A Highly Satisfactory Vitamin B Deficient Ration. ∗

Abstract

For a number of years we have experienced considerable difficulty in securing uniform and reliable results on so-called vitamin B deficient rations. In all of our vitamin B studies we have negative as well as positive controls. Very frequently the negative control, receiving no vitamin, would continue growing for many weeks, which was quite distressing, particularly in the biological assay of concentrates which necessitated discarding appreciable amount of data, thus involving much waste of time and labor. After numerous attempts to arrive at the source of the troubles we finally discovered 2 main factors responsible for animals growing after the vitamin reserves have been depleted. First, our method of purifying casein by washing for 7 to 10 days in acidulated water is inadequate and second, subjecting dried baker's yeast to such drastic treatment as autoclaving for 8 hours at 20 pounds pressure did not always insure the destruction of all traces of vitamin B. Furthermore, such drastic autoclaving destroyed appreciable amounts of vitamin G.

The purification of commercial casein, purchased from Atterbury Bros., New York, is carried out as follows: 3 kg. of casein are suspended in 30 liters of tap water to which are added 50 cc. glacial acetic acid and 5 cc. chloroform and 5 cc. toluene as preservatives. The water, acetic acid, and preservatives are changed daily for a period of 14 days. During the last 3 days of this period distilled water is used instead of tap water. The casein is then dried at 100°C. and ground. Five kilograms of the dried acid washed casein are subsequently suspended in 25 liters of 25% ethyl alcohol, by volume, and soaked for 24 hours. During this period the casein is stirred in the alcohol for 12 hours with a mechanical stirrer.