Source: https://www.nexcelom.co.uk/applications/yeast-viability.php
Timestamp: 2019-04-21 14:39:47+00:00

Document:
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.
How Does Yeast Count & Viability by Dual Fluorescence Work?
A highly viscous corn mash sample is mixed with a dilution buffer and stained with nucleus staining dyes.
Live nucleated cells emit green fluorescence when excited by blue light.
Dead cells emit red light when excited by green light.
Live and dead cells are then distinguishable by color and viability is generated as a percentage based on live/total cell count.
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.
Munton's rehydrated yeasts were automated counted using Cellometer in bright-field to measure cell concentration and cell size.
Advanced software de-clustering algorithm can de-cluster yeasts to individual cells to generate more accurate concentration and size measurement.
Advanced software de-clustering algorithm can also de-cluster chain-forming yeasts to individual cells to generate more accurate concentration and size measurement.
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 1. Linear range of accurate yeast concentration measurements and corresponding CV values.
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-660-502). 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.
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.
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 2. Accuracy and reliability of viability measurements of live yeast mixed with various quantities of heat-killed yeast.
In general, yeasts used in the biofuel industry are in complex medium such as corn mash, corn stover, or sugar cane, which can serve as nutrients to produce bioethanol. These complex media increase the difficulties for automated counting using bright-field method due to the debris particles in the sample. Using a combination of acridine orange (AO) and propidium iodide (PI) can perform dual fluorescence detection of live and dead cells, respectively, which can specifically stain the live/dead cells to accurately determine the concentration and viability of the complex sample.
Above shows 3 complex samples of corn mash, corn stover, and sugar cane. The bright-field images contain many debris particles, but in the fluorescent images, only the yeast cells fluoresce brightly that can be automatically counted.
The brewing yeasts were stained for 1 hour with CFDA-AM (using FOM VB-535-402) to measure enzymatically active yeasts. After incubation, the cells are immediately analyzed using Cellometer to measure the number of enzymatically active yeasts to determine the vitality of the sample. The percent of yeasts that are actively fermenting during production can be determined and used to optimize the fermentation process. The fluorescent linear gating was set to measure the population percentage of high CFDA-AM fluorescence.
Yeast glycogen is a macromolecule that provides the energy and carbohydrates required for yeast sterols and lipid synthesis, which ensures yeast metabolism during fermentation. Glycogen content can be measured by staining with acriflavine (using FOM VB-535-402) and analyze fluorescence intensity of individual cells in a fluorescent histogram. Glycogen has been shown to correlate to vitality and is an essential physiological parameter to be monitored.
Neutral lipids are energy rich molecules that are stored in yeasts, which cannot be metabolized. However, it can protect yeast cells from toxic substances like ethanol, which can prolong survival capacity. In order to measure neutral lipids, the yeasts can be stained with Nile Red (using FOM VB-595-502), and analyze fluorescence intensity of individual cells in a fluorescent histogram. The fluorescent linear gating was set to measure the population percentage of high Nile Red fluorescence.
Trehalose is a sugar disaccharide that supplies energy during cell cycle, or cell proliferation. Similar to neutral lipids, trehalose can protect yeast cells against stress, high ethanol content, heat, dehydration, oxidation, pH change. In order to measure trehalose content, the yeast cells are stained with concanavalin A-FITC (using FOM VB-535-402), and analyze fluorescence intensity of individual cells in a fluorescent histogram. Trehalose are also known to increase survival capacity during fermentation. The fluorescent linear gating was set to measure the population percentage of high FITC fluorescence.
Green fluorescent proteins expression in yeasts can be measured using Cellometer (using FOM VB-535-402). Bright-field images are analyzed initially to count all the cells and the fluorescence intensities within each cell are measured and plotted in a histogram for analysis. The fluorescent linear gating was set to measure the population percentage of high GFP fluorescence.
Abbott, D. A., and Ingledew, W. M. (2004). "Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production." Biotechnology Letters, 26, 1313-1316.
V. Anton-Leberre, E. Haanappel, N. Marsaud, L. Trouilh, L. Benbadis, H. Boucherie, S. Massou, and J. M. François, "Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae," Bio Electro Magnetics, vol. 31, pp. 28-38, 2010.
Antoni, D., Zverlov, V. V., and Schwarz, W. H. (2007). "Biofuel from microbes." Applied Microbiology Biotechnology, 77, 23-35.
Argueso, J. L., Carazzolle, M. F., Mieczkowski, P. A., Duarte, F. M., Netto, O. V. C., Missawa, S. K., Galzerani, F., Costa, G. G. L., Vidal, R. O., Noronha, M. F., Dominska, M., Andrietta, M. G. S., Andrietta, S. R., Cunha, A. F., Gomes, L. H., Tavares, F. C. A., Alcarde, A. R., Dietrich, F. S., McCusker, J. H., Petes, T. D., and Pereira, G. A. G. (2009). "Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production." Genome Research, 19, 2258-2270.
M. Arlorio, J. D. Coïsson, and A. Martelli, "Identification of Saccharomyces cerevisiae in bakery products by PCR amplification of the ITS region of ribosomal DNA," European Food Research and Technology, vol. 209, pp. 185-191, 1999.
Basso, L. C., Amorim, H. V. d., Oliveira, A. J. d., and Lopes, M. L. (2008). "Yeast selection for fuel ethanol production in Brazil." FEMS Yeast Research, 8, 1155-1163.
Bauer and R. Kölling, "Characterization of the SAC3 gene of Saccharomyces cerevisiae," Yeast, vol. 12, pp. 965-975, 1996.
J. C. Bouchez, M. Cornu, M. Danzart, J. Y. Leveau, F. Duchiron, and M. Bouix, "Physiological Significance of the Cytometric Distribution of Fluorescent Yeasts After Viability Staining," Biotechnology and Bioengineering, vol. 86, pp. 520-530, 2004.
R. Boyd, T. S. Gunasekera, P. V. Attfield, K. Simic, S. F. Vincent, and D. A. Veal, "A flow-cytometric method for determination of yeast viability and cell number in a brewery," FEMS Yeast Research, vol. 3, pp. 11-16, 2003.
G. Cahill, P. K. Walsh, and D. Donnelly, "Determination of Yeast Glycogen Content by Individual Cell Spectroscopy Using Image Analysis," Biotechnology and Bioengineering, vol. 69, pp. 312-322, 2000.
L. L. Chan, E. J. Lyettefi, A. Pirani, T. Smith, J. Qiu, and B. Lin, "Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method," J Ind Microbiol Biotechnol, vol. 38, pp. 1109-1115, 2010.
L. L. Chan, X. Zhong, A. PIrani, and B. Lin, "A novel method for kinetic measurements of rare cell proliferation using Cellometer image-based cytometry," Journal of Immunological Methods, vol. 377, pp. 8-14, 2012.
L. L. Chan, X. Zhong, J. Qiu, P. Y. Li, and B. Lin, "Cellometer Vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping," Cytometry Part A, vol. 79A, pp. 507-517, 2011.
L. L.-Y. Chan, N. Lai, E. Wang, T. Smith, X. Yang, and B. Lin, "A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry," Apoptosis, vol. 16, pp. 1295-1303, 2011.
W. L. Chang, H. C. v. d. Heyde, and B. S. Klein, "Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays," Journal of Immunological Methods, vol. 211, pp. 51-63, 1998.
M. Ciani, I. Mannazzu, P. Marinangeli, F. Clementi, and A. Martini, "Contribution of winery-resident Saccharomyces cerevisiae strains to spontaneous grape must fermentation " Antonie Van Leeuwenhoek, vol. 85, pp. 159-164, 2004.
D. Deere, J. Shen, G. Vesey, P. Bell, P. Bissinger, and D. Veal, "Flow Cytometry and Cell Sorting for Yeast Viability Assessment and Cell Selection," Yeast, vol. 14, pp. 147-160, 1998.
Foglieni, C., Meoni, C., and Davalli, A. M. (2001). "Fluorescent dyes for cell viability: an application on prefixed conditions." Histochemical Cell Biology, 115, 223-229.
Gibbons, W. R., and Hughes, S. R. (2009). "Integrated biorefineries with engineered microbes and high-value co-products for profitable biofuels production." In Vitro Cellular & Developmental Biology - Plant, 45, 218-228.
Gordon, G. W., Berry, G., Liang, X. H., Levine, B., and Herman, B. (1998). "Quantitative Fluorescence Resonance Energy Transfer Measurments Using Fluorescence Microscopy." Biophysical Journal, 74, 2702-2713.
M. J. Henry-Stanley, R. M. Garni, and C. L. Wells, "Adaptation of FUN-1 and Calcofluor white stains to assess the ability of viable and nonviable yeast to adhere to and be internalized by cultured mammalian cells," Journal of Microbiological Methods, vol. 59, pp. 289-292, 2004.
Hernlem and S.-S. Hua, "Dual Fluorochrome Flow Cytometric Assessment of Yeast Viability," Current Microbiology, p. Published Online, 2010.
S. Y. L. Hsu, H. F. Hsu, P. Isacson, and H. F. Cheng, "Schistosoma mansoni and S. japonicum: Methylene Blue Test for the Viability of Schitosomula in Vitro," Experimental Parasitology, vol. 41, pp. 329-334, 1977.
Hu, X. H., Wang, M. H., Tan, T., Li, J. R., Yang, H., Leach, L., Zhang, R. M., and Luo, Z. W. (2007). "Genetic Dissection of Ethanol Tolerance in the Budding Yeast Saccharomyces cerevisiae." Genetics, 175, 1479-1487.
L. M. King, D. O. Schisler, and J. J. Ruocco, "Epifluorescent Method for Detection of Nonviable Yeast," Journal of American Society of Brewing Chemists, vol. 39, pp. 52-54, 1981.
Koksch, M., Rothe, G., Kiefel, V., and Schmitz, G. (1995). "Fluorescence resonance energy transfer as a new method for the epitope-specific characterization of anti-platelet antibodies." Journal of Immunological Methods, 187, 53-67.
Ling, E., Shirai, K., Kanekatsu, R., and Kiguchi, K. (2003). "Classification of larval circulating hemocytes of the silkworm Bombyx mori, by acridine orange and propidium iodide staining." Histochemical Cell Biology, 120, 505-511.
P. Malacrinó, G. Zapparoli, S. Torriani, and F. Dellaglio, "Rapid detection of viable yeasts and bacteria in wine by flow cytometry," Journal of Microbiological Methods, vol. 45, pp. 127-134, 2001.
Mascotti, K., McCullough, J., and Burger, S. R. (2000). "HPC viability measurement: trypan blue versus acridine orange and propidium iodide." Transfusion, 40, 693-696.
R. McCaig, "Evaluation of the Fluorescent Dye 1-Anilino-8-Naphthalene Sulfonic Acid for Yeast Viability Determination," Journal of American Society of Brewing Chemists, vol. 48, pp. 22-25, 1990.
Michelson, A. D. (1996). "Flow Cytometry: A Clinical Test of Platelet Function." Blood, 87(12), 4925-4936.
J. W. Millbank, "The Action of Acriflavine on Yeast Protoplasts," Antonie Van Leeuwenhoek, vol. 28, pp. 215-220, 1962.
Mills, D. R. (1941). "Differential Staining of Living and Dead Yeast Cells." Journal of Food Science, 6(4), 361-371.
Y. Miura, N. Wada, Y. Nishida, H. Mori, and K. Kobayashi, "Chemoenzymatically synthesized glycoconjugate polymers," Biomacromolecules, vol. 4, pp. 410-415, 2003.
Nikolić, S., Mojović, L., Rakin, M., Pejin, D., and Nedović, V. (2009). "Effect of different fermentation parameters on bioethanol production from corn meal hydrolyzates by free and immobilized cells of Saccharomyces cerevisiae var. ellipsoideus." Journal of Chemical Technological Biotechnology 84, 497-503.
M. Nikolova, I. Savova, and M. Marinov, "An Optimised Method for Investigation of the Yeast Viability by Means of Fluorescent Microscopy," Journal of Culture Collections, vol. 3, pp. 66-71, 2002.
J. Novak, G. Basarova, J. A. Teixeira, and A. A. Vicente, "Monitoring of Brewing Yeast Propagation Under Aerobic and Anaerobic Conditions Employing Flow Cytometry," Journal of the Institute of Brewing, vol. 113, pp. 249-255, 2007.
K.-B. Oh and H. Matsuoka, "Rapid viability assessment of yeast cells using vital staining with 2-NBDG, a fluorescent derivative of glucose," International Journal of Food Microbiology, vol. 76, pp. 47-53, 2002.
S. C. d. L. Paulillo, F. Yokoya, and L. C. Basso, "Mobilization of Endogenous Glycogen and Trehalose of Industrial Yeasts," Brazilian Journal of Microbiology, vol. 34, pp. 249-254, 2003.
Periasamy, A. (2201). "Fluorescence resonance energy transfer microscopy: a mini review." Journal of Biomedical Optics, 6(3), 287-291.
Pirani, A. (2010). "Yeast Concentration and Viability using Image-Based Fluorescence Analysis." Nature Methods, Application Notes(6), Online Version.
Raschke and D. Knorr, "Rapid monitoring of cell size, vitality and lipid droplet development in the oleaginous yeast Waltomyces lipofer," Journal of Microbiological Methods, vol. 79, pp. 178-183, 2009.
Rodríguez-Porrata, M. Novo, J. Guillamón, N. Rozès, A. Mas, and R. C. Otero, "Vitality enhancement of the rehydrated active dry wine yeast," International Journal of Food Microbiology, vol. 126, pp. 116-122, 2008.
Schlee, M. Miedl, K. A. Leiper, and G. G. Stewart, "The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast," Journal of the Institute of Brewing, vol. 112, pp. 134-147, 2006.
Selvin, P. R., and Hearst, J. E. (1994). "Luminescence energy transfer using a terbium chelate: Improvements on fluorescence energy transfer." Proceedings National Academy of Science, 91, 10024-10028.
Slater, M. L. (1976). "Rapid Nuclear Staining Method for Saccharomyces cerevisiae." Journal of Bacteriology, 126(3), 1339-1341.
J. C. Slaughter and M. Minabe, "Fatty Acid-containing Lipids of the Yeast Saccharomyces cerevisiae during Post-fermentation Decline in Viability," Journal of Sci. Food Agric., vol. 65, pp. 497-501, 1994.
J. C. Slaughter and T. Nomura, "Intracellular glycogen and trehalose contents as predictors of yeast viability," Enzyme Microb. Technol., vol. 14, pp. 64-67, 1992.
Smart, K. (2003). Brewing Yeast Fermentation Performance, 2 Ed., Blackwell Science Ltd.
Solomon, M., Wofford, J., Johnson, C., Regan, D., and Creer, M. H. (2010). "Factors influencing cord blood viability assessment before cryopreservation." Transfusion, 50, 820-830.
Stengel, A., Goebel, M., Yakubov, I., Wang, L. X., Witcher, D., Coskun, T., Tache, Y., Sachs, G., and Lambrecht, N. W. G. (2009). "Identification and Characterization of Nesfatin-1 Immunoreactivity in Endocrine Cell Types of the Rat Gastric Oxyntic Mucosa." Endocrinology, 150(1), 232-238.
Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., and Loper, K. (2004). "Evaluation of an Automated Instrument for Viability and Concentration Measurements of Cryopreserved Hematopoietic Cells." Laboratory Hematology, 10, 109-111.
Taylor, F., Mcaloon, A. J., James C. Craig, J., Yang, P., Wahjudi, J., and Eckhoff, S. R. (2001). "Fermentation and costs of fuel ethanol from corn with quick-germ process." Applied Biochemistry and Biotechnology, 94(1), 41-49.
Trevors, J. T., Merrick, R. L., Russell, I., and Stewart, G. G. (1983). "A Comparison of Methods for Assessing Yeast Viability." Biotechnology Letters, 5(2), 131-134.
Vairo, M. L. R. (1962). "A Modified Adsorption Method for Determining Percentage of Dead Yeast Cells." Biotechnology and Bioengineering, 4, 247-254.
Vertès, A. A., Inui, M., and Yukawa, H. (2008). "Technological Options for Biological Fuel Ethanol." Journal of Molecular Microbiology and Biotechnology, 15, 16-30.
Wallen, C. A., Higashikubo, R., and Dethlefsen, L. A. (1980). "Comparison of Two Flow Cytometric Assays for Cellular RNA-Acridine Orange and Propidium Iodide." Cytometry, 3(3), 155-160.
S. M. V. Zandycke, O. Simal, S. Gualdoni, and K. A. Smart, "Determination of Yeast Viability Using Fluorophores," Journal of American Society of Brewing Chemists, vol. 61, pp. 15-22, 2003.
T. Zhang and H. H. P. Fang, "Quantification of Saccharomyces cerevisiae viability using BacLight," Biotechnology Letters, vol. 26, pp. 989-992, 2004.
Berkes CA, Chan LLY, Wilkinson A, Paradis B. Rapid quantification of pathogenic fungi by Cellometer image-based cytometry. Journal of Microbiological Methods 2012 Dec:468-476.

References: V. 
 V. 
 V. 
 V. 
 V. 
 v. 
 V. 
 V.