METHODS OF HARDENING. 291 



renders them insoluble. Similar events occur when Flemming's 

 solution is used for fixation, but here the osmic acid also enters into 

 some form of combination with the proteids and fats. It is not 

 unlikely that Orth's fluid owes its value in part to the formic acid 

 which formalin contains as the result of oxidation of the formalde- 

 hyde ; this acid having an action similar to that of acetic acid. 

 These recent analyses of the action of fixing solutions tend to render 

 many of the old formulae superfluous. Probably, all that is desirable 

 may be obtained by the use of three fixing solutions : first, Flem- 

 ming's solution, which fixes admirably, but penetrates with difficulty 

 owing to the slow diffusion of the osmic acid, and renders the tissues 

 relatively refractory to dyes ; second, a formula containing potassium 

 bichromate or corrosive sublimate and acetic acid, such as Zenker's 

 fluid, which penetrates well and leaves the tissues in a condition 

 favorable for staining ; third, diluted formalin to which about 5 per 

 cent, of acetic acid has been added. This solution penetrates rela- 

 tively larger pieces of tissue readily and does not weaken their 

 affinity for dyes, but the fixation of the tissue elements is less 

 perfect owing to the comparatively slight coagulating action of the 

 formalin. 



Methods of Hardening-. 



Solutions of chromates, as Muller's fluid, will, after a time, con- 

 fer a pretty firm consistency upon tissues, and even render them 

 brittle. Tissues fixed in corrosive sublimate are also very much 

 hardened. But the usual practice is to harden specimens in alcohol 

 after fixation. To obtain the best results this hardening should be 

 done gradually, since immersion in strong alcohol is apt to produce 

 undesirable shrinkage, affecting the various tissue-elements in dif- 

 ferent degree. 



Seventy per cent, alcohol (736 cc. 95 per cent, alcohol to 264 cc. 

 water) is weak enough to begin with. After the tissues have been in 

 alcohol of that strength for twenty-four to forty-eight hours, accord- 

 ing to the size of the pieces, they are placed in 80 per cent, alcohol 

 (842 cc. 95 per cent, alcohol to 158 cc. water) for an equal length 

 of time, and then in 95 per cent, alcohol. From the 95 per cent, 

 alcohol thev are placed in absolute alcohol, if it be desired to embed 

 them in either collodion or paraffin. If they are not intended for 

 immediate use, they may be kept indefinitely in 80 per cent, alcohol. 



During the hardening it is best not to allow the tissues to rest 

 on the bottom of the vessel containing the alcohol, as they are 



