38 Microscopic Histochemistry 



the ferrocyanide as fast as they are formed, provided that 

 ferrocyanide is really present "on the premises." Since it dif- 

 fuses much more slowly than the acid (and very poorly 

 through collodion), it may arrive too late to bind the ferric 

 ions where formed, and some diffusion of the latter may take 

 place. For this reason, ferrocyanide must be given a head 

 start. The following technique will invariably yield sharp 

 pictures. 



Method 



Fixation in a neutral fluid. Celloidin must be removed from 

 sections of celloidin-embedded material. 



Place slides (or frozen sections) in a fresh and filtered 

 5-10 per cent solution of potassium ferrocyanide. After 5 

 minutes add about M volume of a 10 per cent solution of 

 hydrochloric acid. The latter must be of a good analytical 

 grade and contain no iron (produce no visible greenish or 

 bluish tinge when mixed with the ferrocyanide). Stir the 

 mixture. Keep sections in it for about 20-30 minutes. Wash 

 under the tap. Counterstain with a red nuclear dye (alum 

 carmine, safranine, neutral red, but not lithium carmine, 

 which will bleach Prussian blue), dehydrate, and mount. 

 Hemosiderin iron will show up in an intense blue shade. 

 Balsam of Canada may cause gradual fading of the stain 

 over a period of months or years; the more modern synthetic 

 mounting media are safe. 



Tirmann^^ suggested the combination of the preceding two 

 methods, to utilize the advantages of the absolute specificity 

 of the Prussian blue method and the purportedly higher sen- 

 sitivity of the ferrous sulfide method. He proceeds as follows: 

 The section is first treated with ammonium sulfide, washed, 

 and then subjected to the action of an acidified solution of 

 potassium ferricyanide, which will convert ferrous sulfide 

 into TurnbulFs blue ( ferrous ferricyanide ) , of a shade indis- 



25. Tirmann, J.: Gorbersdorfer Veroffentl., 2:101, 1898. 



