Chromatography and Biological Stains IV. Preparation of a Suitable Fat Stain from Commercial Sudan III

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Abstract
A variety of modifications of the Bensley and Bensley method for the preparation of a fat-staining solution from commercial Sudan III were examined for dye content by paper chromatography and for fat staining efficiency on frozen sections of Musca domestica larvae. These modifications have led us to believe that the most effective staining agent in commercial Sudan III is a bright orange compound with an Rf value of 0.92 in an isoöctane system. This compound could be obtained in good yields by reflux extraction of the commercial dye widi dry petroleum ether (1 g. dye to 100 ml. solvent) for 2 hours, filtering, concentrating to 25 ml., and adding the concentrated solution to a 25 × 200 mm. Celite-silicic acid column. The chromatogram was then developed with petroleum ether for 3 hours, dried by suction and extruded. The orange band was sectioned, air dried and stored. One gram of this dry powder extracted with 10 ml. of 40% alcohol gave an efficient staining solution.