Preparation for Embedding 31 



shorter time. Impregnating with nitrocellulose has distinct advantages 

 when it is desirable to avoid heat and when a tissue becomes hard too 

 readily, or is too large for the paraffin technic. In nitrocellulose, shrink- 

 age is reduced to a minimum, whereas in paraffin it can amount to as 

 much as 8-20% of the original size. Gelatin can be used for extremely 

 friable tissue in the freezing technic, and water soluble waxes are used 

 when alcohols, hydrocarbons and the like must be avoided. 



Before embedding in either paraffin or nitrocellulose, all water must 

 be removed from the tissue. This dehydration usually is achieved in a 

 series of gradually increasing percentages of alcohol in water. Gradual 

 changing through 30, 50, 70, 80, 95% and absolute alcohol is said to 

 reduce some of the shrinkage occurrino" in the tissue. In cases where 

 time does not permit such a series, the 30% and 80% steps, and even 

 the 50% change, may be eliminated without great damage to the tissue. 

 The time required for each step depends on the size of the object — i/^ 

 to 2 hours, maybe 3 hours in extreme cases. A second change of absolute 

 alcohol should be included to insure complete removal of water. But 

 tissue should remain in absolute alcohol only long enough to remove 

 all traces of water; a total of 2-3 hours should be ample, even for large 

 pieces. Too long an exposure to 95% and absolute alcohol tends to 

 harden the material, making it difficult to section. 



There are other agents ^vhich are just as successful dehydrants as 

 ethyl alcohol (ethayiol). The ideal dehydrating fluid would be one that 

 can mix in all proportions with water, ethyl alcohol, xylene and paraffin. 

 Two such solutions are dioxane (page 39) and tertiary butyl alcohol 

 (biitanol). Absolute butyl alcohol is miscible with paraffin, and after 

 infiltration with warm (50°C) butyl-alcohol-paraffin mixture, infiltra- 

 tion with pure paraffin can follow. If isobutyl alcohol is used, there may 

 be some difficulty with impregnation, probably due to the limited mis- 

 cibility of this alcohol with paraffin and water. In both cases — tertiary 

 and isobutyl alcohol— there is more shrinkage than with other alcohols. 

 hopropyl (99%) alcohol (isopropanol) is an excellent substitute for 

 ethyl alcohol, and is sufficiently water free for use as absolute alcohol. 

 Actually isopropyl alcohol has less shrinkage and hardening effect than 

 ethyl alcohol; it is cheaper and free of internal revenue restrictions. One 

 disadvantage must be remembered, isopropyl alcohol cannot be used 

 prior to nitrocellulose embedding, since nitrocellulose is practically 

 insoluble in it. Also dyes are not soluble in it, so it cannot be used for 

 staining solutions. 



For the preparation of dilutions of ethyl alcohol, it is customary to 

 use 95% alcohol and dilute it with distilled water in the following 



