On a Green Leucocytosis in Oysters, 



35 



every reagent was perfectly pure, firstly, with regard to the absence 

 of copper or iron, and, secondly, that no acid was present ; thus, for 

 example, commercial turpentine may give a distinctly acid reaction, 

 and this would be sufficient to remove the copper. If sections 

 were imbedded in gum — and often the best results were obtained 

 by this method — the tissues were allowed to remain for as short a 

 time as possible in distilled water and then transferred to perfectly 

 fresh neutralised solution of gum-arabic, and allowed to remain in it 

 for only a short period. The pigments appeared partially soluble in 

 water. 



Comparatively thick sections were cut, in which the distribu- 

 tion of the green colour could be seen with the naked eye. These 

 were placed in absolute alcohol in every case before proceeding to 

 test. The reagents which we employed were potassic ferrocyanide, 

 1*5 per cent, solution,* freshly prepared ammonium-hydrogen 

 sulphide, and pure hematoxylin. 



Potassic Ferrocyanide. — Sections were taken from absolute alcohol 

 and passed into distilled water for a moment in order to remove the 

 alcohol. They were then placed in the potassic ferrocyanide solu- 

 tion, when the portions previously green assumed a red colour ; this 

 reaction set in immediately. The presence of a 0'5 per cent, solu- 

 tion of hydrochloric acid added in equal quantity to the ferrocyanide 

 solution previous to use (as recommended by Macallum for iron) 

 tended to hasten the reaction, and in some cases was necessary in 

 order to obtain it. 



The sections were then washed in distilled water, dehydrated in 

 absolute alcohol, cleared in cedar oil, and mounted in Canada 

 balsam. The red coloration was found located to the masses of 

 leucocytes, and the individual leucocytes themselves were of a faint 

 yellowish-red colour. In the cases of the very granular pigmented 

 leucocytes the granules assumed a distinct red-brown colour. In 

 this way the distribution of the leucocytes and of the vessels which 

 contained them was mapped out. Very beautiful preparations of 

 the engorged green vessels were obtained by partially dissecting the 

 mantle in the fresh oyster so as to expose the ramifying vessels, then 

 hardening in alcohol, and subsequently treating with ferrocyanide 

 solution, when the vessels assumed a well-marked red colour; beautiful 

 results were also obtained by ammonia. Fresh blood obtained from 

 the heart in which vast numbers of the green leucocytes were 

 present also gave a red reaction with acidulated ferrocyanide solu- 

 tion. Control bloods from white oysters gave an exceedingly faint 

 or no reaction. 



* For the sake of uniformity we finally adopted the strength of solutions given 

 by Macallum in his paper on the " Distribution of Assimilated Iron Compounds," 

 ' Quarterly J ournal of Microscopical Science,' 1896. 



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