CHROMATOLYTK^ CHANGES DURING HIBERNATION 395 



All the animals of both series were killed quickly by trans- 

 fixing the heart through the chest wall. No anaesthetic was 

 used, exce])t in five cases where only suflficient ethei- was given to 

 keep the animal quiet. These five animals were all killed after 

 hibernation while awake and active. The amount of ether 

 given did not seem to have any noticeable effect on the Nissl 

 granules. If, however, these five cases are excluded from con- 

 sideration because of the introduction of this additional factor, 

 the two series involve as strictly comjjarable cases two before 

 hibernation, nhie during hibernation and five after hiliernation. 

 The blood was washed out immediateh' after deatli with normal 

 saline solution by injection through the aorta. The saline was 

 followed by a satui'ated acjueous solution of bichloride of moi- 

 cury to which had been added 10 ]ier cent of formalin. Tluis 

 the central nei'vous system was fixed \-ery (juickly in situ. The 

 whole brain and cord were then removed and cut into transverse 

 secti(ms a few millimeters thick. The desired levels were further 

 fixed in a saturated aciueous solution of bichloride of mercuiy for 

 48 hours, washed in running water 36 hours, and dehydrated in 

 graded alcohols containing iodine in the usual manner. The 

 tissue was cleared in xylol and imbedded in ])araffin melting at 

 54°C. The levels thus imlx'ddcd were: olfactory bulb, motor 

 cortex, mid-thalamiis, midbrain at the level of the superior 

 colliculus, mid-cerebelhmi and ])()ns. nu'dulln oblongata at ex- 

 treme inferior border of fouith Ncntiicle. lijst cei'vical, sixth 

 cervical, sixth thoracic, second lumbar and lowei- Imnbai- seg- 

 ments of the spinal cord. 



Sections were cut five microns in thickness, except in the case 

 of the spinal cord where the sections were six mici'ons thick, 

 and stainetl on the slide for ten minutes in a large quantity of hot 

 (70°C.) solution of 1 per cent methylene blue in water saturated 

 with aniline oil. The excess stain was washed off rapidly in 

 water and decolorization carried on by transferring the slides 

 directly to 95 per cent alcohol for two to ten minutes. Dehy- 

 dration was completed in absolute alcohol and clearing in caju- 

 put oil followed by xylol. The sections were mounted perma- 

 nently in Canada balsam dissolved in xylol. Where the size 



