The vital staining of mitochondria with janus green etc. 269 



The preparations may now be examined, preferably with Zeiss 

 apochromatic lenses, objective 1,5 mm and compensating ocular 4. Almost 

 immediately one of the preparations 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 the preparations last for from one hour and a half to two 

 hours. Evaporation may be reduced by putting a ring of vaseline 

 around the edges of the cover glass. 



Bensley has found that diethylsaf ranin , which may be readily 

 made from janus green, has also a specific affinity for mitochondria 

 and may be used to advantage as a vital dye. Since diethylsafranin 

 is red it is often of use in combinations with other blue and green dyes, 

 where janus green would be useless on account of the similarity in color. 



Diethylsafranin may be conveniently obtained from janus green 

 as follows: 



1. Make a saturated ablution of janus green in distilled water 

 in a flask. 



2. Add a little zinc dust and a few drops of hydrochloric acid. 

 The solution first assumes a bright crimson color and then bleaches, 

 the hydrochloride of the leucobase of the safranin being formed. 



3. Filter. Shake the filtrate in air and thus reoxidize the leucobase. 



4. Precipitate the dye by saturating the solution with sodium 

 sulphate. It is often necessary to use a little heat. A dark red 

 precipitate is formed. 



5. Filter. Collect the precipitate on the filter. Wash with a sa- 

 turated solution of sodium sulphate and dry it. 



6. Dissolve out the dye from the dried precipitate with absolute 

 alcohol. _ 



7. Filter and evaporate the filtrate to dryness. 



8. Dissolve the dye in the required concentration in distilled water 

 or in salt solution. 



It may be used in the same way as janus green but a stronger 

 solution of 1 : 1,000 must be employed. In order to obtain good results 

 it should be made up fresh every two or three days. It does not give 

 so uniformly constant results as janus green. 



