486 E. V. Cowdry, 



gamot oil and imbedded in paraffin (vide steps 3 to 7, method 6). 

 Such a preparation is illustrated in fig. 6. The mitochondria are 

 stained a very deep bhie-black color and their outlines are veiy 

 clear-cut and well defined. 



The important factor in all these methods is the fixation. A good 

 fixation for one method of staining is, in general, good for others. A 

 few exceptions must of course be made to 'this rule. Osmic acid and 

 potassium bichromate are so frequently used, because, in spite of their 

 poor powers of penetration, they are excellent cytoplasmic preser- 

 vatives. Acetic acid is often added in order to facilitate the penetra- 

 tion, and heating the fixative to about 40" C may be of advantage. 

 Too much acetic acid, on the other hand, dissolves mitochondria and 

 this is one of the chief reasons why these structures have been almost 

 completely neglected until quite recently. This explains the non- 

 appearance of mitochondria after fixation in Zenker's fluid. Alcohol and 

 coi'rosive sublimate are in general to be avoided as fixatives. 



It seems unnecessary to mention that the tissues must be abso- 

 lutely fresh at the time of fixation. The pieces should not be more 

 than 3 mm, thick, and equal penetration from all sides sliould be 

 assured, either by frequent agitation, or by placing a few layers of 

 filter paper in the bottom of the bottle. Mechanical injury must be 

 carefully avoided. Transferring tissues should be done by means of 

 forceps the ends of which have a little clean linen bound about them. 

 It is a good plan, in the case of spinal ganglia, to leave a fairly 

 long piece of peripheral nerve attached by which they may be moved 

 in manipulation. 



The nerve cells at the periphery of a block of fixed tissue gen- 

 erally present a condition more like the living state than those more 

 deeply situated. The reason is that the constituents of the best fixing 

 fluids are chosen with a view to balancing their swelling or shrinking 

 tendency, their coagulating or solvent action, to increase the rate of 

 penetration of the mixture, etc.; and they act first upon the outer 

 cells. Thus, when Zenker's fluid is employed, with a reduced acetic acid 

 content, the mitochondria are fixed and preserved in the outer layers 

 of cells only. 



