Lyophilization, or freeze-drying, involves the freezing of a culture followed by its drying under vacuum, which results in the sublimation of the cell water. The technique involves growing the culture to the maximum stationary phase and resuspending the cells in a protective medium such as milk, serum or sodium glutamate. A few drops of the suspension are transferred to an ampoule, which is then frozen and subjected to a high vacuum until sublimation is complete, after which the ampoule is sealed. The ampoules may be stored in a refrigerator and the cells may remain viable for 10 years or more (Perlman and Kikuchi, 1977).
Lyophilization is very convenient for service culture collections (Snell, 1991) because, once dried, the cultures need no further attention and the storage equipment (a refrigerator) is cheap and reliable. Also, the freeze dried ampoules may be dispatched as such, still in a state of 'suspended animation' whereas liquid nitrogen stored cultures begin to deteriorate. However, freeze-dried cultures are tedious to open and revitalize and several sub-cultures may be needed before the cells regain their typical characteristics. Overall, the technique appears to be second only to liquid nitrogen storage and even when liquid nitrogen is used makes an excellent insurance against the possibility of the breakdown of the nitrogen freezer. The technique is considered in detail by Rudge (1991).
Whichever technique is used for the preservation of an industrial culture it is essential to be certain of the quality of the stocks. Each batch of newly preserved cultures should be routinely checked to ensure their quality and such a procedure has been outlined by Lincoln (1960): a single colony of the culture to be preserved is inoculated into a shake flask and the growth of the culture observed to ensure a typical growth pattern; after a further shake-flask sub-culture the broth is used to prepare a large number of storage ampoules. At least 3% of the ampoules are reconstituted and the cultures assessed for purity, viability and productivity. If the samples fail any one of these tests the entire batch should be destroyed. Thus, by the use of such a quality-control system stock cultures may be retained, and used, with confidence.
Was this article helpful?