16 A. KOHN AND M. LION 



aftei storage for 6 months and reconstitution were tested for 

 viability, biochemical reactions, serological specificity, virulence, 

 resistance to antibiotics, motility and pigment formation. Of 

 all the strains tested, only three strains of pneumococci. one 

 strain of meningococcus, one strain of TV. gonorhoeae and four 

 strains of//, influenzae were lost. All the viable strains preserved 

 their characteristics very well. 



Since the most efficient way of preserving micro-organisms in 

 a desiccated state involves a freezing stage, we will briefly 

 consider what happens to the micro-organisms when they are 

 frozen. 



Experiments of Becquerel^ showed that bacteria withstand 

 temperatures as low as 0.05" K. At very low temperatures ( — 190°- 

 — 272°) all cell constituents are vitrified and dissociation and 

 ionization are suppressed. At — 200° chemical reactions are 

 eight miUion times slower than at 20°. The molecular state of 

 some substances such as peroxides, catalase, hemoglobin and 

 cytochrome changes; Keilin^, using this phenomenon of change 

 in adsorption spectra in frozen material, found that the spores 

 of B. subtiUs contained 6% of the cytochrome content of 

 vegetative cells, a fact which he was not able to determine by 

 measuring the respiratory activity of the spores. 



Practically, such low temperatures (below — 196°) are not 

 employed for the preservation of viability in micro-organisms; 

 rather they are frozen and kept at temperatures around — 80°. 



It has been shown by Weiser and Osterud^^ that death by 

 freezing involves two processes, (a) a rapid or immediate death 

 caused by freezing and thawing per se, and (b) a 'storage death' 

 which is a function of time and temperature. Immediate death 

 occurs at a stage of freezing when extracellular ice formation is 

 being completed. As may be seen in Fig. 1, the rate of storage 

 death is not uniform: it is more rapid at temperatures above 

 — 30°, probably because the eutectic points of the solutes in 

 media used for freezing are around this temperature. 



Death of cells due to freezing was attributed by Haines^^ to 

 irreversible changes in the bacterial proteins, leading to their 



