4 



Dehydrating and Clearing 



Any object which is to be mounted in a resinous medium or embedded in 

 paraffin for section cutting will have to be "dehydrated" and "cleared" before 

 either of these processes can take place. Neither balsam nor paraffin are mis- 

 cible with water or even with alcohol. The processes of dehydration and clear- 

 ing involve soaking the specimen in some material which will extract the 

 water from it, and which itself is miscible with some other material in which 

 balsam and wax can be dissolved. The first of these reagents, used to extract 

 water from the specimen, is known as a dehydrating agent. The second reagent, 

 used for the removal of the dehydrating agent and in which balsam or wax 

 must be soluble, is known as a clearing agent, because most of the chemicals 

 used have a relatively high index of refraction and, thus, cause the object to 

 become more transparent. 



The commonest reagent used for dehydrating is ethyl alcohol, which is 

 available in most laboratories both as neutral grain spirits (95 per cent alcohol) 

 and absolute alcohol (100 per cent alcohol). The removal of the last 5 per 

 cent of water from neutral grain spirits is a very expensive operation, so that 

 95 per cent alcohol should be used wherever possible. 



Were the majority of specimens merely to be thrown in 95 per cent alcohol, 

 the violent diffusion currents which would be set up would result in the col- 

 lapse of cavities or in the distortion of the specimen. -It is customary, therefore, 

 to use these alcohols as a graded series. It is conventional today to employ the 

 series of 30 per cent, 50 per cent, 70 per cent, 90 per cent, and 95 per cent and 

 to pass the specimen from one of these strengths to the next, leaving it in 

 each sufficiently long to become impregnated. This series is not reasonable, 

 for there is a much greater and more violent diffusion current when a speci- 

 men is passed from water to 30 per cent alcohol than there is when a specimen 

 is passed from 70 per cent to 90 per cent alcohol. The author much prefers 

 to use the series of 15 per cent alcohol, 40 per cent alcohol, 75 per cent alcohol, 

 and 95 per cent alcohol, and would recommend this for the beginning student. 

 This series more nearly represents the intention of the worker, which is to 

 subject the specimen to a graded series of stresses rather than to a graded 

 series of alcohols. 



It is doubtful whether or not it is necessary even to use a series of alcohols 

 when the object to be dehydrated is a thin section attached to a slide. The 

 only purpose of using an intermediate concentration of alcohol between water 

 and 95 per cent is to avoid the rapid dilution of the latter by the water carried 



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