Quantification methods of determining brewer’s and pharmaceutical yeast cell viability: accuracy and impact of nanoparticles

Abstract

For industrial processes, a fast, precise, and reliable method of determining the physiological state of yeast cells, especially viability, is essential. However, an increasing number of processes use magnetic nanoparticles (MNPs) for yeast cell manipulation, but their impact on yeast cell viability and the assay itself is unclear. This study tested the viability of Saccharomyces pastorianus ssp. carlsbergensis and Pichia pastoris by comparing traditional colourimetric, high-throughput, and growth assays with membrane fluidity. Results showed that methylene blue staining is only reliable for S. pastorianus cells with good viability, being erroneous in low viability (R2 = 0.945; $$\widehat{\sigma }$$ σ ^ = 5.78%). In comparison, the fluorescence microscopy–based assay of S. pastorianus demonstrated a coefficient of determination of R2 = 0.991 at $$\alpha =0$$ α = 0 ($$\widehat{\sigma }$$ σ ^ = 2.50%) and flow cytometric viability determination using carboxyfluorescein diacetate (CFDA), enabling high-throughput analysis of representative cell numbers; R2 = 0.972 ($$\alpha =0$$ α = 0 ; $$\widehat{\sigma }$$ σ ^ = 3.89%). Membrane fluidity resulted in a non-linear relationship with the viability of the yeast cells ($$\alpha \ne 0$$ α ≠ 0 ). We also determined similar results using P. pastoris yeast. In addition, we demonstrated that MNPs affected methylene blue staining by overestimating viability. The random forest model has been shown to be a precise method for classifying nanoparticles and yeast cells and viability differentiation in flow cytometry by using CFDA. Moreover, CFDA and membrane fluidity revealed precise results for both yeasts, also in the presence of nanoparticles, enabling fast and reliable determination of viability in many experiments using MNPs for yeast cell manipulation or separation.

The Effect of a Magnetic Field on the Transport of Functionalized Magnetite Nanoparticles into Yeast Cells

“…The toxic effects of nanoparticles, including iron oxide nanoparticles, are described in the literature [60]. The studies cover both mammalian cells and yeast cells.…”

“…Van der Laan et al [58] characterized the oxidative stress in yeast cells after internalizing fluorescent nanodiamonds proposed as free radical biosensors. Eigenfeld et al [60] described the impact of nanoparticles on yeast cell viability and Moło ń [42] reported the antioxidant properties and toxic effect of the TEMPO spin label. Yeast cells were also applied under various cellular stress impacts, including oxidative, osmotic, and ethanol accumulation [59].…”

Studying the impact of cell age on the yeast growth behaviour of Saccharomyces pastorianus var. carlsbergensis by magnetic separation

“…An advantage of this method is that it is non‐invasive and does not influence cell viability, as it uses functionalised, superparamagnetic iron oxide nanoparticles. [ 39 ]…”

“…The viable and non‐viable cells were classified using a random forest model, [ 39 ] as published previously.…”

“…An advantage of our method is that it is non‐invasive and does not influence cell viability, due to the use of functionalised, superparamagnetic iron oxide nanoparticles (IONs). [ 39 ] Moreover, compared with FACS magnetic separation is both age‐specific and fast, since we used the chitin‐enriched bud scars for age differentiation. [ 40 ] Here, the signals of fluorescent bud scars and yeast cell autofluorescence overlap for differently aged yeasts, so data post‐processing is needed to obtain the cell age distribution.…”