DS 22.20 



DYE STAINS OF SPECIAL APPLICATION 



439 



22.20 TYPICAL EXAMPLES 



Demonstration of mitochondria in 



the pancreas using the acid fuchsin- 



toluidine blue-aurantia stain of Kull 



1914 



There are so many modifications of the 

 method of demonstrating mitochondria 

 here described, that it seems desirable to 

 give a fairly detailed description of the 

 original technique, and to leave to the 

 reader the task of determining by subse- 

 quent experimentation which of the nu- 

 merous modifications he would prefer to 

 employ. The method here described, 

 which is most commonly referred to in the 

 hterature as Champy-KuU, has the ad- 

 vantage that it permits considerable ex- 

 perimentation under standardized condi- 

 tions, and jaelds reproducible results, once 

 the timing on any particular batch of 

 material has been estabhshed. The method 

 involves overstaining in acid fuchsin, 

 which is strongly absorbed by the nuclei, 

 the mitochondria, and any bacteria pres- 

 ent. The acid fuchsin is then differenti- 

 ated in a solution of toluidine blue, which 

 removes the acid fuchsin from the nuclei. 

 These remain stained blue, and finally 

 differentiation is conducted in aurantia 

 which removes the unwanted toluidine 

 blue from all parts of the cytoplasm except 

 the mitochondria. 



A word of warning may be inserted at 

 this point relative to the use of aurantia. 

 This dye is produced from diphenylamine, 

 to the derivatives of which many persons 

 are exceedingly sensitive. Those who have 

 suffered from dermatitis in the handhng 

 of photographic developers should under 

 no circumstances handle solutions of au- 

 rantia, for the dermatitis produced is diffi- 

 cult to get rid of and the sensitivity of the 

 w'orker to this reagent appears to be in- 

 creased by every exposure to it. The dry 

 dye itself is explosive and should always 

 be kept in solution. 



Pancreas has been selected as a demon- 

 stration object because it is both readily 

 obtainable, and also normally contains 

 very large and clear mitochondria. Baker 

 1933, 189 recommends the intestinal 

 mucosa of the white mouse for the same 



reason. The writer prefers the pancreas 

 because it is so much more easily fixed 

 than the mucosa of the mouse; if the in- 

 testine is used it must be split up, and the 

 piece pinned out flat, to enable the fixa- 

 tive to reach the epithehum. 



It does not matter which particular 

 animal is taken, but if mitochondria are 

 to be well demonstrated, it should be in 

 good health and should be killed by a 

 blow on the head rather than by narcotic 

 drugs. The pancreas is most readily ob- 

 tained in the following manner. 



Tie or pin out the animal on its back, re- 

 move the whole of the skin from the ab- 

 domen without breaking through the 

 muscular wall of the abdominal cavity, 

 and then remove the wall of the abdominal 

 cavity by an incision made round its 

 periphery. The very considerable loss of 

 blood which results from this may be ig- 

 nored. Now spread the pancreas on a piece 

 of glass by the following method. Hold the 

 glass by the edges in the right hand, and 

 Uft the stomach and upper portion of the 

 small intestine with the left hand, the in- 

 testine being spread away from the 

 stomach with the fingers. This leaves the 

 pancreas stretched in the mesentery. Now 

 lay the sheet of glass flat against the 

 mesentery, with the pancreas spread on 

 the surface, and then pass the stomach 

 and intestine from left to right, while 

 tipping the glass sUde upward, so that the 

 weight of the stomach and of the intes- 

 tine hanging down keep the mesentery 

 stretched over the surface. Run a sharp 

 knife round the edge of the slide so as to 

 cut through the mesentery, lea^dng the 

 pancreas exposed on the glass surface. It 

 is, naturally, necessary to lower the right 

 hand to prevent the weight of the stomach 

 and intestine from dragging the mesentery 

 off the glass when the first cut is made, 

 but a httle practice will enable one to ob- 

 tain the material spread cleanly on the 

 glass slide with httle trouble. Place the 

 slide, with its adherent mesentery flat in 

 a glass dish (naturally with the pancreas 

 on its upper surface) and flood with the 

 fixative. The classical technique of Kull 

 requires the osmic-chromic-dichromate- 

 pyrohgneous fixative of Champy 1913 

 (Chapter 18, F 1670.0080), which is ap- 



