58 A. B. MACALLUM. 
sections, of 8—10 mm. in thickness, are cut, and then by 
Schallibaum’s method fixed on the slide. After removing the 
paraffin, and washing the slide for some time in absolute 
alcohol, it is put for two or three minutes in a solution of 
saffranine of the strength recommended by Pfitzner: 1 grm. 
saffranine, 100 cc. alcohol, and 200 cc. distilled water. The 
slide is afterwards washed in distilled water to remove the 
excess of the colouring matter, and then put in absolute alcohol. 
The time during which the slide with the sections on it must 
lie in alcohol must be determined by experience, for if left one 
minute too long the whole of the saffranine is removed. The 
sections are cleared up in turpentine, and mounted in dammar 
or balsam. 
In spite of the uncertainty of this method of double staining 
there is always in each section, if it has not been allowed to lie 
toc long in alcohol, a number of places where neither too much 
nor too little of the saffranine has been extracted, and where 
one obtains, consequently, such relative effects of the two stains 
as Is indicated in figs. 1, 2. 
Of the various methods of employing gold chloride to 
demonstrate nerve structures in epithelium I have, after a 
careful comparison of the results obtained by each, adopted and 
followed one for the greater number of my experiments. I did 
not deem it of any value to try the method employed by 
Pfitzner, seeing that it is open to the objection urged by 
Mitrophanow against it, that chromic acid prepares non- 
nervous structures for impregnation with gold. Canini found 
the same method to give no results at all for the epithelium 
of higher Vertebrates. The method which I followed, and 
which was employed by Mitrophanow also, consists simply in 
treating the perfectly fresh tissue with a 1 per cent. solution 
of gold chloride for an hour, then washing it in distilled 
water, and finally placing it in a solution of formic acid, 
made in the proportion of one part of the acid to ten of dis- 
tilled water. Kept for about thirty hours in this fluid with 
complete exclusion of light the tissue will have at the end of 
that time a deep violet colour. To complete the success of the 
