h sirnplc technique is described for diffe-ntial sraining of human chromosomes with Giemsa. T h e procedure involvcs DNA denaturation with a methanol-acetic acid fixative, and subsequent annealing using a saline solution. This rechnique has a number of advantages over quiilacrinc fluorcscmcc, and gives a mere distinct hnding pattern. It is a rapid, inexpensive proccdurc and can h done wirlt existing equipment and material in most cytogcnetic laboratories.The ability to localize defined segments of DNA in chromosome preparations following denaturation and annealing was recently reported by Jones (1970). A few months later, Pardue and Gall (1970) demonstrated that Giernsa stain binds preferentially to the annealed DNA regions in mouse chromosomes. Similar experimenrs have demonstrated Giemsa staining of heterochromatic areas on the human chromosomes complement (Sumner et al., 1971; Arrighi and Hsu, 1971), and a review of the work in this area has been published in a Nature New Biology Editorial (1971).The purpose af this study was to perfect a simple technique for identifying homo1ogous chromosomes more readily and using the existing material in our laboratory. The procedure was as follows: 1. Micromethod leucocyte cultures were initiated using Chromosome Medium 1A (Grand Island Biological Co., Grand Island, NY 14072). The cells were harvested following a 3-hr exposure to one drop of a 0.00125 mg/rnl colchicine solution, which was added to the cultures after a 68-to 70-hr incubation period. The chromosome preparations were made following the usual procedures of treatment %with a hypotonic solution (0.075 M KCL) and fixative (methanol: acetic acid, 3: 1). If the slides were not to be stained the same day they were prepared, they were refrigerated.2. The slides were immersed in a 0.3 M Na C1 and 0.03 M tri-sodium citrate solution for 60 min kept at 60°C in a water bath, followed by a brief rinse in distilled water.3. The slides were stained in a Giemsa staining solunon'for 30 min. The solution was prepared as follow: SO mI of hnffer (56 rnl of 0.15 M Na, HPO* and 104 rnl of 0.15 M KHz PO,; pH 6.R), 30 rnl distilled water, and 1 ml of commerciaE Giemsa stain (Will Corp.).4. After staining, the slides were washed briefly in distilled water, air dried, immersed in x y l m for 5 min, and mounted in Permount.
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