88 THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 



the same mitochondrion differ in this respect. In this way the entire cell assumes 

 a pink color, but the mitochondria are most intensely stained. 



The next change is a further reduction of the diethylsafranin to the leucobase. 

 The tissue bleaches. If the preparation is viewed with a low power, the perijihery 

 of the piece of tissue in contact with the surrounding fluid is colored green (stage 1) ; 

 nearer the center a band of pink is seen (stage 2); the center itself is color- 

 less; so that all stages in the reaction may be seen at one time. When the leuco- 

 base is once formed it is impossible to restain the mitochondria with either diethyl- 

 safranin or janus green. 



The reaction of mitochondria to janus green is essentially similar in other 

 tissues. I have elsewhere (19146, p. 276) described it in detail in human lymiiho- 

 cytes, which may be contrasted with pancreas-cells, since they float freely in a 

 fluid medium. 



All my attempts to make permanent preparations of these vitally stained 

 mitochondria, made on the basis of the fact that both the picrates and molybdates 

 of janus green are relatively insoluble in alcohol, proved futile. 



Frozen sections may be made of the tissues stained with janus green, but the 

 freezing generally brings about a destruction of the mitochondria. 



Certain conditions may be recognized which tend to inhibit the j anus-green 

 reaction. The mucus that is secreted by Planar ia and other invertebrates stains 

 intensely with janus green, but the mucus secreted by leeches does not prevent 

 staining of mitochondria in spermatogenesis. In this way a large amount of janus 

 green is removed from the solution, with the result that the concentration of the 

 solution in actual contact with the cells is much weaker than that originally applied. 

 Moreover, masses of mucus often surround the cells and form barriers against the 

 diffusion of the dye. Albumin acts in a somewhat similar way. When present in 

 sufficient amounts, as in the blood and tissue juices of Linmlus, Callinectes, etc., 

 it becomes coagulated and stained by the janus green. It then presents a very 

 confusing picture which is very likely to lead the unwary astray. A high con- 

 centration of sodium chloride (3 per cent) or potassium nitrate (3 per cent) will 

 prevent the janus green from acting. The greatest obstacle, however, to the gen- 

 eral use of janus green as a vital stain for mitochondria in all cells is its very limited 

 power of penetration. For this reason the staining of mitochondria in intact brain- 

 cells is very diflRcult. Here, however, another factor enters in, namely, the rapid 

 reduction of the dye to its leucobases by the reducing action of the tissue itself, 

 so that when the skull is opened up the brain is found to be colored red instead 

 of blue. I have attempted to prevent this reduction by bubbling pure oxygen 

 through the stain as it was being injected, without much success. It does not help 

 matters to remove the skull-cap. Vasodilators are of no assistance in bringing 

 a larger volume of solution in contact with the cells.- 



Conversely, other conditions favor the janus-green reaction. Of these, ready 

 permeability of the cells is by far the most important. It is highly desirable that 

 the janus green should be brought into immediate contact with the cells them- 

 selves. This indeed is the reason why the best results are always obtained in blood- 



