By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)
Cell tradition ideas permit various molecular and mobile organic inquiries to be addressed, supplying physiological stipulations while averting using laboratory animals. as well as simple options, quite a lot of specialized functional protocols masking the subsequent components are integrated: phone proliferation and demise, in-vitro versions for telephone differentiation, in-vitro types for toxicology and pharmacology, commercial program of animal mobilephone tradition, genetic manipulation and research of human and animal cells in culture.
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Extra info for Animal Cell Culture Techniques
Note: Fading of the Hoechst 33258 stain may occur under the conditions required for analysis of the slides. 1 % (w/v) (Battaglia et al. 1994). 2 Direct Culture Procedure This direct method of mycoplasma detection involves growing the mycoplasma on a selective agar and observing the colonies by microscopical examination. Mycoplasma can reproduce in a cell-free medium where, on agar, they exhibit a characteristic colony morphology, with the center of the colony embedded beneath the surface. Mycoplasma colonies in agar are viewed by microscope.
M. hyorhinis is difficult to grow on agar (Uphoff et al. 1992). The DNA fluorochrome method cannot be used for direct identification of contamination. peR does not differentiate dead from viable mycoplasma and the range of mycoplasma species detectable is limited by the peR probes used. This chapter describes methods of mycoplasma detection by DNA fluorochrome staining and by the direct culture method. These are the two most widely used techniques, can be routinely used in all cell culture laboratories and identify mycoplasma contamination by a broad range of mycoplasma species.
However, as long as the agars and broths are shown to support the growth of a range of common cell culture-infecting mycoplasma and the internal assay controls are as expected, any of these formulations are appropriate. The protocol outlined here is that used within this laboratory and which has been shown to support the growth of both M. arginini and M. orale. Variations to the procedure include the addition of penicillin to the agar and broth (to decrease the risk of microbial contamination) and the inclusion of DNA and different types of serum (horse serum and pig serum in both the standard and heat inactivated forms) .
Animal Cell Culture Techniques by Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)