HARDENING 733 



stand for two hours, after which it is neutralized by the gradual addi- 

 tion of 4 to 6 c.c. of glacial acetic acid, the mixture being conBtantly 

 stirred, and the latter portions of the acid diluted with four volumes 

 of distilled water, and added drop by drop. The acid soon changes the 

 color of the mixture from a purplish carmin to a bright crimson. Care 

 should be taken not to add too much acid. When properly prepared, 

 the sense of smell should detect both ammonia and acetic acid, and the 

 fluid should have a dark crimson color (the addition of too much acid 

 produces a brighter crimson). Should the mixture be slightly over- 

 acidified a few drops of diluted ammonia will restore the proper con- 

 dition. The carmin solution is now added to the gelatin mass, a little 

 at a time and with constant stirring, and the whole is filtered through 

 cotton flannel wrung out of hot water. 



The gelatin mass may be kept for a short time by being covered 

 with methylic alcohol, but is better used at once. 



The pressure required for injection may be obtained by the gentle 

 use of a hand syringe; by the displacement of the confined air in a 

 large bottle or carboy by tap water; or much better by the use of a 

 water blast, of which the small glass type is relatively inexpensive and 

 will furnish a pressure for injection about equal to 180 mm. of mercury. 

 The air outflow of the water blast is connected by rubber tubing with 

 a glass canula of proper size to fit the vessel injected, a Wolff bottle 

 containing the warm injection mass being interposed. I,f a manometer 

 is connected, by means of a T -canula, on the proximal side of the 

 Wolff bottle, a relatively even and accurately measured pressure is 

 assured. The amount of pressure should be at first low (20 to 40 

 mm. of mercury), and should be gradually increased up to, but not 

 much beyond, the normal blood pressure in the vessel injected. 



The injected organ is cooled rapidly in a refrigerator, or by being 

 packed in ice or immersed in ice water. After solidification small pieces 

 are immediately placed in 95 per cent, alcohol for fixation, dehydration, 

 and hardening. 



HARDENING 



After proper fixation nearly all tissues require to be further hardened 

 before satisfactory sections can be cut. This is accomplished by im- 

 mersion in alcohol until dehydration is complete. The process requires 

 from a day to a week, according to the size of the tissue and the volume 

 and strength of the fluid. Various strengths of alcohol are advised. For 



