390 APPENDIX. 



which they existed during life, it is necessary to kill the tissue rapidly, other- 

 wise all evidence of certain phases of cellular activity, as the figures of mitotic 

 division, may disappear during the slow death of the cells. 



This rapid killing of the tissues, known as fixation, is accomplished by 

 plunging the fresh, preferably still warm, material into some suitable fixing 

 solution. The solutions for this purpose are many, since some tissues fix 

 well in certain fluids, while in the same ones other tissues may be only in- 

 differently preserved. The general precautions to be observed in fixing 

 include: material should be in as small pieces as practicable, never over 2 cm. 

 thick and better not more than half as much. . Otherwise the penetration is 

 incomplete with corresponding imperfect fixation. The volume of fixing 

 fluid should be many (fifty or more) times that of the tissue. Further, the 

 fluid should be changed whenever it becomes turbid. This often happens 

 within the first few hours after the introduction of the tissue. The latter 

 should not be washed in water on being removed from the animal, but placed 

 directly, with the minimum handling, in the fixing fluid. A cushion of ab- 

 sorbent cotton affords desirable support and insures access of the reagent on 

 all sides. Among the most useful fixing reagents are the following. 



Zenker's Fluid. 



Potassium bichromate 2.5 gm. 



Sodium sulphate i gm. 



Mercury bichloride 5 gm. 



Distilled water, warm 100 cc. 



Just before using, to each 20 cc. of the above solution add I cc. of 

 glacial acetic acid. 



Place small pieces of tissue in a generous amount of the fluid for 10-24 

 hours ; then wash in running water for 1 2-24 hours ; transfer for a few hours 

 to alcohol of increasing (50, 65, 80) strength, in the strongest of which keep 

 until used. This fluid is an excellent fixative for cell-structure, but, in com- 

 mon with other solutions containing sublimate, has the disadvantage of re- 

 quiring subsequent special treatment to remove the crystals of the mercuric 

 chloride that are commonly deposited throughout the tissue. The removal 

 of the deposits of mercury is conveniently effected by placing the unstained 

 wet-cut sections for 15 minutes in iodine alcohol (3 drops tincture of iodine 

 to 15 cc. 90 per cent, alcohol). An additional 15 minutes in a dilute solution 

 of sodium hyposulphite (made by adding 10 cc. of a 2.5 per cent, aqueous 

 solution of the salt to 100 cc. distilled water) insures, in turn, the removal of 

 the iodine. If, however, the tissue is to be stained in bulk and cut in par- 

 affin, the deposits of mercury must be eliminated by adding the iodine (from 

 .25 to .5 per cent, of Lugol's solution) to the alcohol in which the tissue is 

 kept after fixation. The manipulator must assure himself, preferably by an 

 examination of a free-hand section, of the disappearance of the deposits, 

 otherwise, their removal after the tissue has been embedded in paraffin is 

 troublesome and often impracticable. 



Tellyesnizcky's Fluid. 



Potassium bichromate 3 gm. 



Distilled water 100 cc. 



To which is added just before using 



Glacial acetic acid 5 cc. 



