Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Abstract

This study presents an automated, image-based cytometry method for determination of total counts and viability of yeast cells by using a newly synthesized DNA fluorescent dye PO-TEDM-1 and a new Easycounter YC instrument. The synthesized polycationic asymmetric monomethine cyanine dye PO-TEDM-1 penetrates only into dead cells. The new fluorescent dye has high nucleic acid sensitivity and rapid interaction kinetics. The optimal concentration of the fluorescent dye for staining dead cells was 1 mg mL À1. The Easycounter YC system was used to determine the total cell count and viability of Saccharomyces carlsbergensis. The actual viability measured using the proposed method significantly correlated with the theoretical viability (R 2 of 0.9988). The optimal linear interval was from 1 Â 10 5 to 1 Â 10 7 cells mL À1. The coefficient of variation with Easycounter YC in the optimal range was 2.5-4%, whereas that of the manual hemocytometry method, in the same range was higher, 15-23%. We tested the procedure in a study of the total cell count and viability of yeast cells from the propagators in beer production as a function of the dilution. The proposed method can be used in assays involving simple cell counting and quality assurance in sample bioprocessing.

Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion

“…Flow cytometry. We incubated BY4743 at a concentration of 2.5 Â 10 5 per ml in TEA buffer (pH 8.6) with 0 to 10 mM DTT at 30°C for 0 to 90 min, and at each time point we removed 100 ml, added PI to a final concentration of 2 mg/ml, incubated samples for an additional 5 min to ensure all dead cells take up the dye (62), and measured PI fluorescence on a BD Accuri flow cytometer.…”

“… S. cerevisiae cells were treated as stated, stained with either 2 to 20 μg/ml of propidium iodide (PI) (Sigma), with concentrations within ranges reported for live/dead staining ( 4 , 61 , 62 ), and visualized under fluorescence microscopy using a tetramethyl rhodamine isocyanate filter.…”

Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

“…We incubated BY4743 at at a concentration of 2.5 x 10^5 per mL in TEA buffer (pH 8.6) with 0-10 mM DTT at 30C for 0-90 minutes, and at each timepoint, removed 100 uL, added PI to a final concentration of 2 ug/mL, incubated for an additional 5 minutes to ensure all dead cells take up the dye (61), and measured PI fluorescence on a BD Accuri flow cytometer.…”

“…Micrograms of reducing sugar released by invertase was measured as an A670nm and compared to a glucose curve(59).Propidium iodide stainingS. cerevisiae were treated as stated, stained with either 2 to 20 ug/mL of propidium iodide (PI) (Sigma), concentrations within ranges reported for live/dead staining(4,60,61) and visualized under fluorescence microscopy using a TRITC filter.…”

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy

“…This challenge could be overcome with suitable contrast agents or reporter genes, which would make cells more visible or highlight subcellular features and processes. In fluoresce microscopy, this function is provided by targeted small-molecule dyes and fluorescent proteins, which have revolutionized the utility of this technique in biological research (10)(11)(12)(13). Unfortunately, these same molecules are not effective as phase contrast agents due to their small refractive index difference relative to H2O and its similarity to other intracellular materials (14)(15)(16)(17)(18).…”