LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3.2/2.1
TITLE: INDIRECT HOECHST STAINING OF CELL LINES AND PRODUCTS
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+1oC in a 5%CO2 /95% air atmosphere.
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
© The CABRI Consortium 1999-2013.