INACTIVATION OF VIRUSES 387 



To conclude this section we want to draw attention to studies of the 

 thermal denaturation of the complexes formed between TMV or influenza 

 virus and urea (Stanley and Lauffer, 1939; Scott and Lauffer, 1946b), and to 

 the studies of the thermal denaturation of different viruses at high pressure 

 (see p. 364). These theoretically interesting investigations cannot be discussed 

 in detail here. 



B. Resistance to Low Temperature and to Desiccation 



In practice, the stability of viruses at low temperature becomes a problem 

 in connection with longtime storage in the frozen or freeze-dried form. We 

 shall therefore treat freezing and drying in direct succession and do so mainly 

 by referring the reader to manuals and to a few pertinent publications. 



1. Freezing and Thawing 



The conditions prevailing in a frozen suspension of biological material 

 depend on the way in which cooling is carried out, on the temperature at 

 which it is held, and on the nature and concentration of solutes in the 

 medium at the time of freezing. The physical principles of ice formation at 

 different temperatures and of thawing, with special reference to biological 

 material, have been reviewed by Meryman (1956). Most of our knowledge 

 about the biological effects of freezing and thawing concerns animal cells and 

 bacteria; relatively few viruses have been directly tested for resistance to 

 repeated freezing and thawing. 



From the studies of heat inactivation we know that measurable rates of 

 inactivation are usually not encountered mitil the temperature is raised to 

 about 40-50°C. In a neutral medium most viruses should therefore be ex- 

 tremely stable at temperatures near zero. From studies on cells and bacteria 

 it would seem that damage caused by freezing, storage in the cold, and thaw- 

 ing is a result either of the high salt concentration created when most of the 

 water has crystallized out, or of enzymatic activities which may continue at 

 significant rates if the storage temperature is not too low. The salt effect is 

 difficult to study and to control, since inactivation may depend on transient 

 conditions prevailing only during freezing or thawing; such effects are prob- 

 ably best counteracted by adding glycerol or other protective substances. 

 Inactivation during storage due to enzymatic activity can be dealt with by 

 lowering the storage temperature; temperatures below about — 40°C. are 

 usually safe. 



Rivers (1927) studied the effect of repeated freezing (at — 185°C.) and 

 thawing on several viruses in different suspending media. The vaccinia-like 

 virus III was most susceptible; 12 cycles of freezing and thawing would kill 

 most of the particles, even in a highly protective medium. Vaccinia virus. 



