Nexcelom Bioscience

978-327-5340

  • Cellometer

Yeast Overview

Importance of Yeast Analysis

Yeasts are an economically important organism used for ethanol production, in the beverage and alternative fuels industries as well as a leavening agent in the baking industry. In addition, pathogenic strains of yeasts are involved in both plant and animal diseases. Concentration and viability determinations are routinely performed for quality control purposes in yeast production, fermentation processes, and fungicides research to monitor proliferation of pathogenic yeasts.

The most common method for determining yeast cell number and viability is manual counting on a standard microscope using a hemacytometer and a viability dye. One advantage of this method is that visual inspection of each sample allows the operator to check for contamination, presence of interfering debris, and obvious dilution errors. A major disadvantage is that the manual method is laborious, error-prone and the data acquired is not easily traceable. Although the equipments used to perform manual counting are relatively inexpensive, the cost of human labor and counting errors can be high enough to render manual counting less than practical in a production facility where accuracy, consistency, and record-keeping are highly desirable.

Fluorescence Based Image Cytometry Method for Yeast Concentration & Viability Measurement

1. Pipette 20 µl of sample

Pipette 20 µl of cell sample into a disposable counting slide

2. Insert slide

Insert slide in to cell counter

3. Click count and get results

complex yeast viability data

How Does Yeast Count & Viability by Dual Fluorescence Work?

Highly viscous corn mash sample

A highly viscous corn mash sample is mixed with a dilution buffer and stained with nucleus staining dyes.

Live nucleated yeast cells emit green fluorescence

Live nucleated cells emit green fluorescence when excited by blue light.

Dead yeast cells emit red light

Dead cells emit red light when excited by green light.

dual fluorescence live and dead yeast cells are distinguishable by color

Live and dead cells are then distinguishable by color and viability is generated as a percentage based on live/total cell count.

Yeasts Used in Brewing Industry

In general, yeasts used in brewing are very clean and easily counted using Cellometer image cytometer bright field capability. In addition, in order to measure the viability of the yeast sample, propidium iodide (PI) or oxonol are fluorescent viability dyes that can stain dead cells. Therefore, the total cell count is measured in bright field images and dead cell count is measured in the fluorescent images.

Yeast Concentration Measurement by Bright Field Analysis

Single Cell Count

Yeast single cell count

Automatically measure yeast cell size and concentration using Cellometer image cytometer.

De-clustering of Yeast Cells

Decluster budding yeast cells

Advanced software de-clustering algorithm can de-cluster yeasts to individual cells to generate more accurate concentration and size measurement.

Chain-Forming Cell Count

Yeast Chain-Forming Cell Count

Advanced software de-clustering algorithm can also de-cluster chain-forming yeasts to individual cells to generate more accurate concentration and size measurement.

Cell Concentration Linear Range and Reliability

Serial two-fold dilutions of rehydrated yeast were counted on the Cellometer Auto X4 (10x) to determine the linear range for reliable yeast concentration measurements. The range of concentration that can be measured on the instrument is dependent on the size of the cell being counted (i.e. the range decreases as cell size increases). The Muntons strain is approximately 4.5 µm in diameter when rehydrated, and was selected for this experiment due to its intermediate size when compared to other yeast strains such as those used for lager beer or wine production. The upper limit for accurate yeast counting, as determined by examining counted images, represented a four-fold dilution of the original concentrated yeast suspension (Dilution 2) at 6.6 x 107 cells/mL. Serial dilutions of this sample were counted until the lower limit was reached (Dilution 6) at a concentration of 4.17 x 106 cells/mL.

Plotting of the measured concentration versus the concentration factor (inverse of the dilution factor) resulted in a linear relationship with an R2 of 0.9968. Thus the linear range for counting Muntons yeast (and other similarly sized strains around 4.5 µm in diameter) on the Cellometer Auto X4 (10x) is between 4 x 106 and 6.6 x 107 cells/mL (Figure 1A).

Repeated measurements were performed on the sample to determine the reliability of counting by calculating the Coefficient of Variation (CV). Within the linear range for counting yeast on the Cellometer Auto X4 (10x), the CV was between 4% and 13% (Figure 1B).

Figure 1A

Cell concentration linear range and reliability for counting yeast

Figure 1B

Dilution
Mean Concentration
CV
2
6.58E+07
6%
3
2.99E+07
4%
4
1.55E+07
7%
5
8.84E+06
10%
6
4.17E+06
13%

Linear range of accurate yeast concentration measurements and corresponding CV values.

Yeast Concentration & Viability Measurement by Bright Field & Fluorescence

Viability Measurement Using Propidium Iodide (PI)

Yeast viability by PI

Munton's yeasts were allowed to rehydrate for 30 min, and then stained 1-to-1 with propidium iodide (using FOM VB-595-502 or VB-660-503). The yeasts were immediately analyzed using Cellometer to measure the viability, concentration, and cell size. The bright field images were counted to measure total cell count, while the fluorescent images were counted to measure dead cell count.

Viability Measurement by Oxonol

Yeast viability by Oxonol

Munton's yeasts were allowed to rehydrate for 30 min, and then stained 1-to-1 with oxonol (using FOM VB-535-402). The yeasts were analyzed after 5 min incubation, using Cellometer to measure the viability, concentration, and cell size. The bright field images were counted to measure total cell count, while the fluorescent images were counted to measure dead cell count.

Cell Viability Measurement Accuracy and Reliability

Aliquots of the live yeast suspension were mixed with the heat-killed yeast to generate samples at various levels of viability. The measured viability of the live yeast sample was 78% while the heat-killed sample was 0%. Fractions of live and dead yeast were mixed to analyze intermediate viability levels.

The measured viability was plotted against the percent of heat-killed cells in the final mixture and gives a linear correlation with an R2 of 0.9996. Comparison of predicted and measured viability showed a high degree of agreement and replicates of viability measurements had a standard deviation of ≤ 1.3% (Figure 2A,B). Taken together, viability measurements using PI on the Cellometer Auto X4 (10x) are both highly reproducible and reliable.

Figure 2A

Yeast cell concentration linear range and reliability

Figure 2B

Live/Dead
Predicted
Measured
St Dev
100%/0%
-
78%
0.4%
75%/25%
59%
58%
1.2%
50%/50%
39%
39%
0.6%
25%/75%
20%
19%
1.3%
0%/100%
0%
0%
0.4%

Accuracy and reliability of viability measurements of live yeast mixed with various quantities of heat-killed yeast.

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