22 FIXATION 



Living cytoplasm commonly has a refractive index (r.i.) in the 

 neighbourhood of 1-353;*^^'*^^ that is to say, not very much 

 higher than that of the saline solutions in which cells are commonly 

 immersed for vital study. An aqueous solution of sodium chloride 

 at 0*9% has an r.i. of i*335.^^-"- When cells are examined alive in 

 such media, a w^ater-immersion objective w^ill give almost as good 

 resolution as a first-rate oil-immersion objective, for the high 

 numerical aperture of the latter will be partly wasted. One may 

 surround living cells with innocuous media of the same r.i. as the 

 cytoplasm ** and thus obtain slightly higher resolution (as well as 

 gaining other advantages), but the difference will not be great. As 

 soon, however, as a fixative acts, a profound change occurs. The 

 evidence suggests ** that the protoplasm is now represented by 

 interlacing sub-microscopic fibres having the r.i. of dry protein 

 (about 1*54). These fibres lie in water, if the fixative is aqueous; 

 this can be replaced by media of any desired refractive index. If a 

 medium of r.i. close to that of dry protein is used (Canada balsam, 

 for instance), two results ensue: almost perfect transparency is 

 obtained (which may be modified as desired by the use of dyes), 

 and oil-immersion objectives can be used at their full aperture. 

 This fact should not, however, be too strongly stressed, for the 

 making of a permanent microscopical preparation involves con- 

 siderable shrinkage of the tissues of organisms (p. 76), and this 

 reduces or nullifies the advantage of higher microscopical resolu- 

 tion. The advantage can be fully secured only if the object to be 

 examined happens to be unshrinkable by the processes involved 

 in making a permanent preparation. The valves of diatoms provide 

 an example. 



Some of the constituent parts of organisms do not require 

 fixation, because they are not subject to autolysis and are resistant 

 to bacteria and moulds and to most of the reagents ordinarily used 

 in microtechnique. Examples are chitin, cellulose, scleroproteins, 

 certain inorganic crystals, and amorphous silica. Most such sub- 

 stances not only do not need fixation but are not acted upon by 

 fixatives; or, if acted upon, may be dissolved (for instance, spicules 

 of calcium carbonate by fixatives containing acid). Many fixatives 

 leave droplets of triglyceride untouched ; these do not require to 

 be fixed if no lipid-solvent will be used subsequently. 



Apart from such substances as these, it is the main purpose of 

 fixation to alter the tissue-constituents in such a way as to render 

 them no longer subject to autolysis, to decay through the action 



