Oct 18 2013
The fermentation of yeast is used in the beer, wine, and ethanol production industries. For optimum yeast fermentation performance, percentage cell viability and concentration must be monitored. The labor intensive manual trypan blue viability is automated by the Beckman Coulter Vi-CELL XR.
The Vi-CELL XR software includes the cell type feature. Cell Types are files that store optical settings needed to precisely identify and quantify viable versus nonviable cells. This feature helps in accounting for the fact that cells differ in their optical characteristics.
Yeast cells can be challenging to count as they have a broad size distribution, are difficult to stain, have an increased cell concentration and may contain considerable amount of debris from the fermentation process.
Hence it is essential to optimize the cell type for yeast to obtain precise and accurate concentration results and cell viability. This article demonstrates Beckman Coulter's best practices for the analysis of yeast cell viability.
Equipment Used
The equipment used is:
- Beckman Coulter Vi-CELL XR
- Vi-CELL XR reagent pack
Yeast Sample Preparation
Filtration
It is essential that the yeast fermentation sample is filtered through a 50µm sieve to remove any large debris.
Dilution
It is also important to dilute the concentration between 5 x 104 to 1 x 107 cells/mL using growth media or desired buffer as a diluent.
One rapid way of checking the cell concentration before running on the Vi-CELL is with a Z series or Multisizer series COULTER COUNTER.
Instrument Settings
Creating an Optimized Yeast Cell Type
A yeast sample was analyzed on the Vi-CELL XR with the default yeast cell type. Reviewing the results determined that the yeast sample contained starch granules from the fermentation process and that the cells were not stained completely.
Very small modifications were made to the Yeast Cell Type to correct for this. Table 1 describes the modifications. Figure 1 shows the default yeast cell type and new optimized yeast cell type parameters.
Table 1. Cell type parameter optimization.
| Cell Type Parameter |
Default Value |
Optimized Value |
Reason for Modification |
| Minimum Diameter |
3 |
2 |
Decreased size range to ensure all yeast cells are counted. |
| Maximum Diameter |
20 |
11 |
Decreased maximum size range so that non-yeast cells (debris) would be excluded. |
| Aspirate Cycles |
|
3 |
Increased aspirate cycle to ensure that yeast cells do not stick to the sample cup. |
| Trypan Blue Mixing Cycles |
3 |
9 |
Increased trypan blue mix cycles to increase staining time. |
| Cell Brightness |
85 |
90 |
Increased cell brightness to maximum value of 90 since the yeast cells were very bright in the images. |
| Minimum Circularity |
0 |
0.65 |
Minimum circularity is applied only to nonviable cells. Increasing this value will exclude debris that is similar in size as nonviable cells. |
| Decluster Degree |
High |
Low |
Yeast cells in our sample were well separated, so a decluster degree of low was appropriate for this analysis. |
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Figure 1. Default yeast cell type and new optimized yeast cell type parameters. Image credit: Beckman Coulter
Analysis
Analyzed yeast fermentation samples were taken from three different fermentation time intervals 2h, 18h and 26h.
All samples were diluted 500-fold. Five replicates of each time interval were run consecutively on the Vi-CELL XR using the new optimized yeast cell type.
Table 3 shows the predicted outcomes that were based on feedback from the collaborating bioprocess engineers' experience with these yeast strains.
Results
Tables 2 and 3 summarize the results from the Vi-CELL XR. The cell concentration and the percent viability of the yeast fermentation sample met the predicted outcome. Also the instrument reports mean diameter, cell circularity, cell concentration and size distribution.
One must note the low variability in each Vi-CELL analysis over five replications for each sample, and also the excellent agreement with expectations. Figure 3 shows the Vi-CELL XR results.
Table 2. Results: N=5 Replicates for each sample; Dilution factor = 500.
| Yeast Sample ID |
Fermentation Time |
Total Cell Count Concentration (1 x 106 cells/mL) |
Total Cell Count % CV |
% Viability |
Viability Concentration % CV |
| 1 |
26 hours |
1,133 |
2% |
68% |
1% |
| 2 |
18 hours |
1,146 |
2% |
74% |
2% |
| 3 |
2 hours |
787 |
2% |
72% |
2% |
Table 3. Predicted outcomes
| Yeast Sample ID |
Predicted Outcome |
Demonstrated? |
| 1 |
Sample 1 lower % viability than Sample 2 |
Yes |
| 2 |
Sample 1 higher % viability than Sample 1 |
Yes |
| 3 |
Sample 3 total cell count lower than Sample 1 and 2 |
Yes |
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Figure 2. Vi-CELL XR Software View. Viable cells are circled in green; nonviable in red. Image credit: Beckman Coulter
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Figure 3. Vi-CELL XR results. Image credit: Beckman Coulter
The Vi-CELL XR
The Vi-CELL XR automates the widely accepted Trypan Blue Dye Exclusion method. The Vi-CELL XR integrates advanced imaging technology, proprietary algorithm and fluidics management.
The customized liquid handling system is at the heart of the Vi-CELL XR. This system, which allows sample aspiration, reagent handling and subsequent instrument cleaning, is fully automated.
On aspiration and mixing of the cellular suspension with the Trypan blue dye, it is pumped to the flow cell for imaging. The Vi-CELL XR can analyze up to 100 images for a given analysis increasing total volume from 15 to 30 times over the manual method, with results in less that 2.5 minutes. Results can also be exported to Excel® or printed for archiving.
Vi-CELL XR Superior to Manual Trypan Blue for Yeast Analysis
The standard method for measurement of cell viability is the Trypan Blue Dye Exclusion method. Trypan blue stain (0.4%) is mixed with an equal volume of cells. Viable cells with their intact membranes, exclude the trypan blue stain; nonviable cells, membrane permeable, stain dark blue.
However the manual method needs a technician using a hemacytometer and microscope to enumerate both stained and unstained cells and manually calculate the percent viability. The method has considerable accuracy error because of its subjective nature in addition to being labor-intensive.
The Industrial BioDevelopment Laboratory (IBDL) previously conducted an evaluation of the manual versus automated Trypan Blue Dye Exclusion method for cell counting. IBDL found a significant variability between cell counts determined by different people using the hemacytometer.
The change may be because of variability in sample preparation and/or decisions regarding viability/non-viability of individual cells. Table 4 shows the comparison of the average total and viable cell concentrations determined by the manual and automated methods. Figure 4 shows the comparison of yeast viability stains. Trypan blue as a vital dye has been shown to be equivalent to methylene blue and methylene violet for yeast cells.
Table 4. Comparison of the average total and viable cell concentrations determined by the manual and automated methods.
| Measurement |
Manual (x106 cells/mL) |
Automated (x106 cells/mL) |
| Total cell concentration |
4.71 ± 1.94 |
4.23 ± 0.52 |
| Viable cell concentration |
4.53 ± 1.95 |
4.13 ± 0.51 |
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Figure 4. Comparison of yeast viability stains. Image credit: Beckman Coulter
Conclusion
The Vi-CELL enables automation of the manual method of yeast viability measurements employed in fermentation processes.
The subjective nature of the manual test is removed by the instrument providing objective results for each assay. By optimizing the Yeast Cell Type, it was possible to exclude non-cell objects from the results and target the cells of interest as shown in Figure 5.
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Figure 5. Vi-CELL XR analysis image. Viable cells are circled in green and nonviable in red. Debris is excluded. Image credit: Beckman Coulter
This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc. - Particle Characterization.
For more information on this source, please visit Beckman Coulter, Inc. - Particle Size Characterization.