Analysis of living S. cerevisiae cell states--a three color approach.
BACKGROUND
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.


METHODS
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.


RESULTS
A new and rapid three-color assay was developed and tested under varying microenvironmental conditions.


CONCLUSIONS
Live/dead cell states and the affinity to 2-NBD-glucose vs. proliferation states were determined during respiratory and/or fermentative modes of metabolism.
Department of Environmental Microbiology, Centre for Environmental Research Leipzig/Halle in the Helmholtz association, Leipzig, Germany.
J
J
Achilles
H
H
Harms
S
S
Müller
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D001562:Benzimidazoles / Q000737:chemistry; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D004271:DNA, Fungal / Q000032:analysis / Q000737:chemistry; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011419:Propidium / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014700:Verapamil / Q000494:pharmacology
D001562:Benzimidazoles; D002121:Calcium Channel Blockers; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D007211:Indoles; D011419:Propidium; C007293:DAPI; D003847:Deoxyglucose; D014700:Verapamil; D009327:4-Chloro-7-nitrobenzofurazan; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; C017807:bisbenzimide ethoxide trihydrochloride