Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Abstract

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled 1 . Video LinkThe video component of this article can be found at http://www.jove.com/video/4058/ Protocol 1. Cultivation and harvest of P. putida 1. Prepare a starter culture of Pseudomonas putida by inoculating 100 ml of tryptic soy broth (TSB) with a colony of P. putida cells cultured on agar supplemented with tryptic soy broth (TSA). Keep the culture at 30 °C on a shaking board set at 130 rpm. 2. After 7 hr transfer an aliquot from the starter culture equivalent to 1/10 of the final volume of the main culture to fresh TSB-medium. Keep the cells at 30 °C on a shaking board at 130 rpm for 16 hr. 3. After 16 hr of growth the cells are harvested by spinning the cell culture at 1,500 x g for 20 min at RT. Decant the supernatant and wash the cells by re-suspending the cell pellet is in a NaCl-solution that is isotonic to the growth medium. In this case a 150 mM NaCl solution was used. The cells are then centrifuged once more and the supernatant is decanted before resuspending the cells in the formulation medium, see step 3.2. Cultivation of other species1. To adapt the protocol to other bacterial species the cultivation and harvest procedures should be changed according to the requirements of that species. To avoid osmotic shock when handling the cells during harvest and the washing step(s), the osmolality of the solutions needs to match that of the growth medium at harvest. Formulation of cells1. Prepare the formulation solutions by weighing out the respective matrix components and dissolve them in water. To achieve isotonic conditions, eit...

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