of the Presence of Iron in Chromatin. 281 



required for the production of the reaction varied greatly. A number 

 of conditions seem to assist in, or retard, and even prevent the success 

 of the experiment ; but what are all the favourable conditions I do 

 not know as yet. It is certain, however, that there is a proper pro- 

 portion of glycerine and ammonium sulphide in the mixture to be 

 added, and I am at present endeavouring to determine what that pro- 

 portion is. It is also ascertained that the nucleus must be sur- 

 rounded on all sides by the mixture, otherwise it very rarely shows 

 the reaction. Where the cell body is large, as, for example, in the 

 semi-matured ovarian ova of Necturus, the reaction has not yet 

 appeared ; while in those very small ova, in which the nucleus, rich 

 in chromatin, forms by far the greater part of the cell, the reaction 

 appears as readily as in the testicular nuclei. Again, the preparation 

 may advance in the reaction up to a certain point, showing the 

 majority of its nuclei possessing a light-green, greenish- blue, or 

 slate colour; when a change occurs, the rust-tint replacing these 

 colours in the chromatin. So far as my experience goes, this happens 

 when too little sulphide has been mixed with the glycerine, or when 

 the sulphide used is beginning to turn deep yellow, or is old. 



The deep-yellow sulphide gives no reaction with nuclei, even after 

 eight weeks, while the most active is the freshly prepared reagent. 

 This seems to indicate that the process, by which the iron is set free 

 from the chromatin, is essentially a reducing one, the yellow sulphide 

 having much less reducing power than the colourless, or nearly colour- 

 less, reagent. In this way we can explain why the nucleus must be 

 completely surrounded by the reagent, for the reducing capacity of 

 the latter is limited, and a large quantity of it must be concentrated 

 in its action on some particularly small object. When, however, the 

 cellular elements are in a mass, not even the peripherally placed 

 nuclei are affected in the manner described, but they react when they 

 are teased out and separated. 



In some tissues there is more or less of albuminate iron or of a 

 deposit of inorganic iron compounds. In such a case the addition of 

 ammonium sulphide gives a reaction immediately. The presence of 

 such compounds sometimes offers a difficulty, especially if they happen 

 to occur in the nuclei. I guarded against confusion in all such cases 

 by submitting thin sections of such tissues made with the free hand 

 to the action of a large quantity of Bunge's fluid for eight to ten 

 hours.* Such sections also, if made from alcohol-hardened tissues 

 with a clean steel knife, covered with absolute alcohol, are not in the 



* This fluid consists of ninety volumes of 96 per cent, alcohol and ten volumes of 

 hydrochloric acid, 25 per cent, strength. It, according to Bunge, extracts all inor- 

 ganic iron compounds and albuminate iron from egg-yolk, and I have found that it 

 removes all traces of such from sections of the spleen, liver, and kidney, which 

 "eact immediately on the application of acid ferrocyanide solutions, 



U 2 



