FATTY SUBSTANCES 281 



was fulfilled if histological applications of the Liebermann- 

 Burchardt test gave constantly negativ^e results. The assumption 

 is, however, unjustiliable, as Lison himself had recognised (1933 a, 

 p. 272). 



It is impossible by any stain to establish chemical distinctions 

 between various kinds of fatty substances ; the only methods that 

 can now be recognised as differential are not stains but chemical 

 tests, and those that are generally accepted are exclusively for 

 sterols and their esters. 



654. Methods for Sterols and their Esters. The techniques of 

 Schultz (1925), Romieu (1925 a and b), and Yamasaki (1931) are 

 aj^plications of the Liebermann-Burchardt cholesterol test. 

 Applied to animal tissues they are specific for cholesterol whether 

 in the free or ester form. The only technique in general use is that 

 of Schultz. 



Larson (1933) proposed a test based on the work of Steinle and 

 Kahlenberg (1926). It depends on the formation of an addition 

 product of cholesterol with antimony pentachloride. In view of 

 the similar reactions given by antimony pentachloride with other 

 fatty substances (Steinle and Kahlenberg, 1926) it is doubtful 

 whether Larson's test is specific. 



The techniques of Brunswik (1922), Leulier and Revol (1930, 

 superseding Leulier and Noel, 1926), and Lison (1936) are applica- 

 tions of the ^Vindaus digitonin method and are specific for free 

 sterols. Although sound in principle these techniques need 

 further investigation. 



655. Value of Methods Discussed. 1. Staining with dyes of the 

 Sudan type : the best method for demonstrating fatty substances 

 in general. 



2. Eiiamination of vacuoles in jxiraffin sections : irrational and 

 misleading. 



3. Polariscopic examination : of limited histochemical and 

 morphological value only. 



4. Osmication, Ciaccio's method for " lipoids,'' Smith {Nile blue), 

 Fischler, and Smith-Dietrich methods : these have no histochemical 

 value. 



5. Techniques based on the Liebermann-Burchardt test : when 

 applied to animal tissues, specific for cholesterol whether in the 

 free or ester form : the only technique in general use is that of 

 Schultz. 



6. Techniques based on the Windaus digitonin test for free 

 sterols : specific for free cholesterol in animal tissues, but need 

 further investigation. 



7. Other methods : either need further study or have no 

 advantage over those named. 



656. Methods Recommended. The methods recommended are 

 1. Sudan staining for fatty substances in general. 



