IRON HEMATOXYLIN 



166 



ISAMINE BLUE 



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 5% aq. potassium ferrocyanide 

 and 1/100 N hydrochloric acid. This 

 produces a wheal which turns dark blue 

 in 5 min. A positive reaction can even 

 be obtained after death. (Fishback, 

 H. R., J. Lab. & Clin. Med. 1939-40, 

 25, 98-99). 



In special cases, as in the analysis of 

 small amounts of epidermis, resort may 

 be had to a quantitative polarographic 

 determination of iron, see Carruthers, 

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

 Inst., 1942,3, 217-220. 



Kirk, P. L. and Bentley, J. T., 

 Mikrochemie, 1936, 21, 250^259 advo- 

 cate a titrimetric method for iron. The 

 difficulty of interference by small 

 amounts of copper has been overcome 

 by Ramsav, W. N. M., Biochem. J., 

 1944, 38, 467-469. Click (p. 277) is of 

 the opinion that such methods may be 

 adapted for use with the quantities of 

 material employed in histochemical 

 work. 

 Iron Hematoxylin of Heidenhain is one of 

 the standard stains. It will give excel- 

 lent results after almost any 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. Many 

 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. iron 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. For best 

 general discussion of iron hematoxylin, 

 see Lillie, p. 58. 



Iron Hematoxylin Single Stain — Written by 

 Morris Goldman, Dept. of Parasitology, 

 School of Hygiene and Public Health, 

 Johns Hopkins University, Baltimore. 

 January 29, 1951 — This stain is useful 

 for the purpose of diagnosing intestinal 

 protozoa occurring in fecal smears. It 

 is not intended to replace the longer and 

 more precise iron hematoxylin methods. 

 The stain is prepared from the following 

 relatively stable solutions: Solution A: 

 1% hematoxylin in 95% ale. (best pre- 

 pared from a stock 10% ale. solution of 

 hematoxylin). Solution B: NH^Fe- 

 (804)2- I2H2O, 4.0 gms., glacial acetic 

 acid, 1.0 ml., concentrated H2SO4, 0.12 

 ml., and aq. dest. to make 100 ml. Mix 

 equal parts of Solution A and B, filter 

 after several hours and use. Staining 

 time varies from 30 sec. when stain is 

 fresh to 3 min. after 24 hrs. Discard 

 stain after 3 days. Fecal smears are 

 fixed in Schaudinn's, passed through 

 iodine alcohol to 50% alcohol, stained, 

 washed in running tap water 5 minutes, 

 dehydrated and mounted. The stock 

 solutions used in this technique may 

 also be used in the Heidenhain iron 

 hematoxylin procedure. 



Iron Pigments, see Berlin and Turnbull blue 

 reactions. 



Iron, Radioactive. See Erythrocytes. 



Isamine Blue is described by Conn (p. 137) 



