388 S. GARD AND O, MAAL0E 



thougli more resistant, could be killed by repeated freezing and thawing when 

 diluted in Locke's solution. Herpes virus was stable in brain emulsion, but 

 sensitive if diluted in Locke's solution. Finally, a coliphage was found to be 

 stable hi broth irrespective of phage concentration, but susceptible in salt 

 solutions. 



Instructive data have been collected for freezing of influenza virus at higher 

 temperatures. Penttinen (1950) found that freezing at — 79°C. did not affect 

 the hemaggluting activity of centrifugally purified vaccine, but that subse- 

 quent storage at —10 or — 20°C. gradually destroyed the activity. Virus 

 preparations purified by adsorption elution were stable at these tempera- 

 tures. Greiff e^ al. (1954) made the following statements about the infectivity 

 of allantoic fluids: "... Five freeze-thaw cycles resulted in a fall in titre from 

 10^'^ to 10~"-^ cycles, 2, 3, and 4 causing much greater losses than cycles 1 

 and 5. Eapid cooling to — 40°C. or slow cooling to —80 or — 190°C. did 

 not cause significant titre loss, but rapid coohng to temperatures above 



— 40°C. or slow cooling to temperatures above — 80°C. caused definite 

 titre loss. Loss of titre on storage occurred only at temperatures above 

 -40°C." 



The protective effect of glycerol and other compounds has been studied for 

 various phages by Panijel et al. (1957). The practical aspects of freezing and 

 storage of viruses under different conditions have been reviewed by Harris 

 (1954). 



2. Desiccation 



A dried biological jDrei^aration resembles a frozen one in that both are 

 effectively desiccated; the first by sublimation or evaporation, the second 

 by isolation of the water in the form of ice crystals. By analogy with freezing 

 and tha-wdng, the transitions between the wet and the dry state may be 

 harmful. If effective drying is tolerated, storage at room temperature may be 

 adequate; if drying is less complete, the specimen may have to be kept at low 

 temperature and, from point of view of storage, the advantage of drying is 

 practically lost. 



We have seen that drying of virus preparations may be an essential step 

 in inactivation experiments (see Sections II, B, 1 and 2, and III, A). The 

 work of Lea and co-workers (Lea, 1947) shows that many viruses tolerate 

 drying in thm films if a protective agent like broth is added to the suspension. 

 Pollard and Reaume (1951) observed that the coliphages T3 and T7 could be 

 dried without serious loss of infectivity only if sublimation occurred at 



— 20°C. or less. Phage Tl requires no such precaution and it has therefore 

 been used extensively for studies of inactivation in the dry state. 



The general principles of freeze-drying will not be discussed here; the 

 reader is referred to the recent review by Harris (1954). The results obtained 



