During fed-batch cultivation to produce heterologous protein with yeast cells, cell viability should be determined quickly. Indeed, the early arrest of cell proliferation is a phenomenon often observed in auxotrophic yeast strains such as Saccharomyces cerevisiae BY4741 and ascribable to stressful environmental conditions arising in fed-batch systems. Choosing a cell viability assay can be a challenging task because the growth arrest is not easily attributable to one of the many mechanisms of cell death. In this work, two techniques used to assay cell viability have been compared: the viable cell count on agar plate and the bioluminescence assay based on the luciferase reaction to measure the amount of ATP from viable cells. To compare the methods, either the maximum specific velocity of growth or its specific death rate (kd) at 50 and 53 ◦C of BY4741 strain, were determined. In the first case, both the viable count and the bioluminescence assay gave the same results, showing that the amount of ATP in exponentially growing cells correlates with cell viability. On the contrary, during thermal inactivation the kd value obtained via bioluminescence, resulted always smaller than that obtained by viable cell count even though it was always possible to correlate the kd value obtained by bioluminescence with that obtained by viable count, through a factor. Apparently, in the operative condition examined, cell death either did not lead to the loss of membrane integrity, nor allow the endogenous ATPases to destroy any remaining ATP; thus the ATP levels did not fall precipitously. In the light of the results obtained, due to the ease of use, high sensitivity and action in real-time, ATP-based bioluminescence assay is the natural candidate to replace the cell viable count method which contrarily requires many replicates and extended

Determination of yeast cell viability: viable count vs ATP-based bioluminescence assay

PACIELLO, LUCIA;PARASCANDOLA, Palma
2010

Abstract

During fed-batch cultivation to produce heterologous protein with yeast cells, cell viability should be determined quickly. Indeed, the early arrest of cell proliferation is a phenomenon often observed in auxotrophic yeast strains such as Saccharomyces cerevisiae BY4741 and ascribable to stressful environmental conditions arising in fed-batch systems. Choosing a cell viability assay can be a challenging task because the growth arrest is not easily attributable to one of the many mechanisms of cell death. In this work, two techniques used to assay cell viability have been compared: the viable cell count on agar plate and the bioluminescence assay based on the luciferase reaction to measure the amount of ATP from viable cells. To compare the methods, either the maximum specific velocity of growth or its specific death rate (kd) at 50 and 53 ◦C of BY4741 strain, were determined. In the first case, both the viable count and the bioluminescence assay gave the same results, showing that the amount of ATP in exponentially growing cells correlates with cell viability. On the contrary, during thermal inactivation the kd value obtained via bioluminescence, resulted always smaller than that obtained by viable cell count even though it was always possible to correlate the kd value obtained by bioluminescence with that obtained by viable count, through a factor. Apparently, in the operative condition examined, cell death either did not lead to the loss of membrane integrity, nor allow the endogenous ATPases to destroy any remaining ATP; thus the ATP levels did not fall precipitously. In the light of the results obtained, due to the ease of use, high sensitivity and action in real-time, ATP-based bioluminescence assay is the natural candidate to replace the cell viable count method which contrarily requires many replicates and extended
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11386/3808681
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