Orcein staining and the identification of polytene chromosomes

Acetic orcein staining of polytene chromosomes was introduced in 1941 shortly after the initial studies on aceto-carmine-stained chromosomes by Bridges (2) and has remained a standard method of preparation. Orcein dye can be purchased in both its natural form as extracted from two species of lichens, Rocella tinctoria and Lecanora parella, and a synthetic form. The mechanism of staining is not clearly understood because the stain itself is a variety of phenazones, which may interact at an acid pH with negatively charged groups or possibly interact hydrophobically with chromatin. Acetic acid fixation accommodates stretching of the chromosomes in the interband regions during a squash, thus providing for a higher resolution of the banding structure. The later addition of lactic acid to aceto-orcein (3) kept the glands softer in the fix and allowed for easier spreading of chromosomes. The method and its variations have appeared more recently in several publications (4,5). Drosophila polytene chromosomes are found in a number of larval tissues, including the midgut, hindgut, and the fat body, but the largest chromosomes are found in the salivary glands of the third instar. They are referred to as interphase chromosomes and are structurally more comparable to highly amplified interphase chromatin than to mitotic chromosomes because the gland grows by endoreplication of DNA, thus increasing cell size rather than cell number. Each of the homologs is tightly synapsed in this somatic tissue and undergoes approx 10 rounds of endoreplication, producing 1024 chromatids closely associated in parallel arrays.