PRESERVATION OF SPECIMENS IN PLASTICS 55 



fleshy animal or plant specimens, generally satisfactory results can be 

 obtained, when adequate facilities are available, through the use of a 

 special freeze-drying procedure. The preparation of tissue specimens by 

 freeze-drying and their subsequent embedment in methacrylate plastic 

 blocks was described by Strumia and Hershey in 1944 (19) ; and the 

 equipment used in connection with this work was described by Strumia 

 and McGraw (20) . The method 4 employed for preparing specimen mate- 

 rial in this manner consists of first wetting the specimen thoroughly and 

 then placing it on a base of ice in a metal pan and freezing it in a low- 

 temperature cabinet at between —20° and —25° C. (—4° and —13° F.). 

 As soon as the specimen is frozen, sufficient water is put in the pan to 

 form a layer approximately one-fourth inch deep. After this has frozen, 

 successive layers are added and frozen until the specimen is embedded 

 in a block of ice that covers it to a depth of at least one-fourth inch. It 

 has been found advisable to glaze large specimens by spraying them with 

 an atomizer during the initial freezing before embedment in the suc- 

 cessively frozen layers of ice. Material prepared in this way should be 

 kept in the frozen state until transferred to the drying apparatus, and, 

 if desired, it can be stored in this condition for many months without 

 apparent change. 



Dehydration is carried out in a vacuum system of the type employed 

 for drying biologicals from the frozen state, which must be capable of 

 maintaining a temperature of approximately — 15° C. (5° F.) in the 

 specimens while the ice in them is being removed by sublimation. During 

 dehydration, the ice blocks containing the specimens are kept in loose 

 gauze bags held in wire baskets, and are subjected to continuous drying 

 action until all moisture has been removed. This usually takes from 5 to 

 15 days, depending upon the size of the specimens. 



After being dehydrated, the specimens should be trimmed with a sharp 

 knife to remove all loose portions and then kept in completely dry condi- 

 tion in a desiccator until the time for impregnation and embedment. Best 

 results are insured by carrying out these final steps within a few days, 

 although it is usually possible to store the dry specimens for several 

 weeks without appreciable deterioration. While in the dry state, specimen 

 material prepared in this way appears pale and discolored but much of its 

 original color is restored as soon as the specimen is impregnated with 

 methacrylate monomer or partial polymer. 



In certain cases acceptable results with animal and human tissue 

 specimens can be obtained -by processing them with the Kaiserling or the 

 Klotz fluids (4, 9) , or with suitable modifications of them, and then dehy- 

 drating them in the usual manner, beginning with 70-percent and ending 

 with 100-percent (absolute) alcohol or other suitable volatile water- 

 removing agent The monomer impregnating and embedding operations 

 should be perfomed promptly upon completion of dehydration, and the 

 polymerization of the plastic during embedment should be carried out at 

 the lowest elevated temperature practicable in order to avoid undue 

 alteration of the original color. 



The preparation of tissue specimens by a modification of the Kaiserling 

 • method, which has been found suitable for this application, is described 



4 The procedure to be followed in applying this method is explained in detail in the 

 instruction sheets on embedding biological specimens in plastic, issued by the Rohm 

 ! and Haas Co., Philadelphia, Pa., Aug. 1947. 



