Team:ETH Zurich/lab/bead
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Bacteria are resuspended and diluted in NaCl-solution (0.9 % in H<sub>2</sub>O) so as to achieve the desired cell density. Here, we aimed at a comparably high concentration of 10<sup>7</sup> bacteria per bead (3 mm diameter). The resuspension was added to alginate (2.5%) to reach a alginate concentration of 2%. The viscous solution is filled into a syringe containing an appropriate needle device and extruded at a constant, slow velocity. Ideally, the droplets should fall form a height of approximately 30 cm into a 100 mM CaCl<sub>2</sub> solution. In the CaCl<sub>2</sub> solution gelling of the alginate droplets will occur instantaneously. To avoid excessive salt stress for the bacteria the beads should be transferred to a 10 mM CaCl<sub>2</sub> solution. | Bacteria are resuspended and diluted in NaCl-solution (0.9 % in H<sub>2</sub>O) so as to achieve the desired cell density. Here, we aimed at a comparably high concentration of 10<sup>7</sup> bacteria per bead (3 mm diameter). The resuspension was added to alginate (2.5%) to reach a alginate concentration of 2%. The viscous solution is filled into a syringe containing an appropriate needle device and extruded at a constant, slow velocity. Ideally, the droplets should fall form a height of approximately 30 cm into a 100 mM CaCl<sub>2</sub> solution. In the CaCl<sub>2</sub> solution gelling of the alginate droplets will occur instantaneously. To avoid excessive salt stress for the bacteria the beads should be transferred to a 10 mM CaCl<sub>2</sub> solution. | ||
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Revision as of 20:47, 15 October 2014
Beads
Overview
Properties
Na2+ alginate is a viscous liquid, however, upon addition multivalent ions such as Ca2+ cross-linking of the polysaccharides occurs. Thus gelling of alginate can be achieved by the addition of a Ca2+. For encapsulation the cells or macromolecules are added to Na2+ alginate and subsequently immobilized during the gelling process. In fact, the encapsulation process is mild and compatible with most living cells. The high porosity of the ionically cross-linked polysaccharide lattice allows diffusion of nutrients and other substances into and out of the bead. This property of Ca2+ alginate allows cultivation of bacteria inside beads and does not prevent communication via small molecules between colonies of different beads. Substances such as phosphate or EDTA are sequestrating Ca2+ and thus destabilizing the alginate gel[25]. This fact should be considered when choosing the cultivation medium.
Production
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