392 APPENDIX. 



The mixture is best prepared just before using, although it will keep for 

 some time without serious deterioration, if tightly stoppered. Owing to the 

 cost of the osmic acid, the solution should be used with economy. The 

 tissue, in pieces not over a few millimeters in size, remains in the mixt- 

 ure for 1-2 days, or even longer ; is then washed in running water for 12-24 

 hours and transferred, by ascending strengths, to 80 alcohol, in which it may 

 be preserved, the spirit being changed when cloudy. This reagent, al- 

 though somewhat expensive, is of great value in studies concerning celU 

 structure and mitosis, being one of the most reliable and accurate means of 

 fixation. Its power of uniform penetration, however, is very limited ; it is 

 necessary, therefore, to use pieces as small as possible, otherwise the desir- 

 able action of the osmic acid will be confined to the peripheral zone, whilst the 

 deeper parts of the tissue will be influenced chiefly by the chromic acid alone. 



Absolute Alcohol. This reagent is useful for fixing and hardening 

 certain tissues, as glands and blood-vessels, which remain in it at least 24 

 hours, although the time may be extended to several days. The alcohol 

 should always be changed within 3-4 hours and the tissue supported by a 

 layer of absorbent cotton. It is important that the alcohol be approximately 

 ' ' absolute, ' ' since, even when of 96 per cent, its action is very different. 



The hardening of the tissues preparatory to cutting sections in the 

 usual way, that is, after interstitial embedding, is of much less consequence 

 than accurate fixation, since, unless the material be unduly hardened, the 

 consistence of the mass sectioned is largely determined by that of the sup- 

 porting material the celloidin or paraffin. 



EMBEDDING AND CUTTING SECTIONS. 



Having secured properly fixed and preserved tissues by one or more of 

 the preceding methods, such tissue must be cut into thin sections, stained, 

 rendered transparent and mounted before it can be satisfactorily examined 

 under the microscope. Whether sectioning or staining takes precedence 

 depends upon the character of the object and the end in view. If the object 

 be an embryo or some structure for whose study it is desirable to secure a 

 series of sections in strict sequence, or sections of the least possible thickness > 

 the paraffin method is chosen. If, however, these particular features are un- 

 important and the production of thoroughly good preparations for general 

 histological study is the result in mind, the celloidin method offers advantages 

 and is usually selected. Further, if it is desired to cut serial sections of tissue 

 requiring only a single staple stain, as carmine or hematoxylin, to demon- 

 strate satisfactorily its general structure, much labor is saved and risk of los- 

 ing sections avoided by staining the object in toto before sectioning in par- 

 affin. Unless this is done, the supporting paraffin must be removed by 

 a solvent (xylol) and the sections, previously fixed to the slide, stained in 

 position, a procedure requiring considerable time and care when long series 

 are to be treated. Since, except for embryos and special work, the reten- 

 tion of the sections in strict sequence is not usually necessary, staining in 

 bulk is much less frequently followed than staining after cutting. When cut 

 in celloidin the sections may be conveniently treated in large numbers at one 

 time and contrast dyes employed with little additional trouble. 



Whichever method be chosen, celloidin or paraffin, the fundamental 

 principle is the same the complete impregnation or saturation of the object 

 with the embedding mass, so that when the tissue is cut into thin sections 

 even the most delicate and isolated parts shall be retained in position by the 



