Sep 1 2014
Research conducted by renowned French chemist, Louis Pasteur, revealed that the production of alcohol from glucose was directly related to yeast. The fermentation property of this microorganism is used in the manufacture of ethanol, wine, beer, food and biofuel. Therefore, reliable yeast performance is important during the fermentation process. For optimum yeast performance, it is necessary to monitor percentage cell concentration and viability. Beckman Coulter’s Vi-CELL™ XR cell viability analyzer automates the manual Trypan Blue Dye Exclusion method.
This article describes Beckman Coulter's best practices for studying the viability of yeast cells. The instrument used in this analysis was the Vi-CELL™ XR Reagent Pak.
Vi-CELL™ XR Software
The Vi-CELL™ XR software features many Cell Types, which are files in which the required optical settings are stored to accurately identify and measure viable cells against nonviable cells. Yeast cells have a high cell concentration and exhibit a large size distribution.
They cannot be stained easily and may include considerable amounts of debris from the fermentation process. As a result, yeast cells can be difficult to count. In order to achieve precise cell concentration and viability results, it is necessary to improve the Cell Type for yeast.
Sample Preparation
First, the yeast fermentation sample was filtered via a sieve measuring 50 µm in size. This helps in removing large amount of debris, if any. The sample was then diluted using the preferred buffer or growth media and brought to a concentration between 5 x 104 to 1 x 107 cells/mL. Before running on the Vi-CELL™ XR analyzer, a Multisizer series or Z series COULTER Counter can be used to check the concentration of cells.
Instrument Settings
Next, the yeast sample was examined on the Vi-CELL™ XR instrument by means of the default Yeast Cell Type. Results were reviewed which showed that the yeast sample had starch granules which accumulated from the fermentation process, and also the cells were not entirely stained. This was corrected by making small changes to the Yeast Cell Type. The modifications are shown in Table 1.
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 |
1 |
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. |
Analysis
Yeast fermentation samples which were examined were pulled from three different time intervals: 2h, 18h and 26h. All samples were then diluted 500 times. Following this, five copies of each time interval were run successively on the Vi-CELL™ XR system by means of the new optimized Yeast Cell Type. Table 3 shows the predicted outcomes that were based on the feedback obtained from bioprocess engineers who worked with these yeast strains.
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 2 higher % viability than Sample 1 |
Yes |
| 3 |
Sample 3 total cell count lower than Sample 1 and 2 |
Yes |
Tables 2 and 3 summarize the results obtained from the Vi-CELL™ XR runs. The cell concentration and percent viability of the yeast fermentation sample fulfilled the predicted outcome. Moreover, the system reports cell circularity, viable cell concentration, size distribution, and mean diameter.
Yeast Analysis: Vi-CELL™ XR vs. Manual Trypan Blue Viability Test
As stated above, Trypan Blue Dye Exclusion method is the standard technique used for determining cell viability. An equal volume of cells is combined with trypan blue stain (0.4%). Since viable cells have intact membranes, they exclude the trypan blue stain, while nonviable cells have permeable membrane and stain dark blue.
The manual method uses a microscope and hemacytometer and requires technicians to determine both unstained and stained and also to manually determine the percent viability. This method is not only labor intensive, but also carry significant accuracy error because of its subjective nature.
The Industrial BioDevelopment Laboratory (IBDL) had assessed the manual method against automated method i.e. Trypan Blue Dye Exclusion method for cell counting, and found a considerable difference between cell counts ascertained by different people utilizing the hemacytometer. This difference can be attributed to inconsistency in sample preparation or decisions with regard to viability and non-viability of individual cells.
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Figure 1. Comparison of yeast viability stains. Image credit: Beckman Coulter
The Vi-CELL™ system determined the cell viabilities and concentrations that were reproducible all through the course of the experiment. They were also near to the mean values of the individual cells and did not experience the wide discrepancy of the human counts. This shows that the Vi- CELL™ instrument is reproducible, precise and suitable tool for cell counting. Trypan blue vital dye has been proven to be equal to methylene violet and methylene blue for yeast cells (Figure 1).
Conclusion
The Vi-CELL™ XR cell viability analyzer automates the manual technique of yeast viability measurements used in fermentation processes. The instrument not only eliminates the subjective nature of the manual test, but also delivers impartial results for each assay. By improving the Yeast Cell Type, non-cell objects from the results were removed, targeting only the cells of interest.
Combining proprietary algorithm, advanced imaging technology, and fluidics management, the instrument is fully automated and enables reagent handling, sample aspiration, and subsequent cleaning. The Vi-CELL™ XR is capable of analyzing up to 100 images for a given analysis and increases the overall volume from 15 to 30 times when compared to the manual technique. Results are obtained in less than 2.5 minutes which can be printed for archiving or sent to Excel document.
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.