Preparation of Uniformly Sized Agarose Microcapsules by Membrane Emulsification for Application in Sorting Bacteria

Abstract

In an attempt to develop a high-throughput culture process, bacterial cells were encapsulated in uniform-sized agarose microcapsules prepared by membrane emulsification method. A mixture of diluted bacterial cell suspensions and agarose aqueous solution (40 °C) was used as the water phase, and a mixture of liquid paraffin and petroleum ether that contained hexaglycerin penta ester (PO-500) was used as the oil phase. The water phase permeated through uniform membrane pores into the oil phase under the pressure of nitrogen gas to form a uniform water/oil (W/O) emulsion; the emulsion then was cooled to form uniformly sized gel capsules. The size of microcapsules can be controlled by membranes with different pore sizes, because the mean diameters of the capsules were proportional to the mean pore diameters of the membranes. These microcapsules, which contained bacterial cells, were transferred into a column vessel and were incubated for 14 days to form microcolonies within the microcapsules. The cultured microcapsules were analyzed by flow cytometry, and the microbial colonized capsules were separated from free-living cells and empty capsules, based on the distinctive light-scattering signature of the microcapsules. The result showed that membrane emulsification preserved the viability of cells and this method was effective to create niches for various bacterial cells growing in high-throughput cultivation vessels.