282 FATTY SUBSTANCES 



2. The Schultz test for cholesterol whether in the free or ester 

 form. 



THE SUDAN METHOD 



657. Principle. Sudan colours fat by dissolving in it and the 

 process of staining consists in the passage of dye from one solvent 

 (usually alcohol) to another (fat) in which it is more soluble. 

 Little is known of the various factors that may influence this 

 passage. 



658. Composition of Commercial Sudans. The dye sold under 

 the name Sudan III is reputed to be benzene-azo-benzene-azo-^- 

 naphthol. The dyes sold under the names Sudan IV, scarlet or 

 scarlet R (and corresponding words in foreign languages), and 

 Fettponceau, are reputed to be toluene-azo-toluene-azo-^- 

 naphthol. The powders supplied under these various names 

 differ in both their chemical and staining properties. Sehrt (1927) 

 noticed that different samples of the same brand varied in quality. 

 Romeis (1927) stated that commercial Sudan III contained three 

 dvestuffs, which he termed Sudan red, Sudan orange, and Sudan 

 yellow ; their chemical nature was not determined. Kay and 

 Whitehead (1934) studied three brands of Sudan III, referred to 

 as A, B and C. A furnished excellent fat stains ; later investiga- 

 tion suggests that it was true Sudan IV. B, a widely used German 

 brand, gave fair results ; it was a mixture of dyes only a small 

 proportion of which resembled true Sudan III. C was useless as a 

 fat stain ; it was probably true Sudan III. Kay and Whitehead 

 (1934) found one brand of scarlet R useless for fat staining 

 owing to its ready solubility in water. According to common 

 experience alcoholic solutions of Sudan III are brown or brownish- 

 red ; Daddi (1896), who introduced Sudan III as a fat stain, 

 described its alcoholic solution as scarlet, a word more nearly 

 descriptive of alcoholic solutions of true Sudan IV. True Sudan IV 

 is undoubtedly superior to Sudan III because the red colour it 

 imparts to fatty substances contrasts more sharply with the 

 background of a section than the yellow, orange, or brown of 

 Sudan III. 



659. Solvents for Sudan. The usual solvent is 70 per cent, 

 ethyl alcohol. This has the disadvantage of extracting some of the 

 fat, and various other solvents have therefore been tried. 

 Romeis (1927, 1929) devised a technique in which the dye was 

 dissolved in 40 per cent, alcohol. Objections were raised by 

 Froboese and Sprohnle (1928), but Kaufmann and Lehmann (1929) 

 concluded that the Romeis technique was the best available. 

 Neither the technique nor the interesting discussion it provoked 

 can, however, be regarded as more than suggestive because the 

 chemical nature of the dyestuff used by Romeis was unknown. 



According to Gross (1930) the loss of fat inevitable with alcoholic 



