20 MICROSCOPIC TECHNIQUES 



Macallum's (1908) technique is still employed for this conversion 

 and it consists of a treatment of deparaffinized sections with a solu- 

 tion of either nitric or sulfuric acid in alcohol. The iron liberated 

 is chiefly in the ferric form. In all tests special care must be taken 

 to protect tissues and fluids from dust. Iron will also appear in 

 the tests for lead and copper (see page 22). 



Precautions to prevent diffusion of the iron in aqueous solutions 

 have not been sufficiently exercised in the following procedures. 

 The investigator should modify them accordingly. 



Prussian Blue Test for Ferric Iron and TurnbuU's 

 Blue for Ferrous Iron 



SPECIAL REAGENTS 



Prussian Blue Reagent. 2% potassium ferrocyanide (use fresh 



soln.). 

 TurnbuU's Blue Reagent. 2% potassium ferricyanide (use fresh 



soln.). 

 Acid Alcohol. 1% hydrochloric acid in 70% alcohol. 

 Organic Iron Reagent. Equal vol. of 1.5% potassium ferrocyanide 



and 0.5% hydrochloric acid (use fresh soln.). 

 Organic Iron Conversion Reagent. 3% nitric acid, or 4% sulfuric 



acid, in 95% alcohol. (The sulfuric reagent acts more slowly.) 



PROCEDURE FOR INORGANIC IRON 



1. Fix in 95% alcohol for 24-48 hr. 



2. Prepare paraffin sections as usual ( care must be taken to mini- 

 mize contact with iron — the microtome knife must be free of rust 

 and not freshly honed and glass needles should be substituted for the 

 steel ones) . 



3. After removing paraffin and passing down to distilled water, 

 place sections in either the Prussian or TurnbuU's blue reagent for 

 3-15 min. (If both ferric and ferrous iron are to be visualized, use 

 a mixture of equal vol. of the two reagents.) 



4. Wash in water containing eosin or safranin to counterstain. 



5. Dehydrate, clear, and mount in benzol balsam. 



PROCEDURE FOR ORGANIC IRON 



1-2. Same as inorganic iron. 



3. Liberate iron from the bound forms by treating deparaffinized 



