Nexcelom Bioscience

978-327-5340

  • Cellometer

Introduction

Measuring the count, concentration, viability and vitality of yeast are the most common measurements performed. By using fluorescence based detection methods the Cellometer Vision 10X instrument can also measure the glycogen content, neutral lipid content, trehalose content and GFP transfection efficiency of cultured yeast samples.

Advanced Fluorescent Assays Measuring Yeast Physiologic Parameters

Yeast Vitality Analysis Using CFDA-AM & Propidium Iodide

Yeast Vitality Analysis using CFDA-AM & Propidium Iodide

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 Content Using Acriflavine Fluorescent Staining

Yeast Glycogen Content using Acriflavine Fluorescent Staining

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.

Yeast Neutral Lipid Content Using Nile Red Fluorescent Staining

Yeast Neutral Lipid Content using Nile Red Fluorescent Staining

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.

Yeast Trehalose Content Using Concanavalin A-FITC Fluorescent Staining

Yeast Trehalose Content using Concanavalin A-FITC Fluorescent Staining

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.

Yeast Green Fluorescent Protein (GFP) Transfection

Yeast GFP transfection

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.

References

  • 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.

Cellometer References for Yeast Analysis

Yeast References

  • Liu J, Wisniewski M, Droby S, Tian SP, Hershkovitz V, Tworkoski T. Effect of heat shock treatment on stress tolerance and biocontrol efficacy of Metschnikowia fructicola. Fems Microbiology Ecology;76(1):145-155 2011
  • Liu J, Sui Y, Wisniewski M, Droby S, Tian SP, Norelli J, Hershkovitz V. Effect of heat treatment on inhibition of Monilinia fructicola and induction of disease resistance in peach fruit. Postharvest Biology and Technology;65:61-68 2012
  • Xu L, Du Y. Effects of yeast antagonist in combination with UV-C treatment on postharvest diseases of pear fruit. BioControl;57:451-461 2012
  • Liu J, Wisniewski M, Droby S, Vero S, Tian SP, Hershkovitz V. Glycine betaine improves oxidative stress tolerance and biocontrol efficacy of the antagonistic yeast Cystofilobasidium infirmominiatum. International Journal of Food Microbiology;146(1):76-83 2011
  • Liu J, Wisniewski M, Droby S, Norelli J, Hershkovitz V, Tian SP, Farrell R. Increase in antioxidant gene transcripts, stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure. Fems Microbiology Ecology;80(3):578-590 2012
  • Sui Y, Liu J, Wisniewski M, Droby S, Norelli J, Hershkovitz V. Pretreatment of the yeast antagonist, Candida oleophila, with glycine betaine increases oxidative stress tolerance in the microenvironment of apple wounds. International Journal of Food Microbiology;157(1):45-51 2012
  • Chan LL, Lyettefi EJ, Pirani A, Smith T, Qiu J, Lin B. Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method. Journal of Industrial Microbiology & Biotechnology;38(8):1109-1115 2011
  • 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.
  • Chan LL, Kury A, Wilkinson A, Berkes C, Pirani A. Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae. Journal of Industrial Microbiology & Biotechnology;39(11):1615-1623 2012

Mold References

  • Zhang CF, Wang JM, Zhang JG, Hou CJ, Wang GL. Effects of beta-aminobutyric acid on control of postharvest blue mould of apple fruit and its possible mechanisms of action. Postharvest Biology and Technology;61(2-3):145-151 2011
  • Wang J, Shi X-G, Wang H-Y, Xia X-M, Wang K-Y. Effects of Esterified Lactoferrin and Lactoferrin on Control of Postharvest Blue Mold of Apple Fruit and Their Possible Mechanisms of Action. J. Agric. Food Chem.;60:6432-6438 2012
  • Wang J, Xia X-M, Wang H-Y, Li P-P, Wang K-Y. Inhibitory effect of lactoferrin against gray mould on tomato plants caused by Botrytis cinerea and possible mechanisms of action. International Journal of Food Microbiology;61(3):151-157 2013

Spore References

  • Liu J, Macarisina D, Wisniewskia M, Suia Y, Drobyb S, Norellia J, Hershkovitzb V. Production of hydrogen peroxide and expression of ROS-generating genes in peach flower petals in response to host and non-host fungal pathogens. Plant Pathology;10.1111/j.1365-3059.2012.02683.x 2012
  • Mishra S, Malik A. Nutritional optimization of a native Beauveria bassiana isolate (HQ917687) pathogenic to housefly, Musca domestica L. Journal of Parasitic Diseases 10.1007/s12639-012-0165-5 2012

Parasite References

  • Espinosa A, Paz-Y-Mino-C G. Discrimination, Crypticity, and Incipient Taxa in Entamoeba. Journal of Eukaryotic Microbiology;59(2):105-110 2012
Top