MS 33.0 



METAL STAINS 



571 



washed thoroughly in either tap water or 

 distilled water, dehydrated, and mounted 

 in balsam. 



Demonstration of oligodendria and 



microglia by the method 



of Penfield 1938 



This is one of the most complex of the 

 silver staining techniques, hut tlie com- 

 plexities are justified by tlie relative cer- 

 tainty with which results may be obtained. 

 A relatively large number of reagents are 

 required and it is best to make sure that 

 all of these are available before starting 

 the technique. These reagents are de- 

 scribed below in the order in which they 

 are required. 



First is Cajal 1913 formol-bromide solu- 

 tion (AMS 11.1 Cajal 1913— Chapter 24). 

 This is made by adding 38 c. milUhters of 

 neutrahzed 40% formaldehyde to 212 

 milliUters of triple-distilled water. The 

 formaldehyde must be of reagent grade 

 and should have been neutralized with re- 

 agent-grade borax. The term neutral refers 

 in this instance to any pH between about 

 7 and 7.6. Five grams of reagent-grade 

 ammonium bromide are then added. This 

 solution is stable indefinitely. 



The next three reagents required are a 

 1 % dilution of ammonia, 2% hydrobromic 

 acid, and 5% sodium carbonate. Both the 

 acid and the carbonate must be of the 

 finest grade available; the latter in par- 

 ticular must be chloride-free. 



The silver stain used is a dilution of del 

 Rlo-Hortega 1921 silver carbonate (MS 

 33.1 del Rio-Hortega 1921). This is pre- 

 pared from a 10% solution of pure silver 

 nitrate in triple-distilled water, a 5% solu- 

 tion of reagent-grade sodium carbonate 

 (also in triple-distilled water), and am- 

 monia. Place 30 milliUters of 10% silver 

 nitrate in a 250-miUihter beaker and add 

 to this, with constant agitation, 120 milli- 

 liters of sodium carbonate solution. The 

 solution is now allowed to stand until the 

 silver carbonate has fallen to the bottom. 

 Then as much as possible of the supernatant 

 Uquid is poured from the top. The beaker 

 is then filled with a fresh batch of triple- 

 distilled water, thoroughly agitated, and 

 again allowed to settle. The supernatant 



liquid is poured off and the process re- 

 peated about three times. The precipitate 

 may alternatively be accumulated on a 

 chemically clean filter paper on chemically 

 clean glassware and washed by passing 

 considerable volumes of triple-distilled 

 water through it. Whatever method may 

 be adopted, the silver carbonate is col- 

 lected in approximately 100 milliliters of 

 triple-distilled water; and reagent-grade 

 ammonia is added drop by droj), with agi- 

 tation between drops, until the carbonate 

 is just dissolved. It is essential that the 

 ammonia should not be in excess. At the 

 moment when the precipitate is seen to be 

 clearing up, the rate of addition of am- 

 monia should be reduced to one drop 

 every 10 seconds. The beaker should be 

 observed in a good light against a black 

 background. When the carbonate is 

 dissolved, make up the solution with 

 triple-distilled water to a volume of 240 

 milliliters. For purposes of the present 

 technique, this stain is diluted 56 of the 

 solution just described to 44 of triple- 

 distilled water. 



One requires also a 0.4% neutrahzed 

 formaldehyde solution (that is, one milli- 

 Uter of neutralized formaldehyde diluted 

 with 99 milhhters of water), a 0.2% solu- 

 tion of gold chloride, and the usual 5% 

 solution of sodium thiosulfate. All these 

 reagents except the diluted stain are sta- 

 ble. The latter may usually be kept some 

 weeks in a well-stoppered bottle, prefer- 

 ably in the dark. 



Since the method of Penfield is designed 

 to show both oligodendria and microglia, 

 considerable care must be taken to make 

 sure that the former are present in a nor- 

 mal condition in the brain at the time it is 

 fixed. Ohgodendria (McClung 1929, 362) 

 will be distorted almost beyond recogni- 

 tion if the death of the animal from which 

 they are taken is preceded by coma or 

 deep stupor. Under these circumstances it 

 is best to kill the rabbit used for the prepa- 

 ration by a sharp blow on the occipital 

 region rather than by the more conven- 

 tional method of chloroform or ether. 



Having killed the rabbit, fasten it face 

 down on a convenient dissecting board, 

 skin the head, remove the parietal and 

 frontal bones with bone forceps, flood the 



