Acetic Acid Methods for Chromosome Studies at Prophase and Metaphase in Meristems

Abstract

Monobromobenzene and monobromonaphthalene proved easier to handle than paradichlorobenzene and acenaphthene, and at least as effective for inhibiting spindle formation, for shortening and straightening chromosomes to permit accurate counts and size comparisons. For prophase studies, methyl alcohol pretreatment was found effective in revealing centromeres, heterochromatin, and knobs. The following schedules were found simple and effective. For metaphase chromosomes: (1) Remove root tip from germinating seed and place in saturated aqueous monobromonaphthalene for three hours. (2) Pour solution from vial and replace with a mixture of 70 volumes 95% ethanol and 30 volumes glacial acetic acid. Leave in fixative at least two days. (3) Remove opaque tip from root and macerate in a drop of acetic-orcein. Cover, heat to near boiling, flatten by pressing slide, cover down, onto a thick blotter. For prophase chromosomes: (1) Remove root tip from germinating seed and place in 3% aqueous methanol for three hours. (2) Pour solution from vial and replace with a mixture of 65 volumes methanol, 5 of chloroform, and 30 of glacial acetic acid. Leave in fixative at least two days. (3) Remove opaque tip from root and macerate in a drop of acetic lacmoid. Cover, heat to near boiling, flatten by pressing slide, cover down, onto a thick blotter.