64 * IRON COMPOUNDS IN THE CHLOROPLASTS 



This deep-brown coloration is not simply due to imbibition by 

 the tissue of unaltered hsematoxylin, 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 

 hsematoxylin. 



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 hsematoxylin. 



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

 different from the ordinary use of hsematoxylin as a nuclear stain 

 in histological technique. In the ordinary use of hsematoxylin as a 

 staining reagent a mordaunt is always used either preceding the 

 hsematoxylin as, for example, the iron- alum mordaunt for Heiden- 

 hain's iron-hsematoxylin method or simultaneously as in the use 

 of the hsemalum stain, where the mordaunt alum is mixed with the 

 hsematoxylin. 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 Macal- 

 lum' 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- 

 hsematoxylin 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 

 hsematoxylin, 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 hsematoxylin 

 is weighed out, and washed with the twice distilled water till the 



