MITOCHONDRIA 



211 



MITOCHONDRIA 



bleach in from 30-60 min. to a colorless 

 leucobase. Mitochondria show up bril- 

 liantly in living, particularly tissue 

 culture, cells using dark-ground il- 

 lumination (see Strangeways, T. S. P. 

 and Canti, R. G., Quart. J. Micr. Sci., 

 1927, 71, 1-14). They may be also 

 photographed at high magnification 

 with the electron microscope (R. 

 Claude and E. F. Fullam, J. Exp. Med., 



1945, 81, 51-62; H. U. Zollinger, E.x- 

 perientia, 1950, 6, 16-17). 



Mitochondria can be isolated from 

 tissues by the process of differential 

 centrifugation (R. R. Bensley and N. 

 Hoerr, Anat. Rec, 1934, 50, 251, 499). 

 This technique has been developed by 

 Porter, K. and his colleagues (J. Exp. 

 Med., 1945, 81, 233-246); Claude, A, 

 (Science, 1943, 97, 451-456; J. Exp. 

 Med., 1944, 80, 19); Hogeboom, G. H. 

 and his colleagues (J. Biol. Chem., 



1946, 165, 615-630). These authors not 

 only developed the technique of isola- 

 tion of mitochondria by differential 

 centrifugation and were able to obtain 

 mitochondria which were morphologi- 

 cally identical with those in the living 

 cell, but by a series of chemical studies 

 were able to show that such isolated 

 mitochondria contain the greater part 

 of the respiratory enzymes of the cell, 

 a fact which suggests, not only that the 

 mitochondria may be the main respira- 

 tory centers of the cell but that they 

 can function as synthetic centers as 

 well. The conception of the respira- 

 tor}^ function of mitochondria was sug- 

 gested as long ago as 1912 by Kingsbury 

 A. (Anat. Rec, 1912, 6, 39). 



Isolation of Mitochondria. Hoge- 

 boom, G. H., Schneider, W. C. and 

 Pallade, G. E. (Proc. Soc. Exp. Biol. 

 Med., 1947, 65, 320-321) have described 

 a method of obtaining morphologically 

 intact mitochondria from rat liver. 

 Rat liver is homogenized by the method 

 of Potter, V. R. and Elvehjem, C. A. 

 (J. Biol. Chem., 1936, 114, 495-504) in 

 0.88 M. sucrose. The homogenate is 

 centrifuged 3 times at 600 g. for 10 min. 

 This removes nuclei and intact cells. 

 The supernatant is then centrifuged at 

 24,000 g. for 20 min. This brings down 

 the mitochondria together with a few 

 microsomes. Mitochondria obtained in 

 this way remain stable in form for 

 several days at 4°C. There are a 

 variety of methods for making perma- 

 nent preparations of mitochondria in 

 tissue sections. 



1. Altmann's method. See Carleton, 

 H. M. and Leach, E. H., Histological 

 Technique, Oxford, 1949. Fix in 

 Champy's fluid or in Flemming without 

 acetic. Postchrome for 3 days (trans- 



fer tissue direct to 2§-3% aq. potassium 

 dichromate). Wash 12-24 hrs. in run- 

 ning water, dehydrate, embed and sec- 

 tion. Bring sections to water, then: 

 (1) flood slide with aniline fuchsin 

 (aniline water, made by adding 10 

 cc. of aniline to one half or one litre 

 of hot aq. dest. in a flask and ashing, 

 cooling and filtering, 100 cc, acid 

 fuchsin 12 gm.). Warm slide with 

 bunsen flame till the stain steams (but 

 does not boil). Leave for 5 min. (2) 

 rinse rapidly in aq. dest. (3) differen- 

 tiate in slide jar of picric acid, sat. sol. 

 in ab. ale. 20 cc, 30% ale 80 cc. Dif- 

 ferentiation should be stopped when 

 the red dye has diffused out of the nuclei 

 and cytoplasm and the mitochondria 

 are bright red. (4) rinse in aq. dest. 

 (5) dehydrate rapidly, mount in bal- 

 sam. 



2. Heidenhain's iron hematoxylin 

 method. Tissues may be fixed in 

 formaldehyde, Helly's fluid, Zenker for- 

 maldehyde or Flemming-without-ace- 

 tic, for 24 hrs. Sections are mor- 

 danted in a 5% aq. iron alum for 5 to 

 24 hrs. according to the nature of the 

 tissue. Rinse in water. Stain in 0.5% 

 aq. hematoxylin for a period of time 

 equal to that of the iron alum treat- 

 ment. Rinse in water, differentiate 

 in iron alum solution and control dif- 

 ferentiation with microscope until only 

 nuclei and mitochondria are black. 

 Counterstain if necessary, dehydrate, 

 mount in balsam. 



3. Cain's method (Quart. J. Micr. Sci., 

 1948, 89, 229-231). Fix in Helly's fluid, 

 6 hrs. postchrome 48 hrs. at 37°C. in 

 sat. aq. potassium dichromate, wash 

 overnight in running water, embed in 

 parafRn wax and cut sections about 

 3 n. Bring sections to water treating 

 with iodine (i in 70% ale) and then 

 5% aq. sodium thiosulphate on the way. 

 Dry slide, except sections, flood with 

 aniline fuchsin, (see Altmann's method) 

 and heat until steaming as for Alt- 

 mann's technique. Wash off acid fuch- 

 sin with aq. dest. Irrigate with alka- 

 line solution (one drop of aq. sodium 

 carbonate in 10° cc. of aq. dest.). Dif- 

 ferentiate 30 sec. to I5 min. To stop 

 differentiation dip slide into 1% HCl. 

 Wash in aq. dest., counterstain 1% aq. 

 water-soluble methjd blue. Wash aq. 

 dest., dip into 1% acid 3 sec. only. 

 Wash aq. dest., dehydrate and mount 

 in balsam. 



The Bensley-Cowdry acid fuchsin and 

 methyl green method (Cowdry, E. V., 

 Contrib. Carnegie Inst., Wash., VIII, 

 1918) gives beautiful results. In it the 

 methyl green is used both as differen- 

 tiator and counterstain. 



