298 CYTOLOGICAL METHODS. 



be used alone, because its complete 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 HJEMATOXYLIN METHOD. As an indicator Macallum 

 uses a 0-5 per cent, solution of absolutely ' pure hsematoxylin ' 

 made up in perfectly pure aqua 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 hsematoxylin 

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

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

 hc&matoxylin is unaffected. Such compounds must be unmasked 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 preparation 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 haematoxylin 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 hsomatoxylin 

 seems to be one of oxidation (MAYER, Mitth. Zool. Stat. NeapeL, x, 

 p. 170). 



Extraordinarily small traces of inorganic iron are thus demon- 

 strated. The method is more sensitive than that of Prussian blue 

 or ammonium sulphide. 



