DS 21.20 



DYE STAINS OF SPECIAL APPLICATION 



399 



for a couple of sections) in a glass-stop- 

 pered bottle, and kept at 37°C. The exact 

 temperature is not critical, 37° being 

 quoted because ovens at that temperature 

 are common in most biological labora- 

 tories. After three or four days at 37°C., 

 the sections are transferred, without wash- 

 ing to Weigert's primary mordant. To 

 avoid curhng, it is recommended that 

 the mordant be heated to about 20°C. and 

 allowed to cool wliile the sections are in it, 

 i.e. for a period of 10 to 30 minutes. 

 Neither of the two mordanting processes 

 are critical as to time, nor is there any 

 method of discovering what is the best 

 time for the particular batch, save by trial 

 and error. After the second mordant bath, 

 the sections are washed in several changes 

 of distilled water. Anderson's hematoxylin 

 is then raised, in a beaker or other con- 

 tainer, to a temperature of about 50°C. 

 The sections are dropped into this stain 

 and allowed to remain for one hour. Each 

 section is then taken from the stain (it 

 should be a deep purple) and transferred 

 without washing to Miiller's fixative for a 

 period of 10 to 20 minutes. This is neces- 

 sary to ensure the presence of dichromate, 

 without which differentiation cannot be 

 controlled. This dichromate bath is pe- 

 culiar to Anderson's technique; other 

 " Weigert-Pal" methods depend on the re- 

 tention of dichromate from the fixing and 

 mordanting solutions. The sections are 

 then transferred from Miiller's solution to 

 distilled water, and washed until no fur- 

 ther color comes away. If the sections are 

 now a deep blue, they may be differenti- 

 ated, but if, through some error of tech- 

 nique, they are more brown than blue, it 

 is desirable to transfer them briefly to a 

 weak (0.5%) solution of sodium bicar- 

 bonate until they acquire a deep blue 

 color. Differentiation is the most critical 

 part of the entire method, and the in- 

 experienced worker should proceed by 

 short steps rather than endeavor to con- 

 duct one operation. The sections are taken 

 one at a time and placed in the potassium 

 permanganate solution for about 30 sec- 

 onds. Each section is then removed, rinsed 

 rapidly in distilled water, placed in the 

 potassium sulfite-oxahc acid solution, and 

 watched under the microscope. After two 



or three minutes the white matter will be 

 differentiated from the gray matter, but it 

 is presumed that the differentiation will 

 not have proceeded far enough in this 

 brief period. The section is now returned 

 to the potassium permanganate solution, 

 left for a further period of 20 seconds, re- 

 moved, placed in the bleaching solution, 

 and again watched. The process should be 

 stopped just before the tracts are clearly 

 differentiated, for the differentiation will 

 continue while the sections are being 

 finally washed. This is the next stage of 

 the process. Each section, when differ- 

 entiation is complete, should be passed 

 rapidly through two or three changes of 

 water, then dehydrated, cleared, and 

 mounted in balsam in the customary 

 manner. 



Demonstration of the neuroglial cells 



of the white matter of the cerebral 



cortex using the crystal violet stain of 



Galescu 1908 



Many methods for the demonstration 

 of neurogha are described in Chapter 23. 

 The method of Galescu 1908 (DS 21.22 

 Galescu 1908) is among the more satis- 

 factory of the dye-staining techniques, and 

 has the advantage over the metal-staining 

 techniques that it requires less \agorous 

 attention to detail to secure a passable 

 result. It is not, however, a method which 

 can be recommended for research pur- 

 poses, though it might well prove a useful 

 and interesting demonstration in the 

 hands of a class in microtechnique. 



It is unimportant what animal is used, 

 though a rabbit is a convenient form from 

 which to obtain the brain. In demonstra- 

 tions of neuroglial structures the animal 

 should not be killed by anesthetics, but by 

 a sharp blow on the head, and then tied 

 face downward on a board while the skin 

 is removed from the top of the cranium. 

 The nasal bones should then be broken 

 out with a hammer and chisel, and the free 

 end of the frontal bone thus exposed 

 gripped in a pair of blunt-nosed phers. A 

 sharp upward jerk, with an inwardly di- 

 rected twist of the hand at the same time, 

 will cause the frontal bone to break away 

 cleanly without damage to the underlying 

 brain structures. The parietal bone is then 



