58 THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. . 



IV. TECHNIQUE. 

 EXAMINATION OF LIVING CELLS UNSTAINED. 



There is no longer any question of the actual existence of mitochondria in liv- 

 ing cells. The}' may be studied in cells teased out in serum or physiological salt 

 solution, with direct illumination. It is possible to see them more easily in some 

 cells than in others on account of some variation in their refractive index or that 

 of the cytoplasm in which they are embedded. A remarkable phenomenon is fre- 

 quently seen in the pancreas and other tissues; the mitochondria, instead of being 

 visible with difficulty, flash out sharply and can be seen with the greatest clearness, 

 even though they are quite unstained. This may be due to a decrease in saturation, 

 because we are told that in substances like them outside the organism, a decrease 

 in saturation is accompanied by an increase in refractive index. Guilliermond 

 (1912o, p. 409) thinks that the high refractive index which the mitochondria some- 

 times possess results from the peculiar combination of lipoid with the albuminous 

 substratum of which they consist, which in his opinion may be disposed on the 

 surface, in the form of a fatty membrane. 



Mitochondria may also be examined with the ultra-microscope (as well as by 

 ordinary dark-field illumination) in the manner suggested by Regaud. No sys- 

 tematic attempts have been made to study them with light of different wave- 

 lengths. One of the best ways to study mitochondria in living cells is by means of 

 the tissue-culture method as advocated by the Lewises (1915, p. 339). 



VITAL STAINING. 



It has been claimed by Tschaschin (1912, p. 304) that the mitochondria may 

 be stained vitally with isamine blue (= pyrrol blue); but Scott (1915, p. 835) 

 demonstrated that this was not the case by bringing to Ught the mitochondria in 

 these blue-stained cells with janus green. Nevertheless Levi (1916a, p. 107), 

 still more recently, has come to the conclusion that the mitochondria in the cells 

 of tissue cultures may be stained with pyrrol blue. This confusion is due, in the 

 first place, to mistakes in the identification of mitochondria, and, in the second 

 place, to a lack of any general consensus of opinion as to just what constitutes a 

 staining of mitochondria. Some investigators would speak of mitochondria as 

 being specifically stained when others would be loath to do so; some are content 

 with a mere tingcing of mitochondria, which can with difficulty be seen and which 

 others would overlook. My own experience is that almost any coloring matter 

 will stain the mitochondria if it is used in sufficient concentration, and I am not at 

 all sure that the controversy is a profitable one. 



It is quite possible that in some cases the mitochondria are stained while in 

 others they are not. There is no a priori reason why the mitochondria should not 

 take up these vital dyes. In fact, such action would be in complete accordance 

 with Regaud's electosome theory (p. 118). 



