DEHYDRATION AN13 CLEARING /. ^/JiQ-t 



To save repetition, we shall in treating of stains suppose that, 

 with paraffin sections, the above preliminary steps have already 

 been taken, and further, that sections cut by a freezing microtome 

 are also in spirit and water. 



Dehydration and Clearing. It is convenient, first of all, to 

 indicate the final steps to be taken after a specimen is stained. 

 Dry films after being stained are washed in water, dried and 

 mounted in xylol balsam; wet films and sections must be 

 dehydrated, cleared, and then mounted in xylol balsam. 



Dehydration is most commonly effected with absolute alcohol. 

 Alcohol, however, sometimes decolorises the stained organisms 

 more than is desirable, and therefore Weigert devised the 

 following method of dehydrating and clearing by aniline oil, 

 which, though it may decolorise somewhat, does not do so to the 

 same extent as alcohol. As much as possible of the water being 

 removed, the section placed on a slide is partially dried by 

 draining with fine blotting-paper. Some aniline oil is placed on 

 the section and the slide moved to and fro. The section is 

 dehydrated and becomes clear. The process may be accelerated 

 by heating gently. The preparation is then treated with a 

 mixture of two parts of aniline oil and one part of xylol, and 

 then with xylol alone, after which it is mounted in xylol balsam. 

 Balsam as ordinarily supplied has often an acid reaction, and 

 preparations stained with aniline dyes are apt to fade when 

 mounted in it. It is accordingly often an advantage to use the 

 acid-free balsam supplied by Griibler. Paraffin sections can 

 usually be dehydrated and cleared by the mixture of aniline oil 

 and xylol alone. 



Sections stained for bacteria should always be cleared, at 

 least finally, in xylol, as it dissolves out aniline dyes less readily 

 than such clearing reagents as clove oil, etc. Xylol, however, 

 requires the previous dehydration to have been more complete 

 than clove oil, which will clear a section readily when the 

 dehydration has been only partially effected by, say, methylated 

 spirit. If a little decolorisation of a section is still required 

 before mounting, clove oil may be used to commence the 

 clearing, the process being finished with xylol. With a little 

 experience the process of decolorisation can be judged of by 

 observing the appearances under a low objective. 



THE STAINING OF BACTERIA. 



Staining Principles. To speak generally, the protoplasm of 

 bacteria reacts to stains in a manner similar to the nuclear 



