(54 THE MITOCHONDRIAL CONSTITtJENTS OF PROTOPLASM. 



carefully guarded against. Merely keeping the tissue in salt solution will often 

 cause the mitochondria to assume the most bizarre and perplexing forms. The 

 tissues should be rjuite fresh, particularly the pancreas, in which autolysis takes 

 place very rapidly. But the need for fresh tissue has been so much exaggerated 

 that it has discouraged the study of mitochondria without reason. In the nervous 

 system, for example, where autolysis is slow, the mitochondria may be advantage- 

 ously studied 6 or even 12 hours after death. Material awaiting fixation should 

 be kept in a cool place and precautions taken against evaporation. 



Poor penetration of the fixative is also well known to bring about definite 

 changes in the mitochondria. The mitochondria in the deeper parts of the tissue 

 become spherical and swell up. Indeed, when a fixative like the acetic-osmic- 

 bichromate mixture of Bensley is applied simply by immersion the action of each 

 of the ingredients may be seen. On the very surface the usual filamentous form 

 of mitochondria is maintained through the combined action of osmic acid, bi- 

 chromate, and acetic; a little deeper in, where the osmic acid has not penetrated, 

 the bichromate and acetic alone acting, the mitochondria are swollen up and 

 spherical; while in the central parts of the tissue the mitochondria have been com- 

 pletely dissolved through the ummodified action of the acetic acid. Simultaneous 

 action of the ingredients is satisfactory, but successive action is not. One comes 

 across varying degrees of artifact as one proceeds inward. Naturally, small pieces 

 of tissues alone can be used. They should be of not more than 4 cubic millimeters, 

 and it is better to have even smaller when the tissues are hard and firm and resist 

 the penetration of the fixative. It is unfortunate that investigators do not avail 

 themselves more generally of the method of applying the fixative by injection 

 through the blood-vessels. Naturally this can not be done with osmic acid on 

 account of the expense, but the mixtures of formalin and bichromate advocated 

 by Regaud will give excellent and uniform fixation in very tough and fibrous tissue 

 when applied by vascular injection. It is always necessary to wash out the blood 

 with salt solution before applying the fixative. 



That the concentration of the fixative is also of great importance has been 

 shown by N. H. Cowdry (1917, p. 200), who finds that very dilute solutions of 

 formalin cause a swelling of mitochondria and concentrated ones a shrinkage. It 

 is hkely that most mitochondrial methods bring about a slight shrinkage of the 

 mitochondria. 



Now, the fixative in the right concentration having come in contact with the 

 mitochondria, it becomes necessary to inquire whether it preserves them in their 

 true form without distortion. This is a difficult question to answer. In the 

 majority of cases with care and good fixation the original form of the mitochondria 

 is preserved. This was first shown with striking clearness by the Lewises (1915, 

 p. 347), who studied the living cells in tissue cultures and then actually watched 

 the process of fixation in them. When fixatives do alter mitochondria, they alter 

 them in a definite way. Filamentous mitochondria break up into granules and 

 spherules. Filamentous mitochondria are never formed by the fixative from 

 granules. 



