ALNOOR PIRANI (1), Leo Chan (1), Timothy Smith (1), Emily Lyettefi (1), Ning Lai (1), Bo Lin (1), Jean Qiu (1), Peter Y. Li (1)
(1) Nexcelom Bioscience, Lawrence, MA
Yeast viability is an important parameter that can affect
fermentation performance in a brewery, which can dictate the quality of
the end product. Traditional methods for determining viability involve
either counting cultured yeast colonies on agar or counting methylene
blue-stained yeast cells using a hemacytometer and a microscope. Faster
and more robust technologies such as flow cytometry or absorbance plate
readers involve the use of fluorescent staining rather than colorimetric
stains. Although standard practices, the traditional methods have some
advantages but also some well-recognized drawbacks. The hemacytometer
and microscope are an image-based technology, which allows counting of
single or chain-forming yeasts, but they are labor intensive and prone
to human error when evaluating multiple samples. Flow cytometry is an
automated and high-throughput cell-counting technology, but it cannot be
used for chain-forming yeasts. Previous comparative studies have shown
discrepancies in determining the appropriate yeast viability staining
assay, which may be due to the differences and drawbacks of traditional
and flow cytometry analysis technologies. Here, we present a recently
developed platform for bright field and fluorescence image-based cell
counting and viability measurements that allow for direct comparisons
between different fluorescent staining methods without the previously
mentioned limitations of manual counting and flow cytometry. The system
performs automated cell counting, which reduces assay time and is more
objective, allowing for higher throughput analysis and more robust
results. In addition, the imaging capability allows declustering of
chain-forming yeasts, which improves the accuracy of the results in the
presence of cellular aggregates. This system was used to compare three
fluorescent viability stains: bis-(1,3-dibutylbarbituric acid)
trimethine oxonol (DiBAC4(3), oxonol), propidium iodide (PI), and the
magnesium salt of 8-anilino-1-naphthalenesulfonic acid (MgANS) on
various yeast samples, which resulted in identical viability
determinations for the same sample, in contrast to previous
publications. We propose that this platform can be used for studies on
different yeast strains and various culture conditions to determine the
robustness of various staining methods with the goal of establishing a
standard, optimized method for yeast viability measurements that is more
reliable than the traditional methylene blue stain followed by manual
counting.
Alnoor Pirani received a B.S. degree in molecular genetics and
molecular biology from the University of Toronto and a Ph.D. degree in
cell and molecular biology from Boston University. He is currently
employed at Nexcelom Bioscience as an applications specialist, using his
research expertise and product knowledge to assist customers with their
cell counting and analysis needs. He is also involved in application
development, assisting the R&D team to further develop Nexcelom’s
product offerings for yeast counting and analysis and a variety of other
applications for the brewing, biomedical, and biofuels industries.
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