The test sample is inoculated onto Vero cells which have been grown on coverslips in tissue culture dishes. Both negative and positive controls (Mycoplasma hyorhinis NCTC 10112 and Mycoplasma orale NCTC 10130) are also set up. All samples are then incubated for 3-5 days after which the samples are fixed to the coverslip and stained using the fluorescent dye (Hoechst 33258). The fixed samples are then examined under UV epi fluorescence at x1000 magnification. Positive samples are identified by their characteristic particulate or filamentous pattern of fluorescence on the cell surface or if contamination is heavy in the surrounding area. Negative samples should demonstrate no evidence of fluorescing DNA outside of the cell membrane against the fluorescing Vero cell nuclei.
Stained mycoplasma infected cells viewed under epifluorescence
(x 1000 magnification)
We offer Mycoplasma detection by Indirect Hoechst Stain using a method that meets FDA points to consider in characterisation of cell lines used to produce biologicals (1993).
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