20 A. KOHN AND M. LION 



Among the many processes of drying or dehydration of 

 micro-organisms, freeze-drying has proved to be the least lethal 

 and injurious. It is used nowadays not only for the storage of 

 bacterial and viral strains but also for the preparation of living 

 vaccines for prolonged storage, for preservation of some tissue 

 used in transplantations, of plasma and of many labile biological 

 materials such as enzymes, antibiotics, hormones, etc. 



In the process of freeze-drying of micro-organisms, a suspension 

 of bacteria in a suitable medium is frozen (usually in a shell 

 form on the walls of an ampoule) by dipping the ampoule into 

 a carbon dioxide bath or into liquid air, and the water is then 

 removed from the frozen material by sublimation in a vacuum 

 apparatus. The rate of evaporation of water is such that the 

 energy required for it is provided by the drying of the material, 

 and thus the material remains frozen as long as any water is left 

 in it. When the material is dry, the ampoule may be directly 

 sealed in vacuo, or it may be filled before sealing with an 'inert' 

 gas such as hydrogen or nitrogen. 



This process of lyophilization, — based on the discovery of 

 Wollaston^^ in 1813 that water may be removed from ice by 

 sublimation, — was first applied practically by ShackelU^ in 1909 

 and later introduced for large scale preservation of biological 

 materials by Flosdorf and Mudd^^' ^^. The optimal conditions 

 in freeze-drying were extensively studied by Fry and Greaves^^ 

 and Fry-^. An important contribution to the development of a 

 practical suspension medium was made by Naylor and Smith^^, 

 and their medium, composed of thiourea, ammonium chloride, 

 sodium ascorbate and dextrin, is now widely used. 



Various workers have found empirically suspension media in 

 which micro-organisms were well preserved in a dried state and 

 remained viable after prolonged periods of storage. These media 

 range from skim milk to serum and a mixture of serum and 

 glucose and to Naylor's medium. 



Various theories have tried to explain why these particular 

 substances protect bacteria undergoing drying, but none of them 

 embraces all the materials involved or explains the results. 



