A Universal Stain for the Sex Chromatin Body

For the demonstration of the sex chromatin body in human tissues, fixation in 95% alcohol or modified Davidson's solution (95% alcohol, 30; formalin, 20; glacial acetic acid, 10; distilled water, 30) was best. The staining procedure chosen for most materials is the following: Mounted preparations are coated with celloidin, hydrated, hydrolyzed 20 min in 52V HCl at 20-25°C, rinsed thoroughly in several changes of distilled water and transferred to a buffered thionin solution. This consists of 3 parts: (1) A saturated solution of thionin in 50% alcohol (filtered); (2) Michaelis buffer: sodium acetate (3 H₂O), 9.714 gm; sodium barbiturate, 14.714 gm; CO₂-free distilled water, 500 ml; and (3) 0.1N HCl. To make the staining solution, mix 28.0 ml of the buffer solution with 32.0 ml of 0.1N HCl and bring the total volume to 100.0 ml with the thionin solution. Its pH should be 5.7 × 0.2, and care should be exercised that no acid is carried over from the hydrolyzing solution, since this would progressively lower the pH. The staining time varies from 15 to 60 min, depending on the specimen, but the shortest time consistent with adequate staining gives the clearest preparations. Slides are rinsed in distilled water and 50% alcohol and allowed to remain in 70% alcohol until the heavy clouds of stain cease to appear. Differentiation is completed in 80% and 95% alcohol, followed by dehydration in absolute alcohol, clearing in xylene and applying a cover glass with a synthetic resin (G. T. Gurr's DePeX was used). The sex chromatin is deep blue-violet and sharply contrasted against the lightly colored particulate chromatin of the nucleus. Cytoplasm remains unstained but fibrin and related structures show metachromasia. Chromosomes are well demonstrated if present. The method works on all types of tissues, is simpler and quicker than the Feulgen method, and often yields superior results.