OF THE GREEN CELLS OF PLANTS 63 



but the effect is not very pronounced and is only clear on com- 

 parison 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, demon- 

 strating 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 0-5 per cent, in 

 concentration, and be used in equal volume with the 1-5 per cent, 

 ferricyanide solution so that the concentration of acid acting on 

 the tissue is only 0-25 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 gives a faint blue colour which increases as the mixture 

 is left standing. 



When a solution of haBmatoxylin 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 haematoxylin, 

 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 haema- 

 toxylin 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 appearance of the brown stain characteristic of colloidal 

 iron oxide, while in many of the higher plants (mono- and di- 

 cotyledons) the green leaf at first stains a deep brown, which gradually, 

 in a varying period of a few hours to a day or two, changes to a blue- 

 black, just as is seen in the test-tube when colloidal iron oxide 

 solution is mixed with the reagent. In certain cases, however, the 

 brown colour is found to persist for weeks without change. 



