TECHNIC 503 



dition that is as near as possible to life, and that will withstand change 

 for some little time. Fixation results in a whitened appearance and a 

 firmness, or even a considerable hardening, of the tissue. The fixatives 

 used are usually mineral acids and salts, organic acids, and sometimes 

 heat. A natural death of the tissue would result in its speedy disorgan- 

 ization. 



The second step consists of the use of fluids that will further harden 

 and preserve the tissue, and at the same time will prepare the way for 

 subsequent treatment by removing all unnecessary or injurious sub- 

 stances. The fixatives, especially, must be removed to avoid artifacts, 

 due to crystallization, etc. Alcohol is a fluid admirably adapted to fur- 

 nish these two results, especially when water is first used, in some cases, 

 to remove substances insoluble in alcohol. Lastly, the tissue must be 

 dehydrated for purposes we shall soon see, and here again alcohol is the 

 best reagent. 



This decides the use of alcohol as fulfilling all purposes, and it is 

 used in gradually increasing strengths to prevent too rapid osmosis 

 and consequent distortion. Sometimes additional substances are used 

 hi the alcohol to help remove the fixative, as iodide of potassium after 

 mercury bichloride. 



The third step is decided by our ultimate object of getting the piece 

 of tissue embedded in paraffin (or in celloidin) so that it may be cut in 

 thin sections. Paraffin will not mix with alcohol, so an intermediate 

 fluid, some oil that will mix with both alcohol and with melted paraffin, 

 is employed and substituted for the alcohol. This step is known as 

 clearing. Common clearing reagents are cedar oil, toluol, xylol, etc. 

 Chloroform is also a valuable clearing reagent. 



No obstacle now exists to prevent melted paraffin from being sub- 

 stituted for the clearing reagent. This is done in an oven just warm 

 enough to keep the paraffin melted, and, when infiltrated, the mass should 

 be poured out into a paper box, the bit of tissue oriented, and the mass 

 cooled. When the block is cooled, we find that the tissue is completely 

 embedded in, and infiltrated by, the paraffin. The mass, which is hard, 

 can be cut with a knife into very thin sections without injuring the 

 tissue, or altering its structure or the relative position of its parts. 



The sections should now be caused to adhere to a glass microscope 

 slide, the paraffin melted off with xylol or other clearing reagent, and 

 the specimen then freed from the clearing reagent by alcohol and from 

 alcohol by water. The proper dyes will now stain the cell and its organs 

 with a differential stain that should enable all parts to be seen. When 

 stained, the slide must be dehydrated again, then cleared, and lastly 

 a drop of balsam placed on the section, which is then covered with a thin 

 cover glass. 



