Determine Yeast Concentration and Viability at Your Benchtop
Yeast Viability Measurements With the Tali® Image-Based Cytometer
Fluorescence-Based Viability Assays
Traditional methods to assess yeast viability include determination of the number of colony-forming units (CFUs) or manual counting of stained organisms by microscopic observation. The counting of CFUs requires 24 to 48 hours of incubation, which is too slow for many industries. Alternatively, counting of methylene blue–stained yeast with a microscope is arduous and thus prone to human error.
In recent years the industrial and research communities have been moving toward the use of fluorescent dyes for viability assays. Propidium iodide (PI), the most commonly used fluorescent dye for the determination of mammalian and yeast cell viability, is a membrane-impermeant nucleic acid stain that is excluded from viable cells. PI enters cells with compromised membranes, where it binds DNA and emits red fluorescence upon excitation. Published evidence suggests that, under some conditions, yeast can recover from the loss of membrane integrity that allows PI to enter the cell [1,2]. In such a case, yeast viability calculations that rely on PI staining would overestimate the actual number of dead cells.
Alternatively, the potential-sensitive fluorescent dye DiBAC4(3) has been demonstrated to successfully distinguish between live and dead cells without the ambiguity of PI staining [3,4]. DiBAC4(3) is a small fluorescent molecule that enters yeast cells with disrupted plasma membrane potential (indicating loss of viability). Once inside, DiBAC4(3) binds to intracellular proteins and membranes and exhibits enhanced green fluorescence upon excitation.
Dual Measurement of Yeast Viability
No single parameter fully defines cell viability in all systems. Therefore, it is often advantageous to use more than one approach when making viability determinations. We tested whether the fluorescent dyes DiBAC4(3) and PI can be used for accurate viability measurements in two different species of yeast—Saccharomyces cerevisiae (Baker’s yeast) and Saccharomyces pastorianus (a common yeast strain used by brewers)—using the Tali® Image-Based Cytometer (Figure 1). The bright-field channel of the Tali® cytometer was used to count the cells, and the two fluorescent channels enabled quantitative measurements of the green- and red-fluorescent stained yeast cells. Because of the simultaneous display of cells in the bright-field and fluorescence channels, the Tali® cytometer provides the user with visual confirmation of sample fluorescence, allowing proper setup of fluorescence threshold and therefore higher confidence in the data generated.
We assayed samples containing approximately equal amounts of heat-killed and live yeast cells and found that both DiBAC4(3) staining and PI staining generated results in very good agreement with their expected viabilities of 50% (Table 1). These data suggest that either DiBAC4(3) or PI staining can be employed for accurate yeast viability determinations using the Tali® Image-Based Cytometer.
Figure 1. Yeast viability analysis using the Tali® Image-Based Cytometer. (A) Saccharomyces cerevisiae or (B) Saccharomyces pastorianus was grown overnight in YPD broth and then diluted to 1 x 106 cells/mL in GH medium (2% D-(+)-glucose and 10 mM Na-HEPES, pH 7.2). A sample of each culture was heat-killed by treatment at 100°C for 10 min to generate a dead-cell population. After cooling to room temperature, these samples were mixed with an equal amount of untreated cells to create a population of approximately 50% live and 50% dead cells. A 100 µL aliquot of 1 x 106 cells/mL was incubated in 1 µg/mL (final concentration) DiBAC4(3) for 2 min at room temperature or with 0.4 µM (final concentration) PI. Samples treated with both dyes were handled as follows: To a 100 µL aliquot of 1 x 106 cells/mL, we added DiBAC4(3) to a final concentration of 1 µg/mL; cells were incubated for 2 min at room temperature, and then PI was added to a final concentration of 0.4 µM. To exclude background fluorescence (seen as the large peak to the left in the histograms), the settings for the green- and red-fluorescence thresholds were confirmed by reviewing images from the Tali® cytometer. In the images, blue-circled cells indicate viable, nonfluorescent cells; green- and red-circled cells were recorded as fluorescent in the DiBAC4(3) and PI channels (utilizing the GFP and RFP settings on the Tali® instrument), respectively; yellow-circled cells exhibited both green and red fluorescence.
Table 1. Summary viability data for two yeast strains: Percentage of cells counted in each Tali® cytometer detection channel.
|Detection Channel*||DiBAC4(3)||PI||DiBAC4(3) and PI|
|Green and red (yellow)||0||0||47|
|Green and red (yellow)||0||0||51|
|*The Tali® Image-Based Cytometer was used in conjunction with the green-fluorescent DiBAC4(3) potential-sensitive dye and the red-fluorescent propidium iodide (PI) nucleic acid stain for viability analysis of two yeast strains after mixing equal amounts of live and heat-killed cells, as described in Figure 1.|
More Options for Multiplex Analyses
We have demonstrated that the Tali® Image-Based Cytometer, in conjunction with fluorescent dyes DiBAC4(3) or PI, provides a reliable analytical platform to quantitatively assess yeast viability. Moreover, because either DiBAC4(3) or PI can be used for yeast viability determination, a second parameter can be measured in the available fluorescent channel. For example, if DiBAC4(3) is used for viability measurements, a red-fluorescent probe—such as RFP or an Alexa Fluor® 546, Alexa Fluor® 555, or R-phycoerythrin (R-PE) conjugate—can be monitored in the red channel of the Tali® cytometer. Conversely, when making PI viability measurements in the red channel, the green channel can be used to detect a green-fluorescent probe—such as GFP or an Alexa Fluor® 488 or fluorescein (FITC) conjugate—in the same cell population.
Learn more about the Tali® Image-Based Cytometer.