Factors involved in differential Giemsa-staining of sister chromatids

Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium temperature during UV-exposure and the quality (wavelength)of UV-light influenced the differential staining. Exposure of blacklight of 10(-5) M Hoechst 33528-stained brdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50 degrees C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlender's method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.