DS 21.20 



DYE STAINS OF SPECIAL APPLICATION 



397 



until such a time as the dry crust of the 

 salt collects around the lip of the vessel is 

 to invite a serious accident. Like all other 

 picric compounds, however, this material 

 cannot explode in solution, and is there- 

 fore perfectly safe provided that the neces- 

 sary precautions are observed. 



Now lift the brain on its bed of the 

 parasphenoid bone and place it in a con- 

 siderable quantity of the ammonium 

 picrate solution for about five minutes. It 

 should then be sufficiently hardened on 

 the outside for it to be safe to tip the bone 

 sideways, and to detach the brain from 

 the parasphenoid bone with a sharp 

 scalpel. After another five minutes in the 

 fixative, cut the brain into small pieces 

 (about two or three millimeters long, de- 

 pending upon the region which it is desired 

 to section) and return to the fixative for 

 15 to 30 minutes. 



Now transfer the small pieces, without 

 washing, to the niolybdate fixative se- 

 lected and leave for a period of from about 

 four hours to overnight. A more prolonged 

 immersion will not hurt them, but may 

 tend to make the brain tissue very brittle 

 during section cutting. As soon as the 

 small pieces have been placed in the 

 molybdate fixative, the dish containing 

 the ammonium picrate must be tluM- 

 oughly washed and the residual picrate 

 solution washed down the sink. 



On their removal from the molybdate 

 fixative the pieces are washed thoroughly 

 in running water and then embedded in 

 paraffin. Do not be alarmed that during the 

 course of dehydration in alcohol a certain 

 amount of blue will come from the pieces. 

 The blue which is Uberated during the 

 process of dehydration is that which is not 

 fixed in the nerve cells; this period of dehy- 

 dration therefore serves to differentiate as 

 well as to dehydrate the tissues. Paraffin 

 sections are now cut in the normal manner 

 and mounted on a slide, a process which 

 should present no difficulty with the pres- 

 ent material. It is recommended that sec- 

 tions of about 15 to 20 microns in thick- 

 ness be used if only the nerve cells are 

 desired, or that sections about ten mi- 

 crons thick be used if it is intended to 

 counterstain the sections to show nuclei. 



If it is only desired to show the brain 



cells and their processes, the sections may 

 l)P dewnxod, and then mounted rlircctly 

 in balsam. It is usually better, however, 

 to counterstain these sections with some 

 carmine formula, and it is conventional to 

 use for this purpose one of the alum 

 carmines given in Chapter 20 (DS 11.2). 

 The author's preference is for the formula 

 of Mayer (DS 11.21 Mayer 1892) in which 

 the sections are stained in the following 

 manner. After dewaxing, pass the sections 

 down through the customary alcohols to 

 distilled water and place them in the 

 carmine solution. This is a slow acting, 

 but very safe reagent, and the sections 

 may without danger remain in it over- 

 night. They must, however, be watched 

 to make sure that no overstaining takes 

 place, or the process of differentiation 

 may remove the blue from the nerve tis- 

 sues at the same time as it removes the 

 carmine. On removal from the carmine 

 stain the sections should be rinsed brief!}' 

 in a 5% solution of potassium alum to re- 

 move the adherent stain, then washed 

 thoroughly in tap water until they are 

 free from potassium alum. They are then 

 dehydrated, cleared, and mounted in 

 balsam. 



Preparation of a section of spinal 



cord using the " Weigert-Pal " 



stain of Anderson 1922 



All of the methods given under section 

 DS 21.212 below may loosely be referred 

 to as "Weigert-Pal" techniques, and that 

 of Anderson 1922 is selected as the e.x- 

 ample for the reason that in the author's 

 hands it has always given the most satis- 

 factory results. The principle difficulty 

 which will be encountered is the prepara- 

 tion of the staining solutions, and this 

 must first be briefly described before pass- 

 ing to a description of the technique itself. 

 First it is necessary to prepare Weigert's 

 primary mordant, the formula for which 

 will be found in Chapter 22 (ADS 12.1 

 Weigert 1896). Raise 100 milliliters of dis- 

 tilled water to the boil and throw in 5 

 grams of reagent grade potassium di- 

 chromate, and dissolve it with rapid stir- 

 ring. While the fluid is still boiling vigor- 

 ously, add 2 grams of chromium fluoride, 



