242 W. J. M. SCOTT 



2) 1 per cent aqueous solution of potassium permanganate 30 seconds; 

 but this time must be determined experimentally. 



3) 5 per cent aqueous solution of oxalic acid also about 30 seconds. 



4) Rinse in several changes of distilled water about a minute. In- 

 complete washing prevents the staining with fuchsin. 



5) Stain in Altmann's anilin fuchsin, which is to be made up as 

 follows: Make a saturated solution of anilin oil in distilled water by 

 shaking the two together (anilin oil goes into solution in water in about 

 1 per cent). Filter and add 20 grams of acid fuchsin to 100 cc. of the 

 filtrate. The stain should be ready to use in about 24 hours. It goes 

 bad in about a month. To stain, dry the slide with a towel, except 

 the small area to which the sections are attached. Cover the sections 

 on the slide with a small amount of the stain and heat over a spirit 

 lamp until fumes, smelling strongly of anilin oil, come off. Allow to 

 cool. Let the stain remain on the sections for about 6 minutes. Re- 

 turn the stain to the bottle. 



6) Dry off most of the stain with a towel and rinse in distilled water, 

 so that the only stain remaining is in the sections. If a large amount 

 of the free stain remains it will form a troublesome precipitate with the 

 methyl green; on the other hand, if too much stain is removed the 

 coloration of the mitochondria will be impaired. 



7) Again dry the slide with a towel, except for the area covered 

 by sections. Allow a little 1 per cent methyl green, added with a 

 pipette, to flow over the sections, holding the slide over a piece of white 

 paper so that the colors may be seen. Apply the methyl green for 

 about 5 seconds at first and then modify the time to suit the needs of 

 the tissue. 



8) Drain off excess of stain and plunge the slide into 95 per cent 

 alcohol for a second or two, then rinse in absolute for the same time, 

 clear in toluol, and mount in balsam. 



Several difficulties may be met with: 1) The methyl green may 

 remove all the fuchsin, even when it is only applied for a short time. 

 This is due to incomplete mordanting of the mitochondria by the chrome 

 salts in the fixative. It may often be avoided, either by omitting the 

 treatment with permanganate and oxalic acid, or by treating the sec- 

 tions with a 2 per cent solution of potassium bichromate for a few 

 minutes immediately before staining (as advised by Benseley). The 

 action of the permanganate and oxalic is to remove the excess of bichro- 

 mate. 2) Or the fuchsin may stain so intensely that the methyl green 

 removes it very slowly or not at all. This, on the other hand, is due 

 to too much^ mordanting. It may be corrected by prolonging the 

 action of the 'permanganate and osalic. 3) Sometimes, after obtaining 

 a good differentiation, the methyl green is washed out before the slide 

 is placed in toluol. This may be avoided by omitting the 95 per cent 

 alcohol, by passing from the methyl green to the absolute direct. 4) 

 Unfortunately the stain is not very permanent. Under favorable con- 

 ditions it will last for 3 or 4 years. The fading in color is hastened by 

 light and b}^ heat, and it proceeds very rapidly in a damp atmosphere. 



