FIXING AND HARDENING 25 



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. 



Use liberal quantities of liquid for washing. 



For almost any objects which are to be washed out in water, 

 it is convenient to use wide-mouthed vessels containing the 

 animals or pieces of tissue. Gauze, or mosquito netting of a 

 mesh smaller than the objects, is tied over the mouth and the 

 vessel is placed under a running tap. 



Very minute objects may be tied in a special phial and placed 

 in a larger vessel, or alternatively distilled water may be pipetted 

 on them at intervals. 



The process of washing out is 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 temperature (Fol.). 



The correct period for washing out Golgi apparatus and mito- 

 chondrial material is extremely important (see § 688). 



36. Fixation of Marine Animals.* The tissues of marine 

 organisms are as a general ride more refractory to the action of 

 reagents than those of corresponding fresh-water or terrestrial 

 forms, and fixing solutions should in consequence be stronger 

 (about two to three times). It is generally recommended when 

 possible to make up such fixing solutions, using sea water instead 

 of aqua pura. 



Marine animals ought to he freed from the sea water adherent to 

 their surface before treating them either with alcohol or any fixing 

 reagent that precipitates the salts of sea water. If this be not 

 done, the precipitated salts will form on the surfaces of the 

 organisms a crust that prevents the penetration of reagents to 

 the interior. Fixing solutions for marine organisms should there- 

 fore be such as serve to keep in a state of solution, and finally 

 remove, the salts in question. If alcohol be employed, it should 

 be acidified with hydrochloric or some other appropriate acid. 

 Picro-nitric acid is a fixing reagent that fulfils the conditions here 

 mentioned. (On this subject see Mayer, in Mitth. Zool. Stat. 

 Neapel, ii (1881), pp. 1 et seq., and Allen and Browne in " Science 

 of the Sea," John Murray, 1912). 



37. Hardening. The process of hardening is distinguished from that 

 of fixing as being directed to the attainment of a degree of consistency 

 sufficient to allow of soft tissues being cut into sections without imbed- 

 ding. It is an after-process, and only ranks as a special method. 



INIethods of imbedding have now been brought to such a degree of 

 perfection that the thorough hardening of soft tissues that was formerly 

 necessary in order to cut thin sections from them is, in the majority of 

 cases, no longer necessary. But there are some exceptions. Such are, 



* Not yet properly studied. The subject is bound up with the matters 

 raised in § 31 . 



