60 THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 



In this way the mitochondria are stained a beautiful crimson color against a 

 bright yellow background. It is the oldest and in many respects the best of mito- 

 chondrial methods; but it has two disadvantages — the fixative penetrates badly 

 and the colors fade rapidly. Accordingly, neutral balsam or damar should be 

 used, the specimens should not be exposed to direct sunUght or to heat, and they 

 should be kept in a dry place. 



This method has been greatly modified by Galeotti (1895, p. 466), Schridde (1905, 

 p. 696), Bensley (1911, p. 308), Freifeld,' Kull (1913, p. 153), Schirokogoroff (1913, 

 p. 523), Cowdry (19166, p. 30), Duesberg (1917, p. 469), and others. 



Bensley proceeds as follows: 



Fixation: 



(1) Fix in acetic, osmic, bichromate mixture (2.5 per cent potassium bichromate 8 c.c, 2 per cent osmic acid 



2 c.c, glacial acetic acid 1 drop) for 24 hours. 



(2) Wash, dehydrate, clear, and embed (p. .^9), except that bergamot oil is substituted for xylol. 



Staini?ig: 



(1) Pass down to water. 



(2) Dip in 1 per cent potassium permanganate about 1 minute. 



(3) Rinse in 5 per cent oxalic acid same time and wash in water. 



(4) Stain with anihn fuchsin as indicated (p. .iO). 



(.5) Differentiate in a 1 per cent aqueous solution of methyl green. 



(6) Rinse rapidly in 9.5 per cent alcohol. Pass through several changes of absolute into xylol and mount in 

 balsam. 



The use of permanganate and oxalic acid corrects excessive mordanting with 

 the osmic and bichromate. It may sometimes be dispensed with. The methyl 

 green, which was first used in this way by Galeotti, is a much finer contrast stain 

 than the picric acid and is also more permanent. The precautions already men- 

 tioned against fading should be observed. 



I make use of the following modification : 



Fixation: 



(1) Fix in Regaud's mixture (4 parts of 3 per cent potassium bichromate and 1 part of commercial formahn). 



The commercial formalin may profitably be neutralized by saturation with magnesium carbonate. The 

 mixture may be applied by immersion or injection, the latter being recommended for large objects. It 

 should be changed every day for 4 days and be kept in an ice-box (though this is not essential). Mordant 

 for 8 days in 3 per cent potassium bichromate, changing every second day. 



(2) Wash in running water over night. 



(3) Dehydrate, clear, and embed as indicated (p. 59). 



Staining: 



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



(2) 1 per cent potassium permanganate 30 seconds, but time must be determined experimentally. 



(3) 5 per cent oxalic acid 30 seconds. Note. — Steps (2) and (3) may usually be dispensed with. 



(4) Rinse in several changes of distilled water about 1 minute. Incomplete washing prevents staining with fuchsin. 



(5) Stain in Altmann's anilin fuchsin made up as follows: Make a saturated solution of anihn oil in distilled 



water by shaking the two together. Filter and add 10 gm. of acid fuchsin (Duesberg) to 100 c.c. 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 the stain and heat over a spirit lamp until fumes, smelling strongly of anihn oil, come off; 

 allow to cool; let the stain remain on the sections about 6 minutes; return the stain to the bottle. 



(6) Dry off most of the stain with a towel and rinse in distilled water, so that the only remaining stain is in the 



sections. If a large amount of the stain is left it will form a troublesome precipitate with methyl green; 

 on the other hand, if too much stain is removed the coloration of the mitochondria will be faint. 



(7) Allow a little 1 per cent methyl green, added with a pipette, to flow over the sections, holding the shde over a 



piece of white paper, so that the colors may be seen. Apply the methyl green for about 5 seconds at first 

 and modify as required. This is the crucial point of the method. 



(8) Drain ofT excess of stain, plunge into 9.5 per cent alcohol for a second or two. Then rinse in ab.solute alcohol, 



clear in toluol, and mount in balsam. 



Difficulties. — (1) The methyl green may remove all the fuchsin, even when 

 applied only for a short time. This is due to incomplete mordanting of the mito- 



'For which see Naegeli (1912, p. 30). 



