In the 1930s, the discovery of a simple method for the isolation and detailed microscopic observation of the banded structures that lie within the nuclei of salivary gland cells of Drosophila melanogaster was soon followed by the realization that these structures were in fact a highly amplified form of interphase chromosomes (1). The method involved squashing the salivary gland, immersed in 45% acetic acid, between a coverslip and a microscope slide. Aqueous acetic acid dissolves the cell and nuclear membranes and generally disperses cellular contents except for the chromosomes which are toughened by “acid fixation.” The acid-squashing procedure has, in general, served as the basis for the isolation of D. melanogaster polytene chromosomes for cytological study since that time. It is ideal for the rapid preparation of salivary gland chromosomes for morphological observation in the light microscope and as targets for in situ hybridization (see, however, Note 1).