398 



METHODS AND FORMULAS 



DS 21.20 



a little at a time, with brisk stirring be- 

 tween each addition. When the whole 

 two grams have been added, cool the solu- 

 tion, leave overnight, and filter. This 

 primary mordant of Weigert may be kept 

 indefinitely. When required for use in the 

 method of Anderson, convert it to Ander- 

 son's mordant (ADS 12.1 Anderson 1922) 

 by adding 10 milhhters of a 2% solution 

 of calcium hypochlorite to 90 miUihters 

 of primary mordant; this mixed solution 

 is not stable and must be prepared im- 

 mediately before use. Weigert's primary 

 mordant, as such, is also required in the 

 technique. Anderson's hematoxylin stain 

 (DS 21.212 Anderson 1922) is easily pre- 

 pared. Take 10 milhhters of absolute 

 alcohol and dissolve in it 0.5 gram of hema- 

 toxylin. When the hematoxylin is in solu- 

 tion, add three milhhters of 2% calcium 

 hypochlorite and then shake the vessel 

 vigorously for about two minutes. Filter 

 the mixture and make the filtrate up to 

 100 milhliters before adding three milh- 

 hters of acetic acid; the solution is then 

 ready for use. The technique also calls for 

 the dichromate-sodium sulfate fixative of 

 Muller (Chapter 18, F 7000.0000 Muller 

 1859). Differentiation requires the differ- 

 entiating solutions of Pal (Chapter 22, 

 ADS 21.1 Pal 1887). These are a freshly 

 prepared 0.25% solution of potassium 

 permanganate, and a solution containing 

 0.5% each of potassium sulfite and oxahc 

 acid. Both solutions are relatively un- 

 stable and should be prepared immedi- 

 ately before use. 



With these solutions on hand for the 

 staining process, it is necessary to con- 

 sider the fixative which will be used for 

 the spinal cord. Opinions are widely di- 

 vergent as to the best fixative to employ, 

 the majority of authors preferring merely 

 to use a 5 % solution of potassium dichro- 

 mate. Kultschitsky 1898 (766, 4:223) 

 recommends the cupric-dichromate mix- 

 ture of Erhtzky (F 4700.0000 Erhtzky 

 1877). The writer does not consider the 

 cupric addition necessary, since sufficient 

 copper mordanting occurs in Weigert's 

 primary mordant. 



Having selected the fixative, it remains 

 only to secure the spinal cord, which may 

 be taken from any animal available to the 



investigator. Though the rabbit is widely 

 used, it is somewhat small for demonstra- 

 tion purposes. If access can be had to a 

 slaughterhouse it is very much better to 

 secure a piece of the spinal cord of a pig, 

 though this latter is almost invariably 

 destroyed in the process of pithing with 

 which the commercial killing of pigs is ac- 

 companied. It does not matter in the pres- 

 ent instance that considerable time should 

 elapse between the killing of the animal 

 and the fixation of the cord, since post- 

 mortem changes in the myelin sheaths, 

 which are shown by this process, are gen- 

 erally very slow. Whatever cord is finally 

 selected should be cut into pieces about 

 twice as long as they are broad, and sus- 

 pended in a loosely woven cloth bag in a 

 very large volume of the fixing solution. 

 If, for example, 5 % potassium dichromate 

 has been selected, it is by no means un- 

 reasonable to use a liter of the solution for 

 three or four half-inch lengths of the 

 spinal cord of the pig. The time of fixation 

 does not matter and should be determined 

 by the physical condition of the spinal 

 cord. A simple method of judging fixation 

 in the dichromate (which will not be ap- 

 plicable to the copper-dichromate mix- 

 ture) was suggested by Pal 1887 (23632, 

 6:92), who took pieces from the fixative 

 from time to time and cut them with a 

 sharp knife in order to view the surface. 

 If they are underfixed, the white matter 

 of the cord will be lighter than the gray 

 matter; fixation and hardening are com- 

 plete when the white matter is a darker 

 brown than the .gray matter. Overfixation 

 is not particularly harmful but underfixa- 

 tion will result in unsuccessful prepara- 

 tions. When fixation is complete the speci- 

 mens are washed in running water until 

 no further dichromate comes away, and 

 may then be stored in 70 % alcohol in the 

 dark. If they are to be used immediately 

 they must be dehydrated and embedded 

 in celloidin (Chapter 18) before being cut 

 into sections of from 15 to 20 microns in 

 thickness by any of the techniques there 

 given. 



The sections are accumulated in dis- 

 tilled water, and when a sufficient number 

 have been obtained, are transferred to 

 Anderson's mordant (about 50 millihters 



