Frozen storage of Escherichia coli O157 in buffered peptone water and its detection on bovine carcasses

The adaptation of a standard Escherichia coli O157 isolation method involving immunomagnetic separation and a period of frozen storage was investigated. A series of experiments was designed to test the recovery of a bovine strain of E. coli O157 from buffered peptone water after a period of frozen storage at -80degreesC. The effects of the addition of glycerol at 5 and 10%, freezing time, the number of freeze-thaw cycles, the method of freezing and the method of thawing, the inclusion of a resuscitation-and-incubation step, and the sensitivity of the isolation method were investigated. The most effective method of storing frozen samples for 6 months and recovering strains of E. coli O157 after storage was found to involve 6 h of incubation of sample material in buffered peptone water at 37degreesC before frozen storage at -80degreesC with 10% glycerol, a rapid thaw after frozen storage, and resuscitation at 27degreesC for 1 h and incubation at 37degreesC for 1 h to allow freeze-injured and stressed bacteria to recover with a period of growth prior to immunomagnetic separation isolation. There was no significant decrease in log counts of a bovine strain E. coli O157 over 6 months of frozen storage in buffered peptone water with 10% glycerol. With this method, it was possible to isolate E. coli O157 from naturally infected bovine carcasses after a period of frozen storage.