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Method Article
Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.
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 controlled1.
1. Cultivation and harvest of P. putida
2. Cultivation of other species
3. Formulation of cells
4. Freeze-drying
5. Rehydration
6. Enumeration
7. Characterization of Formulation of Freezing Behavior
8. Surface Tension Measurements of the Hydrated Formulations
9. X-ray Analysis of the Dry Formulations
10. Electron Microscopy
Table 1 displays data on formulation composition, thermal events recorded by DSC during heating of the frozen formulations, structure of the dry samples and the surface tension of the formulation solutions. The Tg' of sucrose has been determined to -40 °C 2, 3and can be difficult to detect for sucrose concentrations below 20% w/w. The thermal event at -35 °C is probably related to the onset of ice dissolution2. The crystalline structure detected by X-ray ...
The motive for this study was to investigate some formulation properties that may be of importance for cell survival during freeze-drying. Although the intrinsic drying tolerance varies between different species, as illustrated in Figure 1A, the trend on how well the different formulations support cell survival is similar. It is informative to start with a comparison of sucrose and Ficoll. It is believed that a key factor for a formulation to support cell survival is the capability of the formulation ing...
No conflicts of interest declared.
The work was supported by The Swedish Foundation for Strategic Environmental Research (MISTRA) through the DOM program and Grant 211684 (BACSIN) from the European Union Community FP7 Framework program. We thank J. Engstrand for assistance in filming the X-ray analysis and L. Tang for helping with the conceptual narrative.
Name | Company | Catalog Number | Comments |
Name of the reagent | Company | Catalogue number | Comments (optional) |
Ficoll PM 400 | GE-healthcare | 17-0300-10 | |
HEC (Natrosol-M Pharm Grade) | Ashland | Gift from Ashland | |
HPMC (Methocel F4M) | Dow | Gift from the Department of Pharmacy, Uppsala University | |
Sucrose | Sigma-Aldrich | S2395 | |
NaCl | Sigma-Aldrich | 71376 | |
Tryptic Soy broth | Merck | 105459 | |
Tryptic Soy Agar | Merck | 105458 | |
Lyostar II | FTS Kinetics | N.A. | |
Pyris Diamond DSC | Perkin-Elmer | N.A. | |
Bruker AXS SMART CCD 1k Diffractometer | Bruker | N.A | |
Dual-beam FEI Strata DB235 FIB/SEM | FEI | N.A | |
Krüss Educational Tensiometer | Krüss | N.A. |
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