Abstract
Biosyntheses often fluctuate with the state of the cell in the cell cycle and on the capacity of the cell to access and metabolize a carbon source. Visualization of substrate uptake by individual cells, together with the simultaneous analysis of proliferation activity and the proportion of dead cells, facilitate reliable and quasi-online process optimization. Flow cytometry and Hoechst 33342 staining were used to follow proliferation activity of living Saccharomyces cerevisiae cells, whereas 2-NBD-glucose was employed to analyze the cells' substrate affinity. Propidium iodide was used to determine the proportion of dead cells. Calibration and verification experiments were performed with cells grown batch-wise as well as in transient state regimes. A new and rapid three-color assay was developed and tested under varying microenvironmental conditions. Live/dead cell states and the affinity to 2-NBD-glucose vs. proliferation states were determined during respiratory and/or fermentative modes of metabolism.
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