210 • Technologies To Maintain Biological Diversity 



micro-organism is one way to present it, but 

 it is expensive both in materials and in labor 

 and does not ensure that the genetic stability 

 of the micro-organism will be maintained. Con- 

 tinuously subcultured organisms can adapt to 

 the specialized conditions of the laboratory and 

 take on characteristics different from those for 

 which they were originally isolated. Long-term 

 storage techniques that minimize such effects 

 have been developed. 



Storage of micro-organisms involves reduc- 

 ing metabolic rates and, thus, the rate at which 

 micro-organisms multiply and use nutrients 

 (19). All methods that reduce metabolic rates 

 cause loss of a certain percentage of the sam- 

 ple. Methods need to be developed, therefore, 

 that not only reduce metabolic rates but also 

 prevent decline in viability in order to prevent 

 loss of the strain. 



An additional time-consuming but crucial 

 task associated with storage technologies is 

 authentication (19). This task involves the main- 

 tenance of accurate records about the culture 

 history and diagnostic characteristics of the 

 strains in a collection. It also involves periodi- 

 cally recovering and culturing stored organisms 

 to determine their viability and to confirm pu- 

 rity and genetic stability. 



The majority of micro-organisms currently 

 preserved in culture collections are held by 

 freeze-drying or by ultra-freezing (cryogenic 

 storage) (16,19). These two methods permit stor- 

 age for extremely long periods of time (currently 

 as long as 30 years) (16). Other special meth- 

 ods for storage of micro-organisms are immer- 

 sion in mineral oil, low-temperature freezing, 

 and desiccation (16). 



Freeze*Drying 



Freeze-drying, or lyophilization, is now the 

 most commonly used storage technique for cul- 

 ture collections. Healthy microbial cells, grown 

 under optimal conditions, are dispensed in 

 small, sterile vials or ampules at a relatively high 

 concentration (e.g., 10** to 10' cells per mil- 

 liliter of solution). The vials are then quickly 

 frozen in a super-cooled liquid solvent bath or 

 in a mechanical ultra-freezer (at —60" C), and 



these frozen suspensions are placed under 

 vacuum to remove the water in them. The vials 

 are then heat-sealed under vacuum by melting 

 the glass tops with an air-gas torch and stored 

 at temperatures lower than 5° C. Lower stor- 

 age temperatures ( — 30° to — 70° C) may result 

 in lengthened viability. 



Chemical agents (cryoprotectants) that pro- 

 tect cells from damage caused by ice-crystal for- 

 mation during the initial freezing are commonly 

 added to cells before freeze-drying. The Amer- 

 ican Type Culture Collection (ATCC) routinely 

 uses 10 percent skim milk or 12 percent sucrose 

 for such purposes. Curators at the Northern Re- 

 gional Research Laboratory of the USDA's Agri- 

 cultural Research Service, in contrast, prefer 

 bovine or equine serum as a cryoprotectant for 

 all microbial species (19). 



Recovery of the lyophilized cells is simple and 

 straightforward. The sealed vial is opened by 

 scoring, and a small amount of liquid-nutrient 

 medium is added to rehydrate the cells. The 

 contents, once rehydrated, are transferred to 

 a culture vessel containing a medium appro- 

 priate for growth. 



The initial cost of equipping a laboratory to 

 undertake lyophilization is as much as $25,000 

 (11). The expense of actually preparing lyophi- 

 lized cultures, however, is low. The long-term 

 viability of such materials is excellent, and this 

 procedure is thus probably the most cost-effec- 

 tive means of microbial preservation in use 

 today (19). Unfortunately, some microbial spe- 

 cies do not fare well under these techniques, 

 and other storage methods must be used. 



Ultra-Freezing 



Fastidious microbial species (i.e., those with 

 complex nutritional requirements) that do not 

 retain viability under other preservation meth- 

 ods (e.g., plant pathogenic fungi) frequently can 

 be preserved by ultra-freezing (2,19). In this pro- 

 cedure, cells sealed in vials or ampules are fro- 

 zen at a slow cooling rate (1" C per minute) un- 

 til they reach — 150° C. The vials are then stored 

 at —150° to —196° C using liquid nitrogen 

 freezers. 



