376 W. H. POOLE ON DOUBLE STAINING, 
mine. Of these the latter proved useful for detecting nuclei, 
but, the protoplasm of the cells remaining almost uncoloured, 
it was impossible to distinguish the shape of the different 
cells, a matter of the greatest importance where, as in the 
brain, cells are met with of such various shapes and sizes. 
Another great deficiency in the carmine-stained sections was 
the indistinctness of the fibres. In all cases a long time 
was required for the carmine to take any effect, sixty to 
seventy hours being insufficient to stain deeply. 
Hematoxylin produced much more successful results. In 
the first place, the fibres were almost always brought out 
distinctly ; and secondly, the cells with their processes 
were in many cases clearly defined. But still the cortical 
substance was frequently insufficiently stained even after 
twenty-four hours’ immersion in the staining fluid, which, 
owing to the use of alum, is sufficient to render the pre- 
parations too brittle to be easily mounted. ‘The special 
value of hematoxylin consists in the clearness with which it 
brings out the nuclei of the medullary substance and the 
fibres and cell-processes of the cortical substance; its fault 
is a want of depth in the colour of the cortical substance. 
Having found aniline-blue useful for staining some hard- 
ened tissues, I was led to try it in this case. The only 
virtue it had was that it stained the protoplasm of the me- 
dullary cells very darkly and always attacked them first; 
that is to say, its strongest point exactly agreed with the 
weakest point in hematoxylin. 
This led me to try double staining, and the results were 
fully up to my expectations. The following is the method 
of staining which I finally adopted. After from twenty 
to twenty-four hours’ immersion in hematoxylin I washed 
the preparation in weak spirit, and then in distilled water 
till all the spirit was driven out. I then immersed it in 
aniline for from half to three quarters of a minute; again 
washed it in spirit, and after the usual treatment mounted 
it in Dammar. 
The preparation of hematoxylin used was that recom- 
mended by Frey, i.e., a few drops of an alcoholic solution of 
the pure crystals added to a solution of alum in water. The 
latter I have used in the proportion of from 2°4 grains of 
alum to an ounce of water. The more alum there is in the 
solution the more rapid is the staining, but there is great 
danger of making a thin section too brittle by the use of 
much alum. The aniline I diluted sufficiently to be able 
to see through it pretty easily. 
The results obtained by this method are most satisfactory. 
