MITOCHONDRIA 



156 



MITOCHONDRIA AND BACTERIA 



janus green and cover eacli immediately. 

 Do not wait to cover until blood has been 

 added to all of them. The weight of the 

 cover glass is sufficient to spread the 

 mixture. If the right amounts of stain 

 and blood have been used the cover glass 

 will settle down on a very thin film of 

 fluid. If too much of either has been 

 used it will float on the fluid and it will 

 not be possible to see clearly. After 

 about 5 or 10 minutes the mitochondria 

 will be seen colored deep bluish green, 

 first in the lymphocytes and later among 

 the granules in the other white cells. 

 To study the preparations at leisure it 

 may be desirable to prevent evaporation 

 by ringing with warm vaseline. For 

 colored illustrations see Cowdry, E. V., 

 Internat. Monatschr. f. Anat. u. Phy- 

 siol., 1912, 29, 1-31. 



Another satisfactory method is to 

 supravitally stain the mitochondria in 

 the pancreas by vascular injection as 

 described originally by Bensley, R. R., 

 Am. J. Anat., 1911, 12, 297-388. About 

 1 liter of solution is put in a bottle, from 

 the bottom of which a glass tube leads 

 off, or from which the fluid is syphoned 

 through a bent glass tube. About 6 

 feet of rubber tubing connect this with 

 a glass cannula. The rubber tube is 

 supplied with a clamp. Artery forceps 

 do nicely. A guinea pig, or other animal 

 of suitable size, is killed and bled from 

 the throat because removal of a good 

 deal of the blood facilitates the injection. 

 The cannula is inserted into the aorta 

 through the left ventricle, or into the 

 thoracic aorta directly, and tied in 

 place. In the former case the branches 

 going up toward the head and arms must 

 be ligated. When all is ready hoist the 

 injection bottle about 4 or 5 feet above 

 the animal and remove the clamp. Open 

 the right auricle so that blood and solu- 

 tion can flow out. In about a minute 

 open the abdomen by a long medial 

 incision but do not display the pancreas. 

 To make sure that all the vessels in the 

 pancreas are being perfused by the solu- 

 tion it is desirable to momentarily clamp 

 the superior vena cava and thus let the 

 solution back up a little under pressure. 

 Now lay bare the pancreas. When 

 the optimum staining is obtained, 

 usually about 10 minutes after the be- 

 ginning of the injection, it should be 

 slightly swollen, owing to separation of 

 lobes and lobules by increase in tissue 

 fluid, and of a uniform fairly dark bluish 

 green color. Remove the pancreas and 

 place it in salt solution. For examina- 

 tion it is essential to take very small 

 pieces not more than 1 mm. in diameter. 

 Mount them in a little salt solution on 

 slides so that they will flatten by the 



pressure of the cover glasses, one piece 

 per slide. Study at low magnification 

 shows irregular masses of small deeply 

 stained cells. These are the islands of 

 Langerhans. It is in the acinous tissue, 

 which is less deeply colored, that search 

 should be made for the mitochondria. 

 Identify first the distal poles of the 

 cells cliarged with zyomgen granules. 

 Then look for greenish blue stained 

 mitochondria in the proximal parts of 

 the cells. After a time the oxygen in 

 the center of the tissue is used up, the 

 dye becomes bleached to a leucobase and 

 then to a pink colored base (diethyl- 

 safranin). This method of supravital 

 staining of mitochondria with janus 

 green can be used for any tissue in the 

 body. It is particularly recommended 

 for the pancreas because its lobules are 

 thin, and easily separated without 

 mechanical injury. 



Other supravital stains for mito- 

 chondria are Diethylsafranin, Janus 

 Blue, Janus Black 1, Pinacyanol and 

 Rhodamin B, which see. When a very 

 dilute solution of methylene blue is 

 applied to mitochondria in tissue cul- 

 tures they can be stained a brilliant 

 blue (Ludford, R. J., Arch. f. exp. 

 Zellf., 1935, 17, 339-359). It is not 

 unlikely that, in conditions difficult to 

 define, a considerable number of dyes 

 will color mitochondria supravitally. 

 When fixed tissues are to be used the 

 choice of method is important. The 

 difficulty with the osmic acid containing 

 fixatives is that they penetrate poorly. 

 The best fixative is the formalin bichro- 

 mate fluid of Regaud followed by mor- 

 danting with 3% potassium bichromate 

 and the best stain is probably Anilin- 

 Fuchsin Methyl Green as used by 

 Bensley. See methods of Altmann, 

 Benda, Champy-Kull, Regaud and Vol- 

 konsky, and AlcClung (pp. 265-274). 

 Mitochondria can now be collected by 

 Centrifugation and subjected to direct 

 chemical analysis (Bensley, R. R. and 

 Hoerr, N. L., Anat. Rec, 1934, 60, 

 251-266; 449-455). Valuable technique 

 for study of isolated mitochondria by 

 Electron Microscope has been intro- 

 duced by Claude, A. and Fullam, E. F., 

 J. Exper. Med., 1945, 81, 51-62. 

 Mitochondria and Bacteria. Demonstration 

 in the same cells. See Cowdry, E. V. 

 and Olitsky, P. K., J. Exper. Med., 

 1922, 36, 521-533, Cowdry, E. V., Am. J. 

 Anat., 1923, 31, 339-343. Stain as for 

 mitochondria with Anilin Fuchsin and 

 Methyl Green. Mitochondria are col- 

 ored crimson. When the bacilli are acid 

 fast as in leprosy they are colored a dark 

 reddish purple ; but when they are not 



