Proportionality between nuclear DNA-content and Feulgen dye-content

Abstract The various methodological aspects of the Feulgen technique were investigated. Within wide limits the duration of the staining as well as that of the subsequent dehydration were found not to influence the intensity of staining. The passage through alcohol, however, renders the slides sensitive to a second treatment (after de-mounting) with the sulphurous acid bleaching solution (“SO 2 water”). The results of several series of measurements covering a total of up to nearly nine hours of such treatments can be interpreted as follows: During this whole period a “loss” of aldehyde groups which are available for staining takes place at a constant relative rate (half time: three hours and 26 minutes). Within half an hour after the first passage through alcohol (which is essential for this effect) a considerable fraction of stain is removed which, however, can be replaced by sufficiently prolonged re-staining. Later the nuclear dye-content decreases apparently only because of the continuous loss of aldehyde groups. The SO 2 experiments provided clear evidence of proportionality errors. The ratio of dye-contents of nuclei of different mitotic stages, as also of the same stage, is apt to change somewhat both during what seems to be a mere loss of dye and a loss of aldehyde groups. This demonstration of proportionality errors does not directly apply to the standard Feulgen technique but there is certainly no reason to suppose that the hydrolysis affects all nuclei in a slide in precisely the same way, if the treatment with SO 2 water in the above experiments does not.