484 E. V. Cowdry, 



tions do not stain well with the acid fuchsin, or when the methyl green 

 or toluidin blue bleaches it ont, it is often advisable to treat them with 

 a 2,5 "/o aq. soin, of potassium bichromate for about 80 sec. and rinse 

 them in water after bleaching before staining in the acid fuchsin. i. e. 

 between steps 3 and 4.) 



When this method is used (fig. 1) the mitochondria appear stained 

 brig-ht red, the Nissl substance green or blue according to whether 

 metliyl green or toluidin blue has been used in the differentiation, 

 and the neurofibrils in the axone hillock light brown. The canalicular 

 system may also be seen winding in and out in the cytoplasm. In 

 such preparations the morphology of the mitochondria may be studied 

 Avith great precision. They are seen to be very minute straight or 

 curved rods, about three times as long as they are broad, distributed 

 equally throughout the cytoplasm, but oriented with their long axes 

 parallel to tlie neurofibrils in the axone and axone hillock, elsewhere 

 irregularly. 



8. Safranin-acid violet (neutral safranin), ijyronin-methijl Mue, 

 and safranin-methyl Mue. — The best fixations for the five mito- 

 chondrial methods already enumerated all contain osmic acid; but the 

 neutral dyes may be employed to best advantage after chrome-sublimate 

 and other fixations in which it has been omitted. 



The neutral safranin method as used by Bensley consists of: 



I. Fixation. 



1. Fix spinal ganglia of the pigeon for "24 hrs. at 40° C in chrome-sublimate; 



2,5 7ü aq. soin, potassium bichromate 100 ccm, 

 mercuric chloride 5 g. 



2. Wash, dehydrate, clear, imbed and section as indicated in method 6. 

 II. Staining. 



1. Preparation of the stain. Add slowly sat. aq. soin, of the color acid, 

 acid violet, to a sat. aq. soin, of the color base, safranin 0., contained in 

 a flask, until a precipitate no longer forms. The point of neutralization 

 may be roughly determined by dropping a little of the mixture on filter 

 paper from time to time until the outside red ring of safranin disappears 

 and the whole blot takes on a neutral tint. Filter. The filtrate should 

 be as nearly colorless as possible. Dry the precipitate on the filter paper 

 for 12 hrs. Collect it and make a sat. soin, of it in abs. ale. 



2. Pass sections down through two changes of toluol and abs. ale. in order 

 to remove all traces of paraffin or toluol which might interfere with the 

 staining. Then through 95 7o, 70 «/o and 50 "/o to aq. dist. 



3. Chrome and osmium fixed material must be bleached in potassium per- 

 manganate and oxalic acid (vide method 7), and sublimate fixed tissues 



