290 MICROCHEMICAL TESTS 



detectable with the ordinary reagents, and which, for conveni- 

 ence, may be designated Inorganic Iron Comjiounds. 



The nature of many of the compounds of iron found in placentas, 

 blood-organs, the liver, etc., is obscure ; many of them appear to be 

 formed as degeneration or excretion products, from the breaking down 

 of haemoglobin. See below. 



Most of our knowledge of the methods for the detection of iron in 

 tissues and cells is due to A. B. Macaixum (Quart. Journ. Micr. Sci., 

 xxxviii, 1895 ; Journ. Physiology, 1897 ; Ergebn. d. Physiol. Wies- 

 baden, 1908). Macallum has shown that, to detect organic iron, one 

 must convert it into inorganic. This can be done by allowing sulphuric 

 or nitric acid alcohol to act upon sections, or a piece of tissue, for from 

 one to twenty-four hours at 35° C, according to the strength of acid 

 and the size of the object. When masked iron is liberated in the tissues 

 by acid alcohol, most of it is in the form of ferric salts, particularly 

 when the oa;idising nitric acid is used, and a small part occasionally of 

 ferrous compounds. Inorganic iron compounds in tissues are usually 

 ferric, more rarely ferrous salts. 



The commonest tests for iron in tissues are the Prussian blue 

 reaction, Turnbull's blue, and Macallum's haematoxylin. The 

 latter test should never be used alone, because its cotnplete specificity 

 is somewhat doubtful. 



It is hardly necessary to point out that proper precautions 

 should be taken to avoid contamination of the tissue by vessels 

 or chemicals which may contain iron compounds. Glass needles 

 should be used instead of steel, and the water used should have 

 been distilled from a clean glass retort. 



The tissues should either be fixed in redistilled formalin (10 per 

 cent.), chemically pure ethyl alcohol or pure 90 to 95 per cent, 

 alcohol, or in redistilled methylated spirit. Bouin's fluid, 

 Flemming, and such mixtures should not be used, as such a practice 

 is almost certain to introduce error. Material should be fixed or 

 hardened for several days in strong alcohol. Sections are made 

 either freehand with a bright rust-free razor wetted with absolute 

 alcohol, or by the paraffin method with a dry rust-free knife. 



Macallum's Hematoxylin Method. As an indicator Macallum 

 used a 0-5 per cent, solution of absolutely " pure haematoxylin " 

 made up in perfectly pure aq. dest. The solution should look 

 brownish-yellow, but when alkalies or alkaline earths are added, 

 the colour becomes violet or red. When such a pure haematoxylin 

 is brought into contact with a salt of iron, the yellow colour 

 becomes blue-black, or bluish-black ; with organic iron compounds 

 the haematoxylin is unaffected. Such compounds must be un- 

 masked by sulphuric or nitric acid alcohol as above mentioned. 



When the compounds of iron to be investigated are found in tissues, 

 the latter are well hardened in alcohol (purified, vide supra), sections 

 prepared and washed in aq. dest., or the tissue simply teased out, and 

 then the haematoxylin solution is added. Those parts which go blue- 

 black or blue-violet contain inorganic iron ; the remainder of the pre- 



