Compounds in Chloroplasts of Green Cells of Plants. 567 



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. 



This deep brown coloration is not simply due to imbibition of the tissue 

 with unaltered haematoxylin, for it is far too deep for this, and, moreover, is 

 not removed by washing with a mixture of equal parts of alcohol and ether 

 as recommended by Macallum. It is a true staining of colloidal iron, 

 present in those parts of the tissues where the brown occurs, and possesses 

 just the same dark brown colour that is obtained on mixing colloidal iron 

 oxide solution and haematoxylin. 



In contrast with vegetable tissues, such a direct staining (either brown or 

 blue-black) is only found in the embryonic condition in the tissues of higher 

 animals, for the iron in the majority of such animal tissues is firmly bound 

 organically and gives no coloration with haematoxylin. 



It is to be remarked that this staining as a test for iron is quite different 

 from the ordinary use of haematoxylin as a nuclear stain in histological 

 technique. In the ordinary use of haematoxylin as a staining reagent 

 a mordaunt is always used either preceding the haematoxylin, as, for example, 

 the iron alum mordaunt for Heidenhain's iron-haematoxylin method, or 

 simultaneously as in the use of the haemalum stain, where the mordaunt 

 alum is mixed with the haematoxylin. But in Macallum's use of the stain 

 no mordaunt whatever is used, but instead a simple aqueous solution in 

 pure distilled water. This solution only strikes a colour where a mordaunt 

 is naturally present in the tissue. Now with iron in colloidal form the 

 colour struck is the deep brown mentioned above, with iron in crystalloidal 

 form the colour struck is blue-black. Thus Macallum's method resembles 

 Heidenhain's staining, but with the previous iron treatment naturally 

 provided in the tissues, and the blue-black effect obtained closely resembles 

 in many cases a Heidenhain iron-haematoxylin stain. 



In order to use the method effectively, it is not merely necessary to avoid 

 all minute traces of iron in the water and other fluids used, but also all 

 traces of alkali and acid, since these interfere with the delicacy of the 

 reaction. Alkali gives a rose-red colour with the haematoxylin, and acid 

 inhibits the development of the blue-black when the amount of iron is 

 small. In making up the stain itself, water twice distilled from glass 

 vessels must be used as the solvent, the second distillation having been 

 made immediately previous to use. To make the staining solution, 0*3 grm. 

 of pure haematoxylin is weighed out, and washed with the twice distilled 

 water till the crystals are colourless, and the wash-water is only pale 



