Long-term preservation of Pastuerella piscicida by freezing or freeze-drying methods.

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This report describes the survival and pathogenicity of Pasteurella piscicida strain 5866, the pathogen of yellowtail, after frozen or freeze-dried storage. In the frozen storage, effect of freeze-thawing on the survival and maintenance of pathogenicity of the strain 5866 were also examined. Cultured cells of strain 5866 were suspended in glycerol (10, 30 or 50% V/V), defibrinated horse blood (10, 30 or 50% V/V), 20% skim milk (10, 30 or 50% V/V), horse or calf serum (50% V/V) or dimethyl sulfoxide (10% V/V) and then dispensed in sterile screw-capped vials. These suspensions were stored at -80°C. For assessment of the stress by freeze-thawing, the frozen cells with the above protective additives were subjected to repeated thawing in running tap water and again freezing at -80°C up to 11 times. For freeze-drying storage, freeze-drying media with serum or skim milk base were assessed. Harvested cells were suspended in freeze-drying media and stored at 4°C after freeze-drying. For assessment of pathogenicity, fish were infected by waterborn infection in sea water.Results of frozen storage experiments showed that none of the additives were sufficient to protect viability and pathogenicity of the strains longer than 52 months. After eleven repetitions of freeze-thawing in the presence of 10% glycerol, which was useful for good survival, the pathogenicity of the strain was maintained as low as that frozen and thawed only once. As for the freeze-drying storage, freeze-drying media containing skim milk or calf serum gave sufficient preservation of the pathogenicity with some high survivals rates. Finally, the medium consisting of 10% skim milk, 0.5% NaCl, 1% sodium glutamate and 5% sucrose proved to be the one conferring the highest survival and maintenance of pathogenicity.

Long-term preservation of Pastuerella piscicida by freezing or freeze-drying methods.

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