268 Chemical Differentiation of the Brain 



MATERIAL AND METHODS. 



The rat material was obtained from the Wistar Institute of 

 Anatomy, which supplied the brains of rats of known age, from 

 animals which had been raised under constant conditions; two 

 factors which are absolutely essential for such a study. The 

 material was collected by Dr. Hatai and the method used was that 

 adopted by the Institute. This in brief is as follows : the rat was 

 chloroformed; the skull opened from the dorsal side; the division 

 between the brain and the cord made at the tip of calamus scrip- 

 torius; and the brain removed. The meninges of the brain were 

 left intact. Such blood as it contained was, therefore, included 

 in the weight. Immediately after removal the brain was placed 

 in a closed weighing bottle, quickly weighed to within 10 mgms. 2 

 and transferred to a wide mouthed bottle of 300 cc. capacity con- 

 taining absolute alcohol. The weighing bottle was weighed back 

 and the difference recorded as the weight of the sample. As the 

 weight of one brain from rats at this early age is 0.2 0.3 gram 

 and as it takes at least from 25 to 50 grams to make one sample 

 for analysis, a large number of brains had to be collected (100 

 brains of the rat at birth for one 25-gram sample). As this cov- 

 ered a period of several weeks it was necessary to heat the sample 

 from time to time in a water bath kept at a temperature of 70C. 

 to insure a thorough penetration of the alcohol and sterilization. 

 The amount of alcohol was so adjusted as to make the final concen- 

 tration not less than 80-85 per cent. A well fitting cork stopper 

 covered with tin-foil was now inserted and the bottle carefully 

 shaken to insure a uniform mixture. The dates of collection of the 

 samples were recorded, as the time a sample has been kept in some 

 cases influences the analytical results. 3 The tightly corked bottles 

 were then shipped to the Laboratory of Biochemistry of the Uni- 

 versity of Chicago, where the samples were analyzed according to 

 Koch's methods of tissue analysis. 4 



2 A coarse weighing of the brain was permissible in this instance as it was 

 not the exact brain weight that was sought but merely data for indicating 

 roughly when the required amount of material had been obtained. 



3 W. Koch: Methods for the Quantitative Chemical Analysis of Animal 

 Tissue, Journ. of the Amer. Chem. Soc., xxxi, p. 1340, 1909. 



4 Ibid., pp. 1329-64. 



