570 



METHODS AND FORMULAS 



MS 33.0 



the beaker and add to it 2.4 grams of 

 reagent-grade silver nitrate. When the 

 solution is complete add one milliliter of a 

 40% solution of sodium hydroxide pre- 

 pared from analytical reagent-grade NaOH 

 dissolved in triple-distilled water. Mix 

 thoroughly until the curdlike precipitate 

 is uniformly distributed through the mass. 

 From the buret add reagent-grade am- 

 monia to this curdy material, stirring 

 vigorously after the addition of each drop. 

 As soon as the curdy precipitate is seen 

 to be clearing up, add one drop about 

 every ten seconds, stirring vigorously be- 

 tween additions. It is as well to put the 

 solution in a good light against a black 

 background so as to detect the exact 

 moment at which the last of the precipi- 

 tate has vanished. It is fatal to the tech- 

 nique to add too much ammonia, but it 

 does not particularly interfere with the 

 stain if a slight excess of the precipitated 

 silver hydroxide is present. Some authors 

 have accordingly recommended that after 

 the end point of the reaction has been 

 reached (that is, when the precipitate has 

 been exactly redissolved) one or two drops 

 of a very weak solution of silver nitrate 

 should be added so that the faintest 

 opalescence is produced. The solution is 

 then transferred to a graduated flask and 

 made up to a total volume of 250 milli- 

 liters with triple-distilled water. This is 

 the stock solution which in the present 

 technique is diluted 40 stock to 60 triple- 

 distilled water before use. 



This staining solution is used after pre- 

 treatment with 3% silver nitrate, immer- 

 sion in which is the next step. This 3% 

 silver nitrate should be in a chemically 

 clean stoppered bottle. To it the well- 

 washed pieces of tongue are removed from 

 triple-distilled water, and there incubated 

 at 36°C. for three days. This reaction is 

 best conducted in the dark, and like reac- 

 tions in all other silver techniques must be 

 watched closely to make sure that no im- 

 purities present in the solution or in the 

 bottle cause a precipitate of metallic silver 

 or of silver salts. If the least cloudiness is 

 seen in the solution, or if a black precipi- 

 tate appears to be accumulating on the 

 bottom, the pieces should immediately be 

 removed to a fresh solution. 



After three days, each piece of tongue is 

 removed from the silver nitrate solution 

 and rinsed briefly in a large volume of 

 triple-distilled water. The object of this 

 rinse is not so much to remove silver ni- 

 trate from the interior of the material as 

 to prevent carrying over excess silver ni- 

 trate solution into the silver stain. When 

 the pieces have been freed from the sur- 

 face-adherent silver nitrate solution, they 

 are transferred to the diluted staining so- 

 lution, where they remain at room temper- 

 ature for 24 hours. They are then washed 

 in running distilled water for at least 24 

 hours, or in three changes of large vol- 

 umes of triple-distilled water changed at 

 intervals of about 12 hours, before being 

 placed in a fresh batch of 8% neutralized 

 formaldehyde. 



The washing which precedes immersion 

 in formaldehyde is one of the most critical 

 steps of the entire technique, for if silver 

 stain or silver nitrate is carried over into 

 the reducing solution, the preparation will 

 be spoiled through a general deposition of 

 silver proteinates. 



After remaining in the neutral formalde- 

 hyde overnight, the pieces are washed 

 thoroughly in tap water. Sections are then 

 prepared by the ordinary parafhn tech- 

 nique. When deaUng with materials as 

 strongly muscularized as is tongue, how- 

 ever, it is desirable to use benzene as a 

 clearing agent in order to prevent the mus- 

 cle from becoming brittle and thus inter- 

 fering with subsequent sectioning. After 

 the sections have been mounted on a sUde, 

 they are passed down to water, thoroughly 

 washed, and then examined under a high 

 power of the microscope. 



If the sections clearly display the nerve 

 endings in the taste buds, they may be 

 again upgraded through the successive 

 strengths of alcohol, dehydrated, and 

 mounted in balsam. If, as is frequently 

 the case, the nerve endings are only lightly 

 stained, the sections placed in a very weak 

 solution of gold chloride (0.004%) until 

 the pale brown color of the nervous mate- 

 rial has been replaced by the dark purple 

 of gold. After this treatment the sections 

 are rinsed for a few minutes in 5 % sodium 

 thiosulfate to remove any residual silver 

 which has not been replaced by the gold, 



