MS 33.0 



METAL STAINS 



569 



MS 33 METHODS USING SILVER DIAMMINE 



33.0 Typical Examples 



Demonstration of the nerve endings 



in the taste buds by the method of 



Bielschowsky 1904 



This method is one of the best of tlic 

 silver diammine techniques for the pur- 

 pose of showing nervous structures, the 

 demonstration of which is the sul)je('t of 

 the next two examples, rather than con- 

 nective tissues. 



In every type of metal staining so far 

 described, emphasis has been laid on the 

 necessity of securing pure reagents. This 

 warning is the more necessary in the 

 present instance because such common re- 

 agents as sodium hydroxide and formal- 

 dehyde are included among the materials 

 retiuired. The sodium hydroxide employed 

 must be of the analytical grade (purified 

 by alcohol) and if possible free from all 

 traces of chlorides. The formaldehyde em- 

 ployed must be of analytical reagent grade 

 and must be neutralized by the addition 

 of analytical reagent grade borax. The 

 term nexdrality in this instance may be 

 extended to include any pH between 7 and 

 7.5, but under no circumstances may an 

 acid formaldehyde be employed. 



The first stage in the preparation is the 

 preservation of small blocks of tissue 

 likely to contain taste buds, in 8 % neutral- 

 ized formaldehyde. These may easily be 

 obtained from a rabbit, in which form the 

 taste buds are concentrated on the lateral 

 surface of the ridges which comprise the 

 foliate papillae. Secure a freshly killed 

 rabbit and disarticulate the lower jaw 

 cf)mpletely, cutting with scissors through 

 the disarticulated joint. A large sharp 

 scalpel or cartilage knife is then passed 

 under the tongue to sever its attachments. 

 The lowei' jaw is removed, and the tongue 

 left in place. The latter can now be re- 

 moved entire, washed free of extravasated 

 blood, and placed with the dorsal surface 

 uppermost on any convenient surface. Ex- 

 amination of the posterior region of the 

 tongue will sliow two oval bulges at the 

 lateral portions of the posterior region. 

 Each of these oval bulges (the foliate 



papillae) consists of many parallel trans- 

 vorsel}^ arranged ridges with deep grooves 

 between them. Taste buds are closely 

 concentrated on the lateral surfaces of 

 these ridges. Each ridge should be severed 

 from its base by running a cartilage knife 

 or razor roughly parallel to the surface 

 of the tongue. A sufficient number of 

 ridges thus removed may be placed until 

 recjuired in the neutralized 8% formal- 

 dehyde, which is to be changed after six 

 hours. The length of time allowed for 

 hardening in the formaldehyde is im- 

 material, but should not be less than one 

 month. 



Three or four days before proceeding 

 with the staining technique, the pieces 

 should be removed from formaldehyde 

 and placed directly into pure pyridine for 

 about three da,ys. It is probable that the 

 main function of the pyridine is to cause 

 a differential shrinkage of the nervous 

 elements, which will thus become more 

 apparent on subsequent staining. The 

 success of subsequent operations depends 

 on the removal of every trace of both 

 formaldehj^de and pyridine from the 

 pieces before they are placed in the silver 

 staining solution. Though one could in 

 theory achieve this by washing in running 

 triple-distilled water, the quantities of 

 water required would be so great that the 

 process is not practical. It is therefore 

 recommended that the pieces be washed 

 in running tap water for at least 24 hours 

 (double the time will not hurt them) and 

 then rewashed in triple-distilled water to 

 remove impurities which may have come 

 from the tap water. The second wash 

 should either be in running triple-distilled 

 water, or alternatively in not fewer than 

 five changes of large volumes of triple- 

 distilled water changed daily. 



Before proceeding further it is necessary 

 to make up the Bielschowsky stock silver 

 solution (MS 33.1 Bielschowsky 1902) 

 used in this technique. First secure a 

 chemically clean 500-milliliter beaker, a 

 chemically clean 250-milliliter graduated 

 flask, a chemically clean glass stirring 

 rod, and a chemically clean l)uret. Place 

 120 milliliters of triple-distilled water in 



