24 FIXING AND HARDENING 



to be fixed whole, let openings, as large as possible, be first made 

 in them. 



The penetration of reagents is greatly facilitated by heat. You 

 may warm the reagent and put it with the object to be fixed in 

 the paraffin stove, or you may even employ a fixing agent heated 

 to boiling-point (as boiling sublimate solution for certain corals 

 and Hydroids, or boiling absolute alcohol for certain Arthropods 

 with very resistant integuments). But this should only be done 

 as a last resource. 



On the other hand, very cold reagents are nowadays used for 

 some purposes. 



Let the quantity of fixing agent employed be many times 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. 



Braus and Druener (Jena Zeit. Nnturw., 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 (Anal. Anz., xhi, 1912, p. 47) fixes thus even large mammals 

 (Chimpanzee, Goat). He first washes out with Ringer's solution. 



35. Washing Out. 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. 



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 



