Simple, Fast and Consistent Determination of Yeast Viability using Oxonol

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Introduction

Cellometer Vision incorporates image-based cell counting and fluorescence detection in a compact and easy-to-use instrument for generating yeast count and viability measurements. By providing more consistent results with less effort than manual methods, Cellometer Vision is ideal for use in fermentation processes in the brewing and bio fuel industries.

While a variety of methods exist to assess yeast viability, each one has specific drawbacks that make it less than ideal for use in a production environment requiring consistent and accurate results. Although culture-based methods can be used to determine yeast viability, staining techniques and direct observation are more commonly used in fermentation processes. The most widely used stain in the brewing industry, for example, is methylene blue. Live cells exclude or reduce the dye while dead cells stain blue. Manual counting of both live and dead cells on a hemacytometer is performed under a microscope to determine concentration and viability, but is a time-consuming and labor intensive process. More importantly, the methylene blue assay has been reported to produce inconsistent results and analysis can be subjective. Errors in cell counting, viability measurements, and data recording ultimately lead to inconsistent fermentation performance.

Use of the fluorescent dye oxonol (bis-(1,3-dibutylbarbituric acid)trimethine oxonol(DiBAC4(3)), an anionic membrane potential dye that preferentially stains dead cells, has been reported as a reliable and accurate method to assess yeast viability. Cellometer Vision automatically counts total and oxonol positive dead yeast cells addressing the need for simple, rapid counting and viability testing of yeast cells. By capturing both brightfield and fluorescent images from the same sample, the software determines total cell count and concentration and determines viability, typically in less than 60 seconds.

Method

Running Assay:

Results

Total counted yeast cells are indicated on-screen by green circles in the brightfield image (Figure 1). Dead cells stained with oxonol are indicated as fluorescent positive green circles in the fluorescent image (Figure 2). Cellometer software automatically calculates cell count, concentration, mean cell size, and viability and displays results. Cell size distribution histograms (Figure 6), data files, and cell images can be instantly created, and saved for further analysis or for quality control record keeping.

Figure 1. Total counted yeast cells are indicated on-screen by green circles

Figure 2. Fluorescent image showing oxonol stained dead cells (green circles), and live cells (red circles)

Yeast Type	Viability %
A	88.5
B	89.5
C	73.2
D	78.7
E	77.6
F	5.1

	Viability	Concentration	Mean Size
Yeast type E sample 1
Mean(n=6)	99.6	9.14X106	4.9
CV	0.2%	5.4%	2.4%
Yeast type E sample 2
Mean(n=7)	85.3	5.92X106	5.4
CV	4.1%	13.3%	1.7%
Yeast type E sample 3
Mean(n=7)	79.6	1.2X107	5.4
CV	4.2%	8.8%	1.8%

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