Chapter II: General Statement of Methods 21 



is not uncommon, in fact, to have triple and even multiple staining. 

 In such staining, the stains are sometimes applied consecutively ; 

 in other cases, at different points in the process of general manipu- 

 lation. Sometimes all the stains may be mixed together, so that 

 immersion of the sections in one liquid is all that is required for 

 double or multiple staining. 



A general rule in staining, especially for entire or bulky 

 objects, is that the specimen should be transferred to the stain 

 from a reagent in which the percentage of water is approximately 

 the same as that of the stain. The same is true when the 

 object is removed from the stain. For example, if the stain to be 

 used is an aqueous solution, the object should enter it from an 

 aqueous solution; if the stain is made up in 95 per cent, alcohol, 

 the object should enter from 95 per cent, alcohol, etc. For 

 reasons see "dehydrating." 



CLEARING 



In the vast majority of cases tissues are too opaque for satis- 

 factory examination until they have been treated with certain 

 clarifying reagents or clearers which render them more trans- 

 parent. 



Such reagents as glycerin, glycerin -jelly, etc., ' are used 

 when the object is to be cleared, without alcoholic dehydration, 

 directly from water. Usually, for permanent preparations, the 

 alcoholic dehydration method is employed and it then becomes 

 necessary to use a clarifying reagent which will replace the 

 alcohol and facilitate the penetration of the final mounting-medium 

 (balsam or damar). 



Perhaps the most useful and rapid clearer is xylol. Xylol, 

 however, is very sensitive to moisture and if the preparation has 

 not been thoroughly dehydrated the final mount will appear 

 milky. For this reason the beginner is recommended to use a 

 carbol-xylol mixture (see reagent 9, chap. i). Carbolic acid has 

 a great affinity for water, and the mixture will therefore clear 

 preparations that are not fully dehydrated. Cedar-wood oil, 

 though somewhat slower than xylol, is one of the best clearers. 

 It is also one of the safest, because tissues may be left in it 



