566 Prof. B. Moore. Presence of Inorganic Iron 



small traces of inorganic iron set free from organic compounds in the tissues 

 on long standing cause faint but increasing staining when a preparation is 

 left over for some days. When a blue-black is obtained, however, within 

 a few hours with this reagent it is a decisive proof of loosely combined, or 

 inorganic, iron in the situation where the staining occurs. 



Ammonium hydrogen sulphide when added to the tissues with an equal 

 amount of glycerine, and the whole kept at 36° C. for some hours, produces 

 a distinct blackening as compared with the normal, but the effect is not 

 very pronounced and is only clear on comparison of treated and untreated 

 tissue. 



Potassium ferrocyanide and hydrochloric acid never gave a blue colour, 

 but a blue colour is frequently, and very distinctly, given within a few hours 

 by potassium ferricyanide and acid, demonstrating that the inorganic iron of 

 the chloroplasts is present in the ferrous condition ; this was typically 

 observed in the case of Spirogyra and Vaucheria. 



There is always some doubt, however, about using a reagent which itself 

 contains the element sought for, and moreover is fairly readily broken down 

 in presence of organic matter and acid. 



The hydrochloric acid used should not exceed - 5 per cent, in concen- 

 tration, and be used in equal volume with the l - 5-per-cent. ferricyanide 

 solution so that the concentration of acid acting on the tissue is only 

 - 25 per cent. Then, if a blue stain is obtained with a considerable intensity 

 within 24 hours, it may fairly certainly be attributed to ferrous iron in that 

 particular situation. The result, however, ought always to be confirmed by 

 the Macallum test, for solid starch or casein left for 24 hours in contact with 

 the above reagents each give a faint blue colour which increases as the 

 mixture is left standing. 



When a solution of hsematoxylin in pure distilled water is mixed with 

 a very dilute solution of an ordinary iron salt such as ferric chloride, a deep 

 blue- black coloration is immediately produced. If, instead of an ordinary 

 iron salt solution, a solution of highly colloidal or dialysed iron oxide be 

 mixed with the solution of hematoxylin there is obtained instead a deep 

 chocolate-brown coloration. In the course of some hours to a day or two, 

 this chocolate brown is replaced by the blue-black colour obtained with 

 ionic or crystalloidal iron salts. Similar results are usually obtained when 

 the hsematoxylin solution is used as a detector of iron in the tissue of plants. 

 In certain cases, notably unicellular green plants and algal filaments, a deep 

 blue-black is obtained within a few minutes without any previous appear- 

 ance of the brown stain characteristic of colloidal iron oxide, while in many 

 of the higher plants (mono- and dicotyledons) the green leaf at first stains 



