g6 MICROSCOPIC METHODS. 



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 dehy- 

 drated, cleared, and then mounted in xylol-balsam. 



Dehydration is most commonly effected with absolute alco- 

 hol. Alcohol, however, sometimes decolorises the stained or- 

 ganisms more than is desirable, and therefore Weigert devised 

 the following method for 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 sec- 

 tion 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. 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, for the same reason that xylol-balsam is 

 to be used for mounting films, viz., that it dissolves out anihne 

 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 decolqrisation 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 progress, not only of these processes but also 

 of staining, can be very .accurately judged of by observing the 

 appearance under a low objective. 



The Staining of Bacteria. 



Staining Principles. — To speak generally, the protoplasm of 

 bacteria reacts to stains in a rnanner similar to the nuclear 

 chromatin, though sometimes more and sometimes less actively. 

 The bacterial stains par excellence are the basic aniline dyes. 



