BLEACHING OF FEULGEN STAINED NUCLEI AND ALTERATION OF ABSORPTION CURVE AFTER CONTINUOUS EXPOSURE TO VISIBLE LIGHT IN A CYTOPHOTOMETER

Abstract

Feulgen-stained cells were placed on microscope stages and exposed to monochromatic (570 mµ, 500 mµ) and polychromatic visible light in two different cytophotometers for as long as 113 hours. A bleaching effect was measured in 8 of 11 differemst experiments by scanning and multiple plug cytophotometry. Dye extinctions were reduced by 27 to 81%, varying with the particular slide preparation and conditions of irradiation. The absorption curve is also altered in shape as a result of the light exposure although the absorption maximum remains at 570 mµ. The bleaching effect was found with batches of pararosaniline or basic fuchsin from three different companies. The effect was observed in freshly-stained sections as well as in an old section. Acetic-alcohol fixation may offer some protection against bleaching compared with formalin fixation. The rate of bleaching is rapid at first and then slows down after about three days. It is thought that one or more leuco compounds are produced in this process. Since as little as 2% bleaching occurs after one hour of irradiation, the phenomenon is not considered as a source of major error in routine cytophotometry. Fronn the practical side, it is suggested that light be blocked from the slide during non-measuring periods and that irradiation time be reduced. The gallocyanin-chromalum-DNA complex is not bleached from cell nuclei after prolonged irradiation although the absorption curve is shifted to lower wavelengths. Protection against bleaching is probably afforded by the screening action of the inorganic salt.