Use of Hoechst 33258 fluorochrome for detection of mycoplasma contamination in cell cultures: development of a technique based on simultaneous fixation and staining

Abstract

A simple rapid technique for detection of mycoplasma contamination in cell cultures by a modified Hoechst 33258 staining procedure, is described. After brief alcoholic fixation in situ, cell monolayers were definitively fixed and simultaneously stained by the addition of a 0.001% (w/v) Hoechst 33258 fluorochrome solution in ethanol for a few minutes. This method and conventional mycoplasma isolation techniques resulted in comparable sensitivity. Mycoplasma contaminants were detected in 21 out of 217 cell culture specimens tested. However, two different specimens were positive only by either procedure. A sequential use of both techniques, based on the search for mycoplasma contamination by microbiological methods only in Hoechst 33258 - negative samples, should provide a reasonable degree of sensitivity, being also accessible to virtually every laboratory involved in tissue cuture work.