100 THE VITAMINS 



both vitamins B and G, and designed to be not only adequate but ap- 

 proximately optimal (for growth of rats) in other respects. The diet 

 (Sherman and Spohn, 1923, as modified by Chase, 1928) consists of 

 casein (purified by extraction with 60 per cent alcohol) 18, Osborne 

 and Mendel (1919b) salt mixture 4, butterfat 8, cod-liver oil 2, auto- 

 claved bakers' yeast ^15, and starch 53 per cent. 



The casein is freed from vitamin B by cold extraction with 60 per 

 cent (by weight) alcohol. Four hundred grams of casein are treated 

 with two liters of 60 per cent alcohol and the whole stirred for one- 

 half hour, then allowed to stand 5^^ hours, filtered with suction and 

 thoroughly washed with one liter of 60 per cent alcohol. It is again 

 treated with two liters of 60 per cent alcohol, and stirred for another 

 half hour. After standing 18 hours it is filtered, washed with one liter 

 of 60 per cent alcohol and finally with one liter of 90 per cent (by 

 weight) alcohol to facilitate air drying. Each time, the casein is care- 

 fully freed from the alcoholic solution and washings. To prepare the 

 autoclaved yeast, powdered bakers' yeast is heated for 6 hours after 

 addition of 0.1 molar sodium hydroxide at 15 pounds pressure (120° 

 C), enough alkali being added to make a smooth paste (125 cubic 

 centimeters per 100 grams yeast), and neutralized after autoclaving 

 by adding equivalent amounts of standard hydrochloric acid. The prod- 

 uct is dried at room temperature before an electric fan, and finally 

 ground to an impalpable powder. 



On this diet, practically devoid of vitamin B while containing an 

 abundance of vitamin G and all other known factors required for 

 the nutrition of rats, the experimental animals usually continue to gain 

 in weight for from one to two weeks by virtue of the vitamin B stored 

 in their bodies before they were place(^ on the diet. This is therefore 

 considered a "depletion period" preliminary to the test period proper. 

 At the end of this depletion or fore period, when all gain in weight 

 has ceased, those of the experimental animals which are to serve as 

 "negative controls" are left on this basal diet only. 



Other rats of the same initial age, and so far as possible from 

 the same litters as the negative controls, are given, in addition to the 

 basal ration, graduated daily doses of the food to be tested as their 

 sole source of vitamin B. Any advantage which they show over the 

 negative controls should be attributable to the vitamin B which they 

 obtain from the weighed portions of the food under examination. If 

 the amount of vitamin B thus obtained is very small it results simply 



* The particular yeast used must be investigated as to completeness of destruction of 

 vitamin B (Bi) on autoclaving. 



