THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 59 



SUPRAVITAL STAINING. 



The most satisfactory dyes for this purpose are janus green B, janus bkie, 

 janus black I, and diethylsafranin (Cowdry, 1916a, p. 430). Their action is 

 similar and is described in detail on page 92. They may be applied by immersion 

 or injection. 



IMMERSION. 



The best results are obtained with blood (Cowdry, 19146, p. 267) as follows: 



Janus green B should be employed in a concentration of about 1 : 10,000 in 0.85 per cent sodium-chloride solution. 

 A drop should be placed on a series of six or more slides. A small amount of freshly drawn blood is then 

 added to the dye and a cover-glass i.s immediately dropped on it. No attempt should be made to mix the 

 blood with the stain before covering. 



The preparations should now be examined. Almost inmiediately one of them 

 will begin to show mitochondria, first in the lymphocytes and later in the granular 

 leucocytes. Soon the mitochondria will be stained in all of them. Under favorable 

 conditions they will last for several hours. Evaporation may be reduced by put- 

 ting a ring of vaseline around the edges of the cover-glass. 



INJECTION. 



This method is most satisfactory with the pancreas (Bensley, 1911, p. 304) and 

 salivary glands. 



The animal is killed and janus green B is injected into the left ventricle or 

 aorta in a concentration of 1 : 10,000 of salt solution by gravity pressure. In order 

 to obtain a good penetration the return flow through the inferior or superior vena 

 cava, as the case may be, should be momentarily cut off by artery clamps. After 

 about 10 minutes' perfusion, small portions of the gland may be removed and 

 examined for mitochondria. When the desired intensity of staining has been 

 reached, the entire gland should be placed in salt solution pending examination. 



fied) 



FIXATION AND STAINING. 

 (1) Altmann's (1890, p. 27) anilin-fuchsin-picric acid method (slightly modi- 



Fiialion: 



(1) Fix small fragments of not more than 2 c. nmi. in equal parts of ."> per cent pot.assium bichromate and 2 per 



cent osmie acid for 24 hours. 



(2) Wash in water, 1 hour. 



(3) Dehydrate in 50 per cent, 70 per cent, 95 per cent, and absolute alcohol 24 hours each. 



(4) Half absolute alcohol and xylol, 3 hours. 



(5) Xylol, 3 hours. 



(6) 60° C. paraffin, 3 hours. Embed. Cut sections 4/1, and fix to slides by albumen-waler method. 



.'^taitiing: 



(1) Pass down through toluol, absolute alcohol, 95 per cent, 70 per cent, and 50 per cent alcohol, about .30 seconds 



each, to aq. dest. in staining jars. 



(2) Stain for 6 minutes in .\ltmann's anilin fuch-sin (anilin water 100 cc, acid fuchsin 20 gm.). The stain may 



be poured onto the slide and the whole gently heated over a spirit lamp. 



(3) Blot and differentiate by carefully flooding the section with a mixtuie of 1 part of .sat. ale. solution of picric 



acid and 2 parts of aq. dest., added with a pipette. During this operation the color can be best seen against 

 a white background. 



(4) Rinse rapidly in 95 per cent alcohol. Pass through several changes of absolute into xylol and mount in 



balsam. 



