26 FIXING AND HARDENING. 



the volume of the objects to be fixed. If this precaution be not 

 observed the composition of the fixing liquid may be seriously 

 altered by admixture of the liquids or of the soluble substances of 

 the tissues thrown into it. For a weak and slowly acting fixing 

 agent, such as picric acid, the quantity of liquid employed should be 

 in volume about one hundred times that of the object to be fixed. 

 Reagents that act very energetically, such as Flemming's solution, 

 may be employed in smaller proportions. 



But fixation may also be performed by injection of the fixing liquid 

 into the objects, thus ensuring a more rapid and thorough penetration 

 of voluminous objects. See for this practice the methods of fixation by 

 injection of GOLGI, DE QUERVAIN, MANN, and others, given under 

 Nervous System. 



BRATJS and DRUENER (Jena Zeit. Naturw., Bd. xxix, 1895, p. 435) 

 fix fishes by injection through the bulbus aortce. The vessels are first 

 washed out with normal salt solution, and the fixing liquid is then 

 thrown in. 



KOLMER (Anat. Anz., xlii, 1912, p. 47) fixes thus even large mammals 

 (Chimpanzee, Goat). He first washes out with RINGER'S solution. 



It is well not to leave specimens in fixing liquids longer than is 

 sufficient to "obtain the desired reaction. Sublimate, for instance, 

 soon makes tissues brittle. But long immersion may be neces- 

 sary to produce the desired optical differentiation with some 

 reagents. 



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 impera- 

 tive. 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. 



It is important 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. Objects fixed in alcohol, formol, acetic acid, 

 picric acid, or nitric acid require to be washed out with alcohol, or 

 at least with some hardening liquid, whilst those that have been 

 fixed with osmic or chromic acid, or with one of the other compounds 

 of the heavy metals, require in general to be washed out with water. 

 Sublimate, however, is best washed out with alcohol. 



