipment in the hood will contribute to turbulence in the lami- nar airfl ow thus increasing the risk of contaminating fl asks and culture plates. Avoid unnecessary traffi c in the vicinity of the sterile hood. (2) All surfaces in laminar fl ow hoods, incubators and water baths should be regularly cleaned and disinfected (70 % ethanol or isopropanol). (3) Try to avoid unnecessary and excessive use of antibiotics. Routine work may preferably be carried out without addition of antibi- otics to the medium. Use of antibiotics will camoufl age poor aseptic techniques, and will contribute to worsen the problem. (4) When new cell lines arrive in the labora- tory, they should be cultivated separated from other cell cultures, rigorously tested and found to be mycoplasma free before being admitted to the cell culture labora- tory. (5) Establish a routine for frequent testing of the cultures. This is an absolute requirement for a responsible scientist. If a culture or cell line is found to be infected, cells, culture trays, medium bottles and any other equipment that has been in contact with the contaminated culture should be destroyed by autoclaving.
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There are several ways to detect mycoplasma infection in cell cultures, but today, most people rely on PCR technique which is fast and sensitive. Results can be obtained in hours. Several vendors provide kits with specially designed primers that allow selective amplifi cation of mycoplasma DNA for PCR-detection with a sensi- tivity that is close to the direct culture approach. Thus, for routine purposes PCR represents the method of choice.
Mycoplasma infection of cell lines and particularly clones of valuable primary cells can be a disaster. It may be possible to “cure” the cells with antibiotics.
Typically, antibiotics like ciprofl oxacin, minocyclin or a combination of tiamulin and minocyclin, have been demonstrated to be effective. However, toxic effects causing cell death is not uncommon. In general, cleaning up a contaminated culture should only be considered as an alternative for absolutely irreplaceable cultures provided that the source of the contamination has been identifi ed and removed from the laboratory.
References
Armstrong SE, Mariano JA, Lundin DJ (2010) The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals 38:211–213
Barile MF, Hopps HE et al (1973) The identifi cation and sources of mycoplasmas isolated from contaminated cell cultures. Ann N Y Acad Sci 225:251–264
Drexler HG, Uphoff CC (2002) Mycoplama contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39:75–90
Heeneman S, Deutz NE, Buurman WA (1993) The concentrations of glutamine and ammonia in commercially available cell culture media. J Immunol Methods 166:85–91
Roth E, Ollenschlager G, Hamilton G, Simmel A, Langer K, Fekl W, Jakesz R (1988) Infl uence of two glutamine-containing dipeptides on growth of mammalian cells. In Vitro Cell Dev Biol 24:696–698
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© The Author(s) 2015
K. Verhoeckx et al. (eds.), The Impact of Food Bio-Actives on Gut Health, DOI 10.1007/978-3-319-16104-4_9