400 



METHODS AND FORMULAS 



DS 21.20 



removed piecemeal and the process re- 

 peated on the other side of the brain. The 

 membranes are now dissected away from 

 the freshly exposed brain, preferably with 

 forceps rather than with a knife. A sharp 

 scalpel is then used to remove pieces of the 

 cerebral cortex of about three- or four- 

 milUmeter side. As these pieces will in- 

 evitably adhere to the knife, it is not pos- 

 sible to transfer them directly to the 6% 

 mercuric chloride, which is used as a fixa- 

 tive, because this reagent will destroy the 

 surface of the blade. They are, therefore, 

 best washed from the knife into a tube of 

 normal saline, which is poured off as soon 

 as they have sunk to the bottom and re- 

 placed with 6% mercuric chloride. The 

 tube of fixative with its contained piece of 

 brain is then tipped into a large vessel (at 

 least 500 milliliters) of 6% mercuric chlo- 

 ride, which is agitated gently at intervals 

 for the next five hours. When the pieces 

 have thus been fixed, the mercuric chloride 

 is poured off and the brain pieces, without 

 handling, are tipped with the last of the 

 solution into a vial of about 20-milliliter 

 capacity. The last of the mercuric chloride 

 is then poured off and replaced with 

 Galescu's osmic-mercuric-chromic-acetic 

 fixative (Chapter 17, F 1360.0010 Galescu 

 1918) which serves both to harden them 

 completely and to render insoluble certain 

 of the fatty substances present. This solu- 

 tion is used at a temperature of 37°C., or 

 such approximation of this temperature as 

 can be maintained in an available oven, 

 for about 12 hours. A convenient time 

 schedule is to kill the rabbit in the morn- 

 ing, to commence the fixation in mercuric 

 chloride immediately, to transfer the 

 pieces to the osmic-mercuric-chromic- 

 acetic mixture in the evening, and con- 

 tinue the process the next day. The next 

 morning the osmic-mercuric-chromic-ace- 

 tic mixture is poured off, replaced with a 

 fresh solution, and returned to the oven. 

 In the evening the solution is again re- 

 placed, and this time the specimens are 

 left in the oven for a whole day. The next 

 evening, therefore, the pieces are trans- 

 ferred to running water for an overnight 

 wash. 



It is an integral part of Galescu's process 

 that alcohol not be used in any stage of 

 the technique, and the pieces are therefore 



next treated with an acetone-iodine solu- 

 tion to remove the mercury from the tis- 

 sues. This solution is prepared by adding 

 one milhhter of Lugol's iodine (Chapter 

 19, ADS 12.2 Lugol) to 100 milhhters of 

 acetone. The specimens are treated for 24 

 hours, and about 100 millihters should be 

 used for half a dozen small pieces of brain. 

 The bottle containing the pieces should be 

 gently tipped from side to side at intervals 

 to prevent the accumulation of depleted 

 solution at the bottom. 



The specimens are then dehydrated in 

 pure acetone until no further iodine comes 

 away, and embedded in paraffin, prefer- 

 ably by the dioxane techniques. A high- 

 melting-point paraffin should be used, 

 since Galescu's process depends on cutting 

 very tliin sections, 5 microns being about 

 as thick as can be conveniently handled, 

 and three microns being very much better 

 if the skill of the technician permits. 



The sections are mounted on a slide, de- 

 waxed ^\•ith xylene, and then transferred 

 to acetone for the removal of the xylene. 

 They are next placed in Galescu's crystal 

 violet-oxalic acid stain (DS 21.22 Galescu 

 1908) for ten minutes, or until examina- 

 tion shows they have acquired a dense 

 violet color. They are then transferred to 

 a beaker of fresh Galescu's stain, and 

 warmed gently on a hot plate, or in a 

 water bath, for about five minutes or until 

 the temperature has reached about 50°C. 

 Each slide is then removed individualh', 

 drained, and flooded from a drop bottle 

 with Lugol's iodine. They should under no 

 circumstances be washed between their 

 removal from the staining solution and 

 their treatment with iodine. The iodine is 

 in its turn drained from the slide and the 

 section blotted gently with a rather stiff 

 grade of filter paper. Each slide is then 

 transferred to a mixture of equal parts of 

 xj^lene and aniline, in which it remains 

 until examination under the high power of 

 a microscope shows that the required 

 neuroglial structures are clearly differ- 

 entiated. This may take from 5 minutes 

 to 2 hours, according to the treatment 

 which the pieces have previously received. 

 As soon as they are found to be differ- 

 entiated, the xylene-aniline mixture is 

 washed off with xylene and the sections 

 mounted in balsam. 



