IODINE VIOLET 



165 



IRON 



Iodine Violet, see Hofmann's Violet. 



Iris Blue, see Resorcin Blue. 



Iris Violet, see Amethyst Violet. 



Iron occurs in tissues "masked" in organic 

 compounds which are not ionisable and 

 free in inorganic compounds which are 

 ionisable into ferric and ferrous salts. 



1. Macallum's hematoxylin method 

 depends upon the formation of a blue 

 black iron hematoxylinate. The tissue 

 is fixed in 95% alcohol 24-48 hours, 

 dehydrated, cleared, imbedded in paraf- 

 fin and the sections are passed down to 

 distilled water. Contact with iron is 

 reduced to a minimum. The microtome 

 knife must be free of rust. Treat sec- 

 tions with a freshly prepared straw 

 yellow 0.5% aqueous solution of hema- 

 toxylin which must be of the highest 

 purity. Inorganic iron produces the 

 blue-black compound which is rela- 

 tively insoluble. Dehydrate, clear and 

 mount in balsam in the usual way. 



The technique for organic iron is 

 more difficult because it must be un- 

 masked before it will react in this way. 

 The best account is Nicholson, F. M., 

 J. Comp. Neurol., 1923, 36, 37-87. In 

 studying the cytoplasmic iron contain- 

 ing proteins of nerve cells of the medulla 

 of rats, he fixed in 95% alcohol 48 hours ; 

 dehydrated in absolute alcohol 2-5 

 hours; cleared in cedarwood oil until 

 transparent; imbedded in paraffin (2 

 changes) and cut sections 7/1. After 

 being deparaffinized, the sections were 

 passed through alcohols to 4% pure 

 sulphuric acid in 95% alcohol held at 

 60 °C. for 5-60 minutes. This liberated 

 the iron. The sections were washed in 

 95% alcohol ; passed down through 

 graded alcohols to aq. dest., and placed 

 in freshly prepared 0.5% aqueous 

 hematoxylin, 1-5 minutes in which the 

 blue-black hematoxylinate forms . Then 

 wash in aq. dest. (not tap water). 

 Counterstain in dilute alcohol erythro- 

 sin and mount as usual. As a check the 

 nuclear chromatin of sections not treated 

 with the acid alcohol should not be 

 colored black by this hematoxylin 

 solution . Difficulty may be experienced 

 because the color of the unmasked iron 

 is faint. The reaction is a chemical one 

 of great delicacy and requires practice. 

 Pancreatic acinous cells also afford 

 favorable material. Look for cyto- 

 plasmic iron in the poles distant from 

 the lumen where thechromidial material, 

 which resembles the Nissl bodies, is 

 most concentrated. 



2. Prussian blue reaction. Prepare 

 sections in the same way, deparaffinize 

 and test as described in Lee (p. 291). 

 For ferric salts of inorganic iron wash in 

 aq. dest., 2% aqueous potassium ferro- 



cyanide, 3-15 minutes; Prussian blue 

 is formed, wash, dehydrate, clear and 

 mount. For ferrous salts substitute 

 ferricyanide for ferrocyanide in the test. 

 For both use equal parts of ferrocyanide 

 and ferricyanide. When the iron is 

 organic it is unmasked by treating the 

 sections with 3% pure nitric acid in 95% 

 alcohol for 24-36 hours at room tem- 

 perature or at 35 °C. if necessary. Wash 

 in pure 90% alcohol and in aq. dest. 

 Place in equal parts freshly made of 

 1.5% aqueous potassium ferrocyanide 

 and 0.5% aqueous hydrochloric acid for 

 not more than 5 minutes. Wash well 

 in aq. dest., colored with eosin or 

 safranin, dehydrate, clear and mount. 

 Hemosiderin gives Prussian blue 

 reaction for inorganic iron. The iron in 

 hemoglobin is not unmasked by these 

 acid alcohols. Brown, W. H., J. Exper. 

 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., Marza, E., and 

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

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

 against confusion with carbon. 



4. Hydroxyquinoline 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 gra. 

 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 wash 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 



