Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Abstract

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24–120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log 10 increase in survival after 2 months storage at 4 °C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 °C increased recovery by 1–2 log 10 survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 °C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 °C and re-hydrated at the same incubation temperature (37 °C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability ( K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 °C) predicted a very low K value of 1.5 × 10 −3. These results will be useful to the development of improved reference materials and samples held in culture collections.