SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR 



brilliant yellow colour of the liver, and, teasing some of it out, ex- 

 amined it between the crossed Nicol prisms of a polarising micro- 

 scope. The result was a very large number of droplets showing double 

 refractivity. On adding Sudan III, Hanes observed that the droplets 

 stained a deep yellowish red, from which he concluded that the chick's 

 liver contains a great deal of cholesterol esters towards the end of 

 incubation. This led him to make a histochemical study of the liver 

 throughout the incubation period. As the liver grows, he said, it 

 assumes more and more the colour of the yolk. The liver cells of the 

 6th-day chick contain numerous small fatty globules, but under the 

 polarising microscope these are isotropic. The globules gradually 

 increase in size and number, and about the 14th day of incubation 

 many of them begin to show double refraction. By the time of hatching 

 the liver is very rich indeed in fat droplets, recalling Imrie & Graham's 

 work on guinea-pig livers. Hanes then availed himself of Kawamura's 

 critical study of the differential histochemical methods for identifying 

 the various fatty substances, and applied them to the chick livers. 

 His results were as follows : 



Nicol prisms ... 



Sudan III 



Nile blue sulphate 



Smith's stain ... 



Ciaccio's stain 



Fischler's stain 



Neutral red ... 



Salkovski's test for cholesterol 



Liver from 

 6th to 14th day 



Isotropic 

 Yellowish red 

 Faintly blue 

 Dark bluish black 

 Positive 

 Negative 

 Negative 

 Faintly positive 



Liver from 

 14th to 2 1st day 



Anisotropic 



Yellowish red 



Uncoloured or faintly pink 



Faintly bluish grey or uncoloured 



Negative 



Negative 



Negative 



Strongly positive 



He concluded from these facts that the fat present in the chick 

 liver up to the 14th day of incubation is not pure neutral fat, nor 

 at that time are any soaps or free fatty acids present. This agrees 

 exactly with Mayer & Schaeffer's ratio mentioned above (p. 12 12). 

 Moreover, Smith's stain, which does not colour neutral fats or 

 cholesterol esters, and does colour lecithin-like substances, is very 

 positive in the early stages. The same remarks apply to Ciaccio's 

 method. As incubation proceeds, the droplets in the liver gradually 

 cease to react with Smith's stain, and by 4 or 5 days after hatching this 

 method will not colour them at all. Ciaccio's stain is likewise negative 

 during the last week of incubation, and after hatching. But it is 

 precisely during this time that they give the reactions characteristic 

 of cholesterol esters. Thus, if warmed to about 40°, they lose their 



