1891.] Cell Granules with Methylene- Blue. 257 



of the film of methylene-blue is shewn by the fact that the 

 rose-tint may be developed by granules of sizes varying from mere 

 points to spherules 2 to 4 \x in diameter. Nor does it depend upon 

 the solidity or fluidity of the staining substance; for the contents of 

 large vacuoles in the ectoderm cells of Daphnia frequently stain 

 an intense rose, the rest of the cell appearing blue. Lastly the 

 rose tint may be produced neither in the cell nor in the animal, 

 but (in the case of Daphnia) by the action on the dye of a sub- 

 stance poured by the ectoderm cells into the surrounding water. 



The hypothesis that the reaction is really of a chemical nature 

 is favoured (1) by the general fact that the imbibition of dyes by 

 the fresh unfixed cells is determined by the chemical nature of 

 those dyes — whether the pigment be basic, or acid ; and (2) by the 

 peculiar method of imbibition of dyes by fresh cells. If still living 

 cells, such as basophil blood corpuscles, are treated with either an 

 organic fluid or normal salt solution, in which a small quantity of 

 methylene-blue is dissolved it is noticed that imbibition of the dye 

 is coincident with the onset of death. So long as the cell remains 

 fully alive it resists infiltration by the pigment, and the granules 

 remain uncoloured. This condition may, especially with eosinophil 

 cells, last for hours. With the first onset of death the dye makes 

 its way through the protoplasm and the granules become coloured. 

 Later, when rigor mortis has become thoroughly established, the 

 nucleus and cell body absorb the dye, and appear blue. In other 

 words the first imbibition of the dye occurs at a period when the 

 complex cell protoplasm is commencing to disintegrate, and when 

 therefore profound chemical changes are taking place. 



In order to determine whether granules are of the rose or blue 

 staining varieties it is necessary to apply the stain in some rela- 

 tively innocuous fluid to the living cells; and subsequent treat- 

 ment with fixing reagents entirely obliterates the reaction. This is 

 because all fixing agents with which I have experimented have 

 some action on methylene-blue. Thus corrosive sublimate pro- 

 duces a rose-coloured modification, and converts blue staining into 

 violet or rose. Ammonium picrate produces a violet tint except 

 in the case of very intensely blue granules. Osmic acid converts 

 the rose into a blue tint. 



Rose-staining basophil granules have been found by me in free 

 basophil cells of Astacus, and of Vertebrates, in the ectoderm of 

 Daphnia, and of the Ammoccete larva of Lampreys, and in the 

 alveolar cells of salivary glands. 



The last two instances are of a specially suggestive nature, as 

 affording instances of cells containing at the same time blue and 

 rose-staining granules. In the cells lining the alveoli of the sub- 

 maxillary gland of a rabbit I have seen, after treatment with dilute 

 methylene-blue in normal salt solution, a zone of rose-coloured 



