McCollum and Simmonds described a test procedure which is infallible as an indicator for the presence or absence of the anti-neuritic substance. The procedure is to restrict young rats to a diet consisting of purified protein, dextrin, a suitable salt mixture, and butter fat or other fat which contains the factor, fat-soluble A. On such a food supply the animals are unable to grow, and ultimately die with or without the development of acute symptoms characteristic of polyneuritis. After a period of two or three weeks there is added to such a diet, a substance to be tested for the so-called anti-neuritic substance. If the animals recover their normal nutritive condition and resume growth, the test is positive. If the animals continue, however, to decline the substance tested for is absent from the material under investigation or is present in amounts too small to meet the nutritive needs of the subjects of the test. It is time that those who are devoting their efforts to the isolation and identification of this most interesting substance should adopt the above described test with a mammal. Until this is done we may expect no further progress in this direction.

Unfortunately this test requires moderately large amounts of material of a nature which it is laborious to prepare. Besides a considerable period of time must elapse before the outcome of such test becomes apparent. There is no chemical test yet discovered which in any way assists us in following up the physiologically active substance in our attempts to isolate it. Funk called attention to the fact that his active preparations (tested on pigeons) never failed to give with the Folin-Macallum reagent a test for the phenol group and Voegtlin has extended the use of this reaction in his efforts to isolate the anti-neuritic substance. This reaction is given by proteins, and by a very large number of organic compounds which are regularly met with in biological work, and has, therefore, no specificity, and can hardly contribute toward progress in the further investigation of the problem of the nature of this substance.