34 ' Paraffin Method (chap. 3) 



student is too slow while making his transfers from benzene into paraf- 

 fin, the benzene may evaporate out of the tissue and leave air pockets. 

 These will not infiltrate with paraffin. Of the three reagents toluene 

 probably is the safest to use; it does not harden as excessively as xylene, 

 and it has a higher boiling point (111°C) than benzene, thus eliminat- 

 ing some of the hazards of evaporation. (Boiling point of xylene, 142°C.) 

 In conclusion, benzene produces less shrinkage than either toluene or 

 xylene. 



If tissue hardening does present a serious problem, then one of the 

 clearing oils can be used. Cedarwood oil is well known and is relatively 

 safe for the beginner, but overnight usually is required for complete 

 replacement of the alcohol in the tissue. Also, as is true of all oils, every 

 trace of oil must be removed during infiltration. Sometimes this condi- 

 tion is difficult to judge, since the oil may have a boiling point in the 

 200s and be slow moving out of the tissue. The action may be improved 

 by mixing oil with an equal amount of toluene. The cedarwood oil 

 method is an expensive one involving the use of a costly oil as well as 

 absolute alcohol. In this case the latter fluid may not be eliminated. 



Chloroform is used in many laboratories, but has outstanding dis- 

 advantages. It dessicates some tissues, connective tissue in particular, and 

 has a boiling point of 61 °C, making it highly volatile. Aniline can be 

 used with sfood results but it too is difficult to remove durinsr infiltra- 

 tion. A mixture of equal parts of oil of wintergreen (methyl salicylate) 

 and aniline followed by pure methyl salicylate, and then methyl sali- 

 cylate-paraffin offers quicker and surer results. Methyl salicylate may be 

 used alone; also there are bergamot, clove, creosote, terpinol and other 

 oils. Amyl acetate and cellosolve (ethylene-glycol-monoethyl ether. Car- 

 bide and Carbon Co.) do not harden excessively; the latter, however, is 

 highly volatile. 



If at any time during the clearing process the clearer (xylene, toluene 

 or benzene) becomes turbid, water is present and the tissue is not com- 

 pletely dehydrated. The only remedy is to return the tissue to absolute 

 alcohol to eliminate the water, then place it again in a fresh supply of 

 clearer. The solution originally used may contain water. Embedded 

 tissue containing water can shrink as much as 50%, and it offers diffi- 

 culties in sectioning and mountinsf sections on slides. 



"Clearing" may seem a strange nomenclature for this intermediary 

 step, but it happens to be a special property of the reagents mentioned 

 above. They remove, or clear, opacity from dehydrated tissues, making 

 them transparent. Blocks of tissue appear to deepen in color; also they 

 seem almost crystalline, never milky. 



