IRON 



127 



IRON HEMATOXYLIN 



Med., 1911, 13, 477-485, devised special 

 methods for its demonstration. Test- 

 ing for iron in association with calcium 

 particularly in bone is critically de- 

 scribed by Cameron, G. R., J. Path, 

 and Bact., 1930, 33, 929-955. He em- 

 phasizes the fact that exposure of tissues 

 and fluids to dust in a city like London 

 is an important source of error. 



3. Microincineration yields a mineral 

 residue that contains iron originally 

 both organic and inorganic. Color of 

 the iron oxides, viewed in the dark field, 

 varies according to Policard (C. rend. 

 Acad. d. sc, 1923, 176, 1187) from yellow 

 to deep red. He suggests that perhaps 

 the yellow to brown ash is of organic 

 iron and the red ash is of free iron. See 

 also Marza, V. D., INlarza, E., and 

 Chiosa, L. Bull, d'hist. Appliq., 1932, 

 9, 213. Scott (McClung, p. 758) warns 

 against confusion with carbon. 



4. Ilydroxyquinoline test (Thomas, 

 J. A. and Lavollay, J., Bull. d'Hist. 

 AppL, 1935, 12, 400^402). Fix in alco- 

 hol, trichloracetic acid or neutral forma- 

 lin. Avoid formol with alkaline water 

 and fixatives containing chromium. 

 Make up reagent by dissolving 2.5 gm. 

 8-hydroxyquinoline in 4 cc. pure acetic 

 acid warming gently. Add quickly aq. 

 dest. to make 100 cc. Filter. Wash 

 sections (or smears or cultures) well in 

 neutral aq. dest. Then add few drops 

 of reagent 5-15 min. Pour off reagent. 

 Add to preparation 1 drop 25% aq. 

 ammonia which produces a ppt. Wash 

 in a stream of neutral aq. dest. If 

 large crystals remain w^ash more ener- 

 getically. Stain nuclei with lithium 

 carmine. Examine in neutral aq. dest. 

 or dehydrate in terpinol and mount in 

 vaseline oil. Iron, green black; nuclei, 

 red. Recommended for localization of 

 iron in granules of vitellus, in red blood 

 cells, and in connection with micro- 

 incineration. Said to be better than 

 Prussian Blue reaction for iron. 



5. Dinitrosoresorcinol (Humphrey, 

 H. A., Arch. Path., 1935, 20, 256-258). 

 Treat paraffin sections of formalin fixed 

 tissue with 30% aq. ammonium sulphide, 

 1 min. Rinse in water and immerse in 

 sat. aq. dinitrosoresorcinol (Eastman) 

 6-20 hrs. A counterstain can be em- 

 ployed. Humphrey does not say which. 

 1% eosin in 50% alcohol should be satis- 

 factory because the iron containing com- 

 pounds such as hemosiderin are colored 

 green. Wash, dehydrate, clear and 

 mount. 



Intravenous injections of colloidal 

 solutions of iron in rabbits are described 

 by Duhamel, B. G., C. rend. Soc. de 

 Biol., 1919, 82, 724-726. 



6. A clinical demonstration of iron in 



the skin in hemochromatosis involves 

 intradermal injection of equal parts of 

 sterile 0.5% aq. potassium ferrocyanide 

 and 1/100 N hydrochloric acid. This 

 produces a wheal whicli turns dark blue 

 in 5 min. A positive reaction can even 

 be obtained after death. (Fishback, 

 H. R., J. Lab. & Clin. Med. 19;J9-40, 

 25, 98-99). 



In special cases, as in the analysis of 

 small amounts of epidermis, resort may 

 be had to a quantitativ^e polaro;j;rai>hic 

 determination of iron, see Carruthers, 

 C. and Suntzeff, V., J. Nat. Cancer 

 Inst., 1942, 3, 217-220. 

 Iron Hematoxylin of Heidenhain is one of 

 the standard stains. It will give excel- 

 lent results after almost anj^ good fixa- 

 tion. Zenker's fluid and formalin- 

 Zenker are suggested. Bring paraffin 

 sections down to aq. dest. Mordant in 

 5% aq. iron ammonium sulphate (iron 

 alum, light violet colored crystals, dis- 

 card the brownish material accompany- 

 ing them) 12-24 hrs. Rinse quickly in 

 aq. dest. Transfer to 1% aq. hema- 

 toxylin (made up by diluting 1 cc. sat. 

 sol. hematoxylin in abs. ale. with 99 cc. 

 aq. dest.) for 12-24 hrs. Differentiate 

 under microscope in 1% aq. iron alum. 

 Wash thoroughly in tap water. Alany 

 counterstains can then be used such as 

 1% aq. Bordeaux red, orange G., acid 

 fuchsin, acridine red, or Mucicarmine. 

 Dehydrate, clear and mount. Nuclei 

 dense blue-black in background of color 

 selected. See Centrosomes, Nuclei, 

 Regaud's Method for mitochondria. 



1. Koneff, A. A., Anat. Rec, 1936, 

 66, 173-179 advises use with anilin blue. 

 Mordant sections 5-10 min. in 5% 

 aq. iroii ammonium sulphate. Rinse 

 quickly in aq. dest., stain 3-15 min. in 

 Harris' hematoxylin. Rinse again in 

 aq. dest. and stain in: anilin blue 

 (Griibler) 0.1 gm.; oxalic acid, 2 gm.; 

 phosphomolybdic acid, 15 gm. and aq. 

 dest. 300 cc. Wash in aq. dest., differ- 

 entiate in alcohol, dehydrate (2 changes 

 of absolute), clear in xylol and mount in 

 balsam. If euperal is used for mounting 

 omit the xylol. Nuclei, violet-brown; 

 cytoplasm, light brown; erythrocytes, 

 dark violet ; myelin and muscle brown ; 

 elastic fibers, reddish brown to red. 



2. Lillie, R. D. and Earle, W. R., 

 Am. J. Path., 1939, 15, 765-770 recom- 

 mend employment of a hematoxylin 

 containing ferric and ferrous iron: (A). 

 Ferric ammonium sulphate, violet crys- 

 tals, 15 gm.; ferrous sulphate, 15 gm.; 

 aq. dest., 100 cc. (B). Hematoxylin, 

 1 gm.; 95% alcohol, 50 cc, glycerin, 

 C.P., 50 cc. Mix A and B in equal 

 quantities before using. 



