26 FIXING AND HARDENING 



for instance, the cases in which it is desired to cut very large sections, 

 such as sections of the entire human brain. 



The reagents employed for hardening are for the most part of the 

 same nature as those employed for fixing. But it does not follow 

 that all fixing agents can be employed for hardening. Corrosive 

 sublimate, for instance, would be most inappropriate as a hardening 

 agent. 



The Practice of Hardening. Employ in general a relatively large 

 volume of hardening liquid, and change it very frequently. If the 

 volume of liquid be insufficient, its composition will soon become 

 seriously altered by the diffusion into it of the soluble substances of 

 the tissues ; and the result may be a macerating instead of a hardening 

 liquid. 



Hardening had better be done in tall cylindrical vessels, the objects 

 being suspended by a thread, or muslin bag, or otherwise, at the top of 

 the liquid. This has the advantage of allowing diffusion to take place 

 as freely as possible, whilst any precipitates that may form fall harm- 

 lessly to the bottom ; or, they may be laid on a layer of cotton -wool, or 

 filter-paper, or spun glass. 



In general, begin hardening with a zveak reagent, increasing the 

 strength gradually, as fast as the tissues acquire a consistency that 

 enables them to support a more energetic action of the reagent. 



Let the objects be removed from the hardening fluid as soon as they 

 have acquired the desired consistency. 



38. With regard to Shrinkage it has been known for years that 

 the critical period was mainly at dehydration and dealcoholisation. 

 Whence the use of such oils as cedar wood, followed by a washing 

 out in benzol or xylol. The introduction of fluids for imbedding, 

 which obviate the use of the xylol and ethyl alcohol, or carbon 

 bisulphide-alcohol stages, has done much to get over this shrinkage 

 (§ 125). For many years it has been usual to double-imbed in 

 celloidin and wax. This takes longer, but it does get rid of a 

 great part of the shrinkage. 



39. Fixation by Altmann's Freezing-Drying Method. Altmann 

 froze organs, or blocks of tissue, and dehydrated them at low 

 temperatures (— 15° C. to — 20° C.) in a vacuum, and showed 

 that such material was preserved without shrinkage and was 

 suitable for the localisation of substances normally within the 

 body (Altmann, Die Elementarorganisfyien und ihre Beziehungen 

 zur den Zellen, 1890, Leipzig : Veit). This method has received 

 scant attention until recently when Gersh {Anat. Rec, liii, 1932, 

 p. 309) greatly improved the apparatus required for the successful 

 application of this method. Briefly stated, the method, as 

 developed by Gersh, consists in freezing tissue in liquid air (by 

 simple immersion) and then dehydrating at about — 20° C. in a 

 vacuum. When completely dried the tissue is placed in melted 

 paraffin and after infiltration is imbedded. It may now be 

 sectioned and treated as desired. This method is finding wide 

 application, by Bensley and Gersh and their students, in the study 

 of cell structures which have been unaltered by the chemical and 

 physical reagents commonly employed in microscopic technique. 



