Mitotic Frequency Determinations and Micro-Photometric Feulgen Dye Measurements in Root Tips

Abstract

Treating a Feulgen stained (10 min. acetic-alcohol fixation, 8 min. hydrolysis) onion root tip with 5% aqueous pectinase for 6 hours causes it to disintegrate on shaking in 1 ml. water into a suspension of stained cells, either individual or in small groups. Vicia f aba and pea root tips require 15 min. hydrolysis and 12 hours pectinase treatment. Absence of cell destruction allows absolute cell-number determination by Brown and Rickless'' counting chamber technic. By centrifuging the cells down, resuspending them in 2 drops of a Karo syrup-phosphate buffer mixture (2 parts Karo to 1 part 0.5M, pH 7 buffer) and mounting a small drop of the now concentrated suspension, a semipermanent slide containing a concentration of well-spread, randomized cells suitable for rapid mitotic frequency determination is obtained. Scoring about 1000 cells as to nuclear stage gives a representative, workable statistical sample and takes only 20-30 min. if interphases are recorded on a mechanical tally. Permanent slides can be prepared by carrying the intact, stained, pectinase-treated root through a water wash, 70%, 95%, and absolute alcohol, shaking and performing the centrifuge procedure with Diaphane in place of the Karo mixture. Although the pectinase treatment appears to introduce some error, the Diaphane mounts are adequate for conventional microphotometric Feulgen dye (DNA equivalent) determinations. The three dimensional character of the cells is retained in all preparations.