DAPI staining improved for quantitative cytofluorometry.

DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.