62 IRON COMPOUNDS IN THE CHLOROPLASTS 



Macallum's test. For this reason it is well to remove the lipoids and 

 chlorophyll, and in many cases this is by no means an easy task. 

 In some cases standing in cold alcohol removes the chlorophyll quite 

 effectually and leaves the tissue colourless and ready for staining; 

 but in other cases the tissue may be left at ordinary temperatures 

 for days in alcohol, and this may even be followed by 

 several extractions with ether and still some of the green colour 

 remains. After a good deal of experimentation the best extractive 

 in these latter cases was found to be boiling alcohol. 



The tissue, either partially teased with the glass points if it is 

 bulky like the leaf of a higher plant or a piece of lichen or moss, or 

 left intact if a delicate structure like an algal filament or pleuro- 

 coccus, is placed in water in a watchglass, and then absolute alcohol 

 is gradually added portionwise and pouring away excess of the mix- 

 ture at intervals until the fluid is finally all absolute alcohol. The 

 preparation is then boiled in the alcohol and the greenish extract 

 .poured away, arid this is repeated till the green tissue becomes 

 colourless. The decolorised tissue is then brought back again into 

 distilled water by gradually adding the water to the alcohol, and 

 pouring off. Finally, it is allowed to stand a few minutes in a 

 watchglass in water redistilled from glass, and is then ready for 

 staining. 



In addition to the unmordaunted simple aqueous solution of 

 well-washed hsematoxylin in f per cent, concentration introduced 

 by Macallum, 1 the older histochemical tests for iron were also 

 utilised namely, potassium ferrocyanide and hydrochloric acid for 

 ferric salts, potassium f erricyanide and hydrochloric acid for ferrous 

 salts, and ammonium hydrogen sulphide in glycerine for both. In 

 our opinion the Macallum test surpasses all these in reliability and 

 delicacy. Its only fault is that it is too delicate, and the 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, 



1 Journ. Phijsiol., vol. xxii., p. 92 (1897). 



