CLEARING 487 



For the very numerous other fixing reagents and mixtures now 

 in use, and the manner of their employment, the reader must be 

 referred to Mr. Lee's ' The Microtomist's Vade-mecum.' 



After due fixation and washing, objects may be stained and 

 mounted in an aqueous medium in the manner directed above 

 (p. 481), if it be desired to prepare them in the wet way. But if 

 they are destined to be preserved in balsam, they must first, after 

 staining if required, be dehydrated and cleared. 



Dehydration is performed as follows: 'The objects are brought 

 into weak alcohol, and are then passed through successive alcohols 

 of gradually increased strength, remaining in each the time neces- 

 sary for complete saturation, and the last bath consisting of absolute 

 or at least very strong alcohol.' For instance, alcohol first of 30 per 

 cent, or 50 per cent., then 70 per cent., then U."> per cent., or, if the 

 objects be very delicate, 80 per cent., before the 95 per cent., the 

 last to be changed at least once. 



Clearing 1 . The water having been thus sufficiently removed, the 

 alcohol is in its turn removed from the tissues, and its place taken 

 by some anhydrous substance, generally an e.-sential oil, which is 

 iniscible with the material used for imbedding. This operation is 

 known as clt'iir'nn/. It is very important that the passage from the 

 last alcohol to the clearing agent be made gradual. This is effected 

 by placing the clearing medium under the alcohol. A sufficient 

 quantity of alcohol is placed in a tube (a watch-glass will do, but 

 tubes are generally better), and then with a pipette a sufficient 

 quantity of clearing medium is introduced at the, bottom of the 

 alcohol. Or you may first put the clearing medium into the tube, 

 and then carefully pour the alcohol on to the top of it. The two 

 tin ids mingle but slowly. The objects to be cleared, being now 

 quietly put into the supernatant alcohol, float at the surface of 

 separation of the two fluids, the exchange of fluids takes place 

 gradually, and the objects slowly sink down into the lower layer. 

 When they have sunk to the bottom (and the wavy refraction-lines 

 .at first visible round them have disappeared) the alcohol may be 

 drawn off with a pipette, and the objects will be found to be com- 

 pletely penetrated by the clearing medium. (It may be noted here 

 that this method of making the passage from one fluid to another 

 applies to all cases in which objects have to 1)6 transferred from a 

 lighter to a denser fluid for instance, from alcohol or from water 

 to glycerine.)' From The Microtomist's Vade-mecum.' 



Another method of passing the objects from the alcohol to the 

 clearing agent consists in giving them baths of mixtures of the 

 alcohol and the clearer, made gradually to contain a higher propor- 

 tion of the latter. 



All clearing agents are liquids of high refraction, having indices 

 of refraction not greatly inferior to that of the elements of tissues 



Sections on slides may be bleached in this way. Instead of hydrochloric acid, nitric 

 acid may be taken; in which case the active agent is evolved oxygen instead of 

 chlorine. This method serves also for removing natural pigments, such as those of 

 the skin, or of the eyes of Arthropods. For bleaching chitin of insects, not alcohol 

 but water should be added to the chlorate and acid.' (From 'The Microtomist's 

 Vade-mecum.') 



