602 



METHODS AND FORMULAS 



MS 34.0 



larger of these two ganglia, which is also 

 the one closer to the surface of the neck, is 

 the ganglion nodosum of the vagus nerve ; 

 while the smaller is the anterior (or su- 

 perior) cervical sympathetic ganglion 

 which is the one sought. This ganglion is 

 then cut out from the surrounding tissue 

 and dropped directly into the fixative, 

 where it should remain for ai)proximately 

 three days. It is difficult to gauge the 

 exact time and it might be well to remove 

 the ganglion from the other side and place 

 it in the same solution. One ganglion may 

 then be removed from solution at the end 

 of two days and the other at the end of 

 four days, with reasonable certainty that 

 one or the other will be in a condition 

 suitable for staining. When the ganglion is 

 removed from the fixing solution it should 

 be rinsed very l:)riefly in distilled water, 

 drained of distilled water on the surface of 

 filter paper, and then transferred directly 

 to the staining solution which is of course 

 kept in a chemically clean stoppered 

 bottle. It remains in the staining solution 

 for from two to three days and is then 

 placed in 90% alcohol. Sections are pre- 

 pared either in celloidin or by the freezing 

 technique and are mounted in exactly the 

 manner described for the method of Golgi 

 given in the last example. 



Demonstration of the neurons and 

 dendrites of the brain of a rabbit em- 

 bryo by the method of Windle 1926 



This is a mixed dichromate-osmic-silver 

 method, of the same general type as 

 Golgi's mixed process (MS 34.21 Golgi 

 1900) but better and more certain for em- 

 bryonic material. Only three solutions are 

 required. The first of these is a 3.5% solu- 

 tion of reagent-grade potassium dichro- 

 mate in triple-distilled water. The second 

 is Windle's 1926 osmic-dichromate fixa- 

 tive (see, in Chaiiter IS, F 1700.0000 

 Windle 1926). Tiie third is 0.75% silver 

 nitrate. The brain of a 14-day rabbit em- 

 bryo is used in the following example. At 

 least a liter of the first solution and half a 

 liter of the second will be required. 



Secure a rabbit on the fourteenth day of 

 gestation and kill it, j^referably by a blow 

 on the head. Lay the rabbit, ventral side 



uppermost, on a dissecting board, tying 

 its four legs outward so as to leave the 

 abdomen stretched. Remove the skin with 

 extreme care, washing away the milk with 

 a stream of water, and remove as many as 

 possible of the milk glands from the ab- 

 dominal surface so as to leave the mus- 

 cular layer exposed. Remove the greater 

 part of the abdominal wall, disclosing the 

 two uteri, one on each side. These will con- 

 tain a series of globular expansions, each 

 about the size of the egg of a bantam hen. 

 Two ligatures should be tied between each 

 ])air of these globular expansions and the 

 uterus cut between the Ugatures, the 

 resultant pieces being removed separately 

 to a clean dish. It will be found on exam- 

 ination that the uterus is not uniformly 

 swollen but is extended to one side more 

 than the other. Each piece is pinned down 

 with the largest expansion u])permost. 

 With a sharp knife, cut horizontally across 

 the top of the glol)ular expansion. With a 

 little i)ractice it is by this means possible 

 to remove a large area without liberating 

 much blood. Each embryo will then be 

 seen clearly and should be removed with a 

 spoon to a dish of normal saline, where it 

 is rinsed to remove as much as possible of 

 the extravasated blood before being trans- 

 ferred to a clean dish of saline for the re- 

 moval of the membranes. The embryo is 

 now placed in a bottle which contains a 

 large volume of 3.5% potassium dichro- 

 mate. On the bottom of the bottle there 

 should be a layer of fat-free cotton or fine 

 glass fiber. The bottle should be gently agi- 

 tated at intervals to prevent the accumu- 

 lation of exhausted fixative on the bottom. 

 After about two days in potassium dichro- 

 mate, the embryo will be hard enough to 

 handle with safety. It should be removed 

 to a clean dish of potassium dichromate, in 

 which the skin is carefully dissected away 

 from the upper portion of the head, the 

 rudiments of the skull removed, and the 

 entii'e brain passetl, with extreme care and 

 without any washing at all, into the osmic- 

 dichromate solution. Here it should re- 

 main three daj^s more. It is then removed 

 from the osmic-dichromate solution and 

 ])assed to another large volume of 3.5% 

 ])otassium dichromate. The purpose of 

 this is to wash out the osmic acid without 



