VITAMIN A 257 



following details of procedure in this extraction have been found con- 

 venient and effective : 



Place 200 grams of fine-grained, air-dry, high grade casein in a 

 2-liter Pyrex flask with 500 cubic centimeters of 95 per cent alcohol, 

 boil for one hour on a steam bath under a reflux condenser and then 

 transfer the contents of the flask rapidly without cooling to a suction 

 filter in a Biichner funnel of about 15 centimeters diameter. (A tinc- 

 ture press may be used when working with larger amounts.) After 

 thorough removal of the hot alcohol, return the casein to the flask and 

 add 500 cubic centimeters of fresh 95 per cent alcohol, boil and filter 

 as indicated above ; then repeat the process again. In this way the casein 

 receives three successive one-hour extractions with boiling alcohol. 

 Finally it is dried in the air at room temperature. 



Numerous feeding experiments showed that the casein thus ex- 

 tracted was free from any trace of vitamin A detectable by present 

 methods and was indistinguishable in this respect from other portions 

 of casein which had been subjected to still longer extraction with hot 

 alcohol. 



Cornstarch is used as the chief source of energy. 



Dried brewery yeast is used as the source of vitamins B and G in 

 the basal diet. Since Osborne and Mendel (1921) have fehown that such 

 yeast, even when fed to the extent of 42.5 per cent of the total food 

 consumed, does not furnish any detectable amount of vitamin A, it 

 seemed safe to assume that in feeding about one-fourth of the quan- 

 tities fed by them no appreciable amount of vitamin A would be 

 introduced into the basal diet. This has been confirmed by many years' 

 experience with the use of basal diets of the type here described. How- 

 ever, each laboratory should carefully test the yeast that it uses. 



Mineral elements are supplied by the use of the complete salt mix- 

 ture prepared according to the directions of Osborne and Mendel 

 (1919). Additional sodium chloride to the extent of 1 per cent of the 

 dry weight of the food mixture is also included in the basal diet, as 

 this seemed to result in the diet being somewhat more uniformly well- 

 relished by the experimental animals of our colony. 



As a source of the antirachitic factor, direct irradiation * of the 

 animal body or the inclusion in the diet of irradiated ergosterol in some 

 form appears to be equally effective. We use an irradiated preparation 

 free from vitamin A and of tested antirachitic potency. 



Fat, other than the small amount contained in the yeast, is not in- 



• For convenience we use the simple term "irradiation" meaning, in this connection, ex- 

 posure to the rays of the mercury.-vapor quartz lamp or other suitable source of ultra-violet 

 rays. The relation of wave-lengths to antirachitic effects is discussed in the following chapter. 



