MICROCHEMICAL TESTS 291 



paration may go quite dark yellowish -brown, especially nuclei, and the 

 presence of iron may thus be obscured To remove this excess the 

 preparation is treated in a mixture of equal parts of absolute alcohol 

 and ether, but not for longer than one hour. The unaffected ha;ma- 

 toxylin is extracted, the blue-black compound remains. Clear in oil 

 of cloves, mount in balsam. Such preparations are permanent. 



This reaction of inorganic compounds of iron with haematoxylin 

 seems to be one of oxidation (Mayer, Mitth. Zool. Stat. Neapel, x, 

 p. 170). 



Extraordinarily small traces of inorganic iron are thus demonstrated. 

 The method is more sensitive than that of Prussian blue or ammonium 

 sulphide. 



Organic Iron Compounds. These will not give the iron 

 reactions unless tTie complex iron compound has been broken up, 

 that is, the iron " unmasked " by some reagent : acid alcohol is 

 used for this. Sulphuric acid alcohol (4 per cent, in 95 per cent, 

 alcohol) and nitric acid alcohol (3 jDer cent, in 95 per cent, alcohol) 

 are better than the hydrochloric acid alcohol (Bunge's fluid). 

 Sulphuric acid alcohol acts very slowly, especially on bulk tissues, 

 and even Protozoa take twenty-four hours at 35° C. before their 

 masked iron is revealed. Nitric acid alcohol acts more quickly and 

 extracts very little of the iron it liberates (which is a danger with 

 Bunge's fluid) ; the process is completed in about thirty-six 

 hours. 



Sections are treated with acid alcohol, 90 per cent, alcohol, 

 and aq. dest., and then Macallum's haematoxylin is added ; the 

 sections are washed in aq. dest., stained in safranin, as described 

 in next section, dehydrated and mounted in balsam. 



Prussian Blue Reactions on Organic Compounds. Sections 

 after being treated in the acid alcohol (nitric or sulphuric) are 

 washed in pure 90 per cent, alcohol and then in aq. dest. They 

 are placed not longer than five minutes in the following solution : 

 aq. potassium ferricyanide 1-5 per cent., and hydrochloric acid 

 0-5 per cent, in aq. solution equal parts, freshly made. Again 

 washed carefully in aq. dest. stained in eosin or safranin, dehy- 

 drated in alcohol, cleared in oil of cedar and mounted in benzol 

 balsam. The safranin or eosin is used in 1 per cent, strength in 

 30 per cent, alcohol, for three minutes for eosin, and for one half- 

 hour for safranin, and differentiated in 90 per cent, alcohol. 



Ferric and Ferrous Salts both occur in inorganic iron com- 

 pounds. Ferrous salts may be distinguished from ferric by the 

 fact that only the latter give an immediate reaction with ferro- 

 cyanide of potassium, while the former react with ferricyanide of 

 potassium. Fix material in alcohol of about 90 ])cr cent, for 

 several days. 



Reaction for Ferric Salts. Wash sections in aq. dest., transfer to 2 

 per cent, or stronger aq. sol. ferrocyanide of potassium for from 

 three to fifteen minutes. Bring to acid alcohol (1 c.c. in 70 per 



10—2 



