VII. QUICK-FREEZING AND FREEZING-DRYING 225 



4. Kolc of l*rotective Proteins 



It has been found that a protective protein greatly prolongs the 

 keeping qualities of the organisms, as Avell as greatly increasing the 

 percentage viability. Serum or plasma may be used but first must 

 be inactivated to remove bactericidal activity. Sterile skim milk 

 (autoclaved) provides a most satisfactory medium (6). Cultures 

 grown in liquid medium may be concentrated by centrifugation and 

 then resuspended in milk. Organisms grown on a solid medium 

 may be harvested by scraping into saline or milk. If saline is used 

 the suspension is then added to an equal volume of sterile skim milk. 

 This mixtvu'e is then distributed into small containers. Usually 

 large numbers of containers of culture are dried at a single time. 

 Small quantities per container are ordinarily adequate, as little as 

 0.05 ml. being satisfactory. It is highly important, however, to use 

 all-glass containers rather than containers that carry a rubber stopper 

 exposed to the atmosphere. Otherwise, diffusion of water vapor 

 through the stopper will quickh^ raise the moisture content of the 

 culture to a point where viability of many species is not maintained. 

 Microorganisms preserved in such small amounts are especially sen- 

 sitive to this condition, hence the necessity of using containers that 

 may be sealed by fusion of glass. 



5. Oxidation as a Factor in Preservation 



It is known that some substances after drying from the frozen 

 state may not retain activity in the presence of oxygen of the air, 

 even though completely dry and hermetically sealed. Typical of 

 such products are those of high lipide content. In such a case either 

 sealing under original vacuum or under an inert gas such as nitrogen 

 or argon is necessary. Siedentopf and Green {11) report that this is 

 the case with their distemper virus. It should be pointed out that 

 release of original vacuum with dry air and subsequent evacuation or 

 replacement of air with inert gas does not operate satisfactority in all 

 cases, since oxygen may be absorbed by the highly porous solid matter 

 and cannot be readily swept out for removal. Also, for distemper 

 virus the nitrogen must be purified by passage over hot copper (IS). 



Farr and Hiller (22) found that application of the method of 

 freeze-drying to oxygenated hemoglobin solutions resulted in prepara- 

 tions in which the hemoglobin had lost 25 to 30% of its oxygen-bind- 

 ing capacity, by change to methemoglobin. However, when hemo- 



