42 E. J. STIEGLITZ 



position as was demonstrated by the ammonium-sulphide reac- 

 tion in similar fresh tissues. The suggestion is made that ammo- 

 nium sulphide (NH4SH) be added to the alcohol to insure the 

 precipitation of the ferric salts. Tartakowsky (47) is in agree- 

 ment with the findings of Hall and made extensive use of the 

 sulphide alcohol as a reagent in fixation. In his estimation, 

 formalin is as good a preserver of the original relations of the 

 iron as pure alcohol. Abderhalden (48), on the other hand, 

 states that it is his belief that alcohol destroys the localization 

 of the iron. Such a conclusion has not been found valid in the 

 present work. Macallum (41) states that ordinarily inorganic 

 iron, as it is found in cells, is so slightly soluble in alcohol that 

 the diffusion, if any, is not appreciable. He states that the 

 forms of inorganic iron most frequent in the body are the phos- 

 phate, hydrate, and oxide. In his estimation, the use of ammo- 

 nium sulphide in the alcohol increases the experimental error, as 

 it may cause the liberation of organically combined iron already 

 present (49). 



The staining technique that was employed is very simple. 

 Equal parts of freshly prepared 2 per cent potassium ferro- 

 cyanide and 2 per cent hydrochloric-acid solutions were used in 

 the precipitation of the Prussian blue in the sections. A uni- 

 form counterstain was obtained with acid carmine, or better 

 with the paler alum cochineal. The tissues were all imbedded in 

 paraffin and sections cut with a rotary microtome. The routine 

 technique employed called for two thicknesses of section : one 

 of 6m for the intracellular localization of the iron and the other 

 for sections of 24/x for the anatomic localization of the salt. 



One other method was used with considerable success in mak- 

 ing the Prussian-blue preparations. A mixture of equal parts of 

 green ferric ammonium citrate and sodium ferrocyanide was 

 injected intravenously and the tissues then fixed in 5 per cent 

 trichloracetic acid. The hydrogen ion concentration of this 

 solution is sufficiently high to cause the precipitation of the 

 Prussian blue directly in the block of tissue and the reagent is at 

 the same time a good protoplasmic fixative. Combinations of 

 trichloracetic acid with other reagents were not as successful, 



