Double Staining for Comparative Measurements in Squash Preparations

Abstract

Comparative measurements of nuclei or chromosomes following different treatments are seldom made on squash preparations, since variations which arise during preparation of the slides may easily mask genuine treatment differences. This drawback may be overcome by making use of dyes which, when substituted for basic fuchsin in Schiff's reagent, will give a Feulgen-type reaction with chromatin. By selecting dyes of contrasting colours, it is possible to intermingle cells from different treatments in the same squash preparation, and to perform comparative measurements on adjacent cells.

Suitable dyes which contrast well with basic fuchsin are toluidine blue, or azure A (which stain chromatin blue) and chrysoidin yellow (which stains chromatin yellow). These dyes are made up and used in the same manner as ordinary Feulgen reagent.

Samples of cells from the two treatments to be compared are fixed, washed and hydrolysed in 1 N HCl at 60 C. One sample is stained in regular Feulgen reagent, the other in the contrast dye, then both are macerated and thoroughly mixed on the same slide in a single drop of 45% acetic acid. A coverslip is added, and the preparation flattened to the required amount and made permanent after dry-ice removal of the cover. This technique may also be utilised for comparative grain counts in autoradiography, provided that the contrast dye does not cause chemical fogging of the film.