On the Use of Basic Dyes for the Demonstration of the Hydrolysis of Fat

of the salts of Nile blue and of several other dyes of the oxazine series have the property of producing double staining in fat-containing tissue sections. Neutral fat is stained red and fatty acids blue or bluish purple. The chemistry of the process has been elucidated by Thorpe (1907) who showed that the solution of a Nile blue salt may contain two red substances which are insoluble in water but soluble in alcohol, fat solvents and fat. These red substances are the free oxazine base of Nile blue and an oxazone formed from the oxazine base by oxidation. The oxazone behaves toward fats and fatty acids like the indifferent azo dyes. It stains both red by dissolving in them. The oxazine base, however, behaves in the same way toward neutral fat, but combines with free fatty acids to reproduce a blue salt, a Nile blue soap. Since then, Nile blue has rendered valuable services in histology, cytology and general bacteriology as a reagent for fat and as an indicator of fat hydrolysis. Used in conjunction with members of the Sudan series, it can be used to differentiate lipoids, neutral fats and free fatty acids from one another. In demonstrating lipolysis by microorganisms, it has been the custom to shake an agar medium containing a small concentration of Nile blue sulfate or chloride with a certain amount of the neutral fat to be tested. The microorganisms are then either plated in this medium or grown on poured plates in the form of a streak or of a giant colony. Hydrolysis is indicated by the color