FIXING AND HARDENING. 27 



soon as fixation is judged to be complete water is injected ; lastly, alcohol, 

 and the fishes are thrown into alcohol. Or, if chromic liquids have been 

 employed, the fishes are put direct into solution of Miiller. See also the 

 methods of fixation by injection of GOLGI, DE QUERVAIN, and MANX, given 

 under Neurological Methods. 



As regards the time during which fixing reagents should 

 be allowed to act, it is well, as a general rule, not to leave 

 specimens in them for a longer time than is sufficient to 

 obtain the desired reaction. Sublimate, for instance, soon 

 makes tissues brittle. But long immersion may be necessary 

 to produce the desired optical differentiation with some 

 reagents ; and I now think that the school of Flemming, who 

 sometimes leave tissues for many weeks in solution of 

 Flemming, may be right in their practice, for the special 

 objects that they have in view. 



Careful washing out (by which is meant the removal from 

 the tissues of the excess of uncombined fixative) is necessary 

 in order to get tissues to stain properly. But it is not always 

 equally imperative. Alcohol and formaldehyde do not require 

 washing out before staining ; acetic and picric acid only for 

 some stains ; sublimate will allow of staining even if not 

 washed out, but allows of a sharper stain if well washed out : 

 all osmic, chromic, and platinic liquids require very thorough 

 washing out. 



Be careful to use the appropriate liquid for washing out 

 the fixing agent after fixation. It is frequently by no means 

 a matter of indifference whether water or alcohol be employed 

 for washing out. Sometimes water will undo the whole work 

 of fixation (as with picric acid). Sometimes alcohol causes 

 precipitates that may ruin the preparations. Instructions on 

 this head are given where necessary. 



Use liberal quantities of liquid for washing. 



Change the liquid as often as it becomes turbid, if that 

 should happen. 



The process of washing out is often greatly facilitated by 

 heat. Picric acid, for instance, is nearly twice as soluble in 

 alcohol warmed to 40 C. as in alcohol at the normal tempe- 

 rature (Fol). 



30. Fixation of Marine Animals. In the case of marine 

 organisms it may be stated as a general rule that their tissues 

 are more refractory to the action of reagents than are the 



