Ch. 8— Maintaining Microbial Diversity • 211 



Photo credit. W.H. Siegel 



Cells being dispensed into ampules to be frozen and 



stored in liquid nitrogen (- 196' C). The cabinet contains 



only sterile, filtered air to lessen the chances of 



contamination of the freeze preparation. 



Ptiolo credit: WH Siegel 



Ampules of freeze-drled or frozen living strains can be 



stored in mechanical refrigerators at -60° C (-76° F), 



in walk-in cold rooms at 5° C (40° F), or in vacuum-insulated 



refrigerators (above) automatically supplied 



wth liquid nitrogen at -196° C (-320° F). 



The cryoprotective agents necessary for this 

 procedure differ from the ones used in lyophiH- 

 zation. ATCC routinely uses a mixture of 

 glycerol (10 percent), dimethylsulfoxide (5 per- 

 cent), and nutrient medium for most bacterial 

 strains. These chemicals are taken into the cells 

 and protect the internal membranes from in- 

 jury caused by freezing. 



Cells stored at cryogenic temperatures must 

 be handled carefully when being recovered. Ice 

 crystals can form as vials are warmed and can 

 kill cells that would otherwise survive the tech- 

 nique. Loss of viability is minimal when the 

 sample is thawed rapidly. Sealed vials are thus 

 put in water at 37" C until all ice melts. Then 

 they are opened and the contents transferred 

 to nutrient medium. 



Ultra-frozen cultures must be maintained at 

 very low temperatures at all times during stor- 

 age. Liquid nitrogen freezers are therefore 

 needed. Proper precautions are important to 

 ensure that such freezers operate properly and 

 have sufficient supplies of liquid nitrogen cool- 

 ant over long periods of time. Thus, the tech- 

 nique can cost more than freeze-drying, both 

 in labor and in materials necessary to main- 

 tain storage temperatures. Ultra-freezing is re- 

 served for microbial species that are not amena- 

 ble to other, less costly procedures. 



Other Methods 



Microbial cell cultures can be stored for short 

 periods of time if the culture is overlaid with 

 sterile mineral oil. The oil prevents dehydra- 

 tion and reduces the metabolic rate of the organ- 

 isms (16). Cells are grown on either nutrient 

 gels or in broth cultures. After incubation and 

 growth, mineral oil is added to the culture to 

 a depth of about 2 centimeters. The cultures 

 are then stored at approximately 4° C. Recovery 

 is by procedures similar to those used for rou- 

 tine subculture. Although cultures preserved 

 in this way have remained viable for as long 

 as 3 years, the method is not considered appro- 

 priate for long-term storage of micro-organisms, 

 because cultures have to be recovered, authen- 

 ticated, and restored every few years (19). 



A few micro-organisms, like some of the ac- 

 tinomycetes and some soil-borne spore-forming 

 bacteria, can withstand normal freezing proc- 

 esses and retain both viability and genetic sta- 

 bility. Cultures are stored on a nutrient medium 

 at 0" to —20° C. Cells may remain viable for 

 as long as 2 years, but damage by ice crystal 

 formation is thought to be extensive (19). For 



