308 R. R. BENSLEY 



c. Formalin bichromate sublimate method: 



Fix twenty-four hours in the following solution : 



Neutral formalin 10 cc. 



Zenker's solution without acetic acid 90 cc. 



Stain, with neutral gentian, acid violet-safranin, or acid fuchsin-toluidene 

 blue (see below). 



d. Acetic osmic bichromate method: 



Fix tissues for twenty-four hours in the following : 



Osmic acid 4 per cent 2 cc. 



Potassium bichromate, 2.5 per cent 8 cc. 



Glacial acetic acid 1 drop 



Prepare sections, which must be less than 4 micra in thickness, for staining as 

 follows: The sections fastened to the slide by the water method are freed from 

 paraffin by toluol, then passed through absolute alcohol to water. They are then 

 treated for one minute with a 1 per cent solution of potassium permanganate, 

 then for the same length of time with a 5 per cent solution of oxalic acid, then 

 thoroughly washed in water, after which they may be stained by the acid fuchsin 

 methyl green method, the acid fuchsin toluidene blue method, the neutral gentian 

 method, or the safranin-acid violet method. 



In addition to the foregoing solutions the following were occa- 

 sionally employed: Benda's fluid for mitochondria; Zenker's 

 fluid; Hermann's fluid; Flemming's two mixtures; aqueous mer- 

 curic chloride; trichloracetic and trichlorlactic acids. 



6. Staining methods 



a. The neutral gentian method. Neutral gentian is the name 

 given to the neutral dye obtained when a solution of gentian- 

 violet (crystal violet) is precipitated by its equivalent of a solution 

 of orange-G. If the correct quantity of the orange-G solution 

 be added, practically complete precipitation is obtained, but 

 if excess of the orange-G solution be added, the neutral dye is 

 redissolved. In the latter case it is necessary to add more crys- 

 tal violet to the solution to secure complete precipitation. The 

 neutral dye thus obtained is practically insoluble in water, but 

 is freely soluble in alcohol or acetone, either of which may be used 

 to make the stock solution. For staining add the stock solution 

 of the neutral compound to 20 per cent alcohol until a solution 



