Compact liquid nitrogen storage system yielding high recoveries of gram-negative anaerobes

Abstract

A simple and compact system suitable for the preservation of fragile gram negative anaerobes and other bacteria in liquid N2 has been developed. Polypropylene straws used as specimen containers can be used easily within glove bags of anaerobic chambers, and their small size greatly increases the number of cultures which can be stored. Ancillary equipment and methods developed are described. The overall system was tested, using Streptococcus mutans, Fusobacterium nucleatum, and Selenomonas sputigena. Various basal suspending fluids and cryoprotective supplements were studied. With fast rates of freezing and thawing, survival recoveries of the test microorganisms ranged from 80 to 100 percent of the input colony-forming units in a complex medium broth base without cryoprotective agent addition, and they consistently were 100 percent when 0.4 mM polyvinylpyrrolidine was used. Overall, cryoprotection by polyvinyl pyrrolidine was superior to that from glycerol or dimethyl sulfoxide, the latter yielding recoveries similar to or less than those obtained with no cryoprotectant additive. All microorganisms were recoverable after storage for 1 year.