r II: General Statement of Methods 17 



No ideal single reagent has been discovered which meets all 

 of these requirements, hence it is customary to combine two or 

 more reagents which individually possess certain of these desirable 

 qualities. All of the best fixing reagents are mixtures. For 

 example, acetic acid is very generally used in fixing mixtures 

 because it penetrates well, produces good optical differentiation, 

 and counteracts the tendency of some reagents (e. g., corrosive 

 sublimate) to shrink tissues. Again, osmic acid, which is an excel- 

 lent fixing agent for very small pieces of tissue, penetrates very 

 poorly; consequently for most objects it must be mixed with 

 reagents which penetrate rapidly and thoroughly. 



Some fixing agents (corrosive sublimate, chromic acid, osmic 

 acid, etc.) enter into chemical combination with certain of the 

 tissue elements, others (alcohol, picric acid, nitric acid, hot water, 

 etc.) act by coagulating or precipitating certain constituents of 

 tissues. 



The chief object of hardening is to bring tissues to the proper 

 consistency for cutting sections. The process, although begun 

 ordinarily by the fixing agent, is usually completed in alcohol. 

 Some objects are not sufficiently hardened until they have remained 

 in alcohol for many hours, or even days. As a rule, tissues should 

 remain in alcohol of at least 70 per cent, strength for a minimum 

 of 24 hours after the preliminary operations of fixing, washing, etc., 

 before they are subjected to further treatment. 



WASHING 



Fixing agents ordinarily, with the exception of alcohol, must 

 be washed out thoroughly or they are likely to interfere with sub- 

 sequent processes. Aqueous solutions are washed out usually in 

 water or a low per cent, of alcohol; alcoholic solutions, with alco- 

 hol of about the same strength as that of the fixing agent. Wash- 

 ing usually requires from 10 to 24 hours, with several changes of 

 the liquid. If water is the washing agent it is best where prac- 

 ticable to use running water. 



Chromic acid and its compounds should be washed out in run- 

 ning water. This should be done in the dark in order that pre- 

 cipitation may be avoided. 



