THte MITOCHONDRIAL CONSTITUENTS OP* PROTOPLASM. 61 



chondria by the chrome salts in the fixative. It nyiy be avoided by omitting 

 steps (2) and (3) , or by treating the sections with 2 per cent potassium bichromate 

 for a few seconds just before staining (as advised by Bensley). The action of the 

 permanganate and oxaUc is to remove the bichromate. (2) The fuchsin may 

 stain so intensely that the methyl green removes it imperfectly or not at all. This, 

 on the contrary, is due to too much mordanting. It may be corrected by pro- 

 longing steps (2) and (3). (3) Sometimes, after obtaining a^good differentiation, 

 the methyl green is washed out before the slide is placed in toluol, in which event 

 omit the 95 per cent and pass to absolute direct. 



This fixative is a good penetrator, in which respect it is much superior to 

 Altmann's fluid or Bensley 's mixture; but it makes the tissues very brittle and 

 difficult to cut. The staining is satisfactory and unifornr. 



(2) Benda's (1901, p. 155) crystal violet ahzarin method: 



Fixation: 



(1) Flemming's fluid, 8 days. 



(2) Wash 1 hour, half pyroHgneous and 1 per cent chromic acid, 24 hours. 

 (.3) 2 per cent potassium bichromate, 24 hours. 



(4) Wash in running water 24 hours, dehydrate, and embed in paraffin. Cut sections 5/i. 



Staining: 



(1) 4 per cent iron alum, 24 hours. 



(2) Rinse in water and bring into an amber-colored solution of sodium sulphaUzarinate, made by adding a satu- 



rated alcoholic solution to water, 24 houi's. 



(3) Blot with filter-paper and stain in equal parts of crj-stal violet solution and water. (N. B. — The crystal 



violet solution made of cone. sol. crystal \iolet in 70 per cent alcohol 1 volume, alcohol 1 volume, and anilin 

 water 2 volumes. 



(4) The solution is warmed until the vajior arises and then allowed to cool for ,5 minutes. 

 (.')) Blot, then 30 per cent acetic acid 1 minute. 



(6) Blot, plunge in absolute alcohol until but little more stain comes off, clear in xylol, and mount in balsam. 



A useful modification is given in Meves and Duesberg (1908, p. 573). Success- 

 ful Benda preparations are excellent. The mitochondria are stained a deep violet 

 color against a rose background. They are also much more permanent than 

 Altmann preparations. Unfortunately the method is long, tedious, and difficult. 



(3) Regaud (1910, p. 296) has used the iron-hematoxylin method of Heidenhain 

 after a large variety of fixatives, the best of which is his formahn and bichromate 

 mixture. 



Fixation: 



(1) Fi.\ in 3 per cent potassium bichromate 80 volumes, commercial formalin 20 volumes, for 4 days, changing 



every day. 



(2) Mordant in 3 per cent bichromate for 7 days, changing every second day. 



(3) Wash in running water 24 hours, dehydrate, clear, embed, and section as indicated (p. .58). 



Staining: 



(1) Pass down to water as indicated (p. 59). 



(2) Mordant in 5 per cent iron alum at 3.5° C. for 24 hours. Rinse in aq. dest. 



(3) Stain for 24 hovu-s in hematoxylin made up as follows: Dissolve 1 gm. pure crystals of hematoxyhn in 10 c.c. 



of absolute alcohol and add 10 c.c. of glycerin and 80 c.c. of distilled water. 



(4) Differentiate in 5 per cent iron alum under microscope. 



Note. — The crucial point in the technique is passing from the mordant to hematoxyhn. The slides 

 must be rinsed in distilled water, otherwise the iron alum will form a dense black precipitate in the stain. 

 On the ether hand, if they are rinsed too much, all the iron alum mordant will be removed. It is neces.sary 

 to strike the happy mean in which a darkening of the hematoxylin alone occurs. It is always difficult to 

 get good hematoxylin, and I find it best to keep on hand a ripe alcohohc solution. 



This is the most permanent as well as the simplest of all mitochondrial stains. 

 It may be used in the damp climates of most of our marine biological laboratories, 



