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Mycoplasma can be detected in cell cultures and products using DNA staining methods such as Hoechst staining. After treatment with Hoechst dye infected cultures observed under epi-fluorescence exhibit fluorescent nuclei against a dark background. Cultures infected with mycoplasma exhibit both fluorescent nuclei and extranuclear fluorescence attributable to mycoplasma DNA.

All cultures should be submitted for testing in antibiotic and cryoprotectant free medium. In addition cultures should not be passaged for three days prior to testing.

Vero or other indicator cells should be seeded at 1x10⁴cells/ml in duplicate into tissue culture dishes containing sterile coverslips. These should be prepared 10-24 hours in advance of inoculation with cell cultures for testing. During this time they should be incubated at 36+1^oC in a 5%CO₂ /95% air atmosphere.

1. Using an inverted microscope examine the Vero dishes for the presence of bacterial or fungal contamination.
2. Remove 1ml of culture supernatant from each dish and replace with 1ml of test article (cell suspension of cell culture product). Dishes should be inoculated in duplicate.
3. Inoculate two pair of dishes with positive control organisms (M. orale and M. hyorhinis). In addition leave one pair of dishes un-inoculated as a negative control.
4. Incubate one dish of each pair for 12-24hr and the remaining dish for 60-72hr. All dishes should be incubated at 36+1^oC in a 5%CO₂ /95% air atmosphere.
5. Following the appropriate length of incubation fix the cultures with Carnoy's fixative, by adding 1ml per dish and incubating at room temperature to 5min.
6. Remove the fixative and tissue culture supernatant by pipetting and add a further 1ml of fixative. Incubate for a further 5 min at room temperature.
7. Remove all liquid form each dish and allow to air dry for 15min at room temperature.
8. Add 2ml of Hoechst stain solution (0.2g/ml) and cover with silver foil. Incubate for 5min at room temperature.

Remove excess stain and mount coverslips onto prelabelled slides, prior to examination at 1000x magnification using epifluorescence.

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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