CHANGES IN PANCREAS, PHOSPHORUS POISONING 241 



The best preparations of mitochondria were obtained in pan- 

 creases fixed by injection through the blood vessels with a mix- 

 ture of neutral formalin and potassium bichromate in accordance 

 with instructions given by Cowdry ('16 a). Fixation by in- 

 jection in this way obviates the factor of poor penetration as 

 well as certain other undesirable accompaniments of the more 

 crude method of fixation by immersion only. This is even more 

 necessary in investigations on the central nervous system. I 

 venture to quote this method of technique in detail: 



Fixation. Chloroform the animal. Inject warmed 0.85 per cent 

 NaCl solution into the aorta through the ventricle. Clamp the vessels 

 to the part which it is not desired to fix. Continue the injection until 

 the salt solution is returned uncolored through the veins. Gravity 

 pressure of not more than 6 feet may be employed. 



Follow the salt solution with the formalin and bichromate mixture : 

 3 per cent potassium bichromate, 4 parts; neutral formalin, 1 part. 

 The potassium bichromate acts best when freshly prepared. Neutral 

 formalin is made from the commercial variety by the addition of mag- 

 nesium carbonate, a deposit of which should always remain at the 

 bottom of the formalin bottle. It is important that the pressure should 

 be at the maximum when the mixture is first injected, so that the blood 

 vessels may be fixed in a state of dilation. If the pressure is low when 

 the fixative comes in contact with the vessel walls they will be fixed 

 in a condition of collapse. It will then be difficult, or even impossible, 

 to obtain a complete injection. The injection should be continued 

 for about an hour. 



The organ is then dissected out and immersed in the fluid. It 

 should be cut into pieces not more than 1 cm. cube. The fixative 

 must be changed every day for 4 to 5 days, otherwise it undergoes a 

 change evidenced by a darkening in color. This change is accelerated 

 by light and by heat, so that the tissue should be kept in the dark and 

 in a cool place. Fixation may also be effected by simple immersion 

 of the tissue in the fixative, instead of by injection, but this procedure 

 is not recommended. 



After this prolonged fixation the tissue is mordanted in a fresh 3 

 per cent solution of potassium bichromate in which it remains for 8 

 or 9 days, changing every second day. 



Wash in running water for 24 hours. The object of this careful 

 washing is to remove most of the formalin and bichromate, for other- 

 wise the tissue will be extremely brittle and hard to cut. 



Dehydration and embedding. 50 per cent alcohol 12 hours; 70 per 

 cent and 95 per cent alcohol 24 hours each; absolute alcohol 6 to 12 

 hours; half absolute and xylol 6 hours; xylol 3 hours;- paraflfin 60°C. 

 3 hours; cut in 4 serial sections. 



Staining. 1) Pass the sections, mounted on slides, down through 

 toluol, absolute, 95, 70 and 50 per cent alcohol to distilled water. 



