Materials and Methods
Image-Based Cytometry with Cellometer Vision
The Cellometer Vision has been previously described elsewhere [17-19,21]. The instrument uses bright field and two fluorescent imaging modes to measure yeast cell concentrations and viability. Each yeast sample (20 µL of a mixture of between 5 x 105 and 1 x 107 cells/mL) was added to a Nexcelom counting chamber and bright field and fluorescent images were taken at four locations within the chamber. All images were subsequently analyzed by Cellometer software to count cells, measure fluorescent intensities, and measure cell morphology characteristics such as cell size and circularity.
Preparation of Yeast Cell Cultures
Single colonies of S. cerevisiae strain EBY-100 were inoculated into conical flasks containing YPD media. The cultures were incubated overnight on a shaker and separated into two centrifuge tubes, where one was heat-killed in a boiling water bath for 15 minutes. Fresh and heat-killed cultures were mixed to produce the theoretical viability mixtures of 0, 25, 50, 75, 100% viability.
Methylene Blue Analysis
A working concentration of methylene blue was created (0.01% w/v) and was mixed with five yeast samples of theoretical viabilities from 0 to 100%. A standard hemacytometer was used to count the samples in quadruplicate. All results were compared to those obtained from image cytometry.
Nuclei Acid Viability Stains
PI, EB, DAPI, and 7-AAD stained with the five yeast viability samples from 0 to 100%. PI and EB were analyzed directly after staining. 7-AAD and DAPI were both incubated for 5 minutes at room temperature before cytometric analysis. All samples were measured in quadruplicate and the results were compared to manual methylene blue results.
Viability Analysis using Membrane Potential, Intracellular Conversion, and Enzymatic Stains
CFDA-AM, oxonol, and MgANS stained the five yeast viability samples from 0 to 100%. CFDA-AM was incubated for 45 minutes at 37°C and oxonol and MgANS were both incubated for 5 minutes in the dark at room temperature. All samples were analyzed with the image cytometer, in quadruplicate, and the results were compared to those obtained from manual methylene blue.
Viability Analysis using Dual Stain Methods
AO/PI and CFDA-AM/PI stained the five yeast viability samples from 0 to 100%. AO/PI was analyzed immediately after staining. CFDA-AM samples were incubated for 15 minutes in the dark at 37°C. All samples were imaged in quadruplicate and were compared to manual methylene blue results.
Additionally, AO/PI results were compared in a head-to-head method to methylene blue results over a time course of bioethanol fermentation. Yeast cells were collected at propagation, 2, 5, 10, 25, 30, 40, 50, 60, and 70 hours into the fermentation process. All collected samples were stained with either AO/PI or methylene blue and then analyzed using the image cytometer or hemacytometer, respectively. Yeast cell concentrations and viabilities were compared.
Measuring Viability and Vitality During Lager and Ale Fermentation
Avery Brewery Company used two commercially-available yeast brewing strains, one ale yeast and one lager yeast strain. The yeast cells were propagated in stainless steel conical vessels with 2-4 ppm of oxygen and perpetual aeration for 48 hours in a 10°P wort. After this time, the tank was cooled, yeast cells were pitched into conical stainless steel fermentation vessels with 13.8 and 10.3°P wort for the ale and lager yeast, respectively. Ale yeast fermented for 6 days and lager yeast fermented for 14 days. The initial cell count for both yeast strains were 1 x 106 cells/mL/°P. Viability was measured with PI and vitality by CFDA-AM every 24 hours throughout the study. Initial measurements were taken when the yeast arrived from the supplier, then once every day through 2-3 days of yeast propagation, again at the “first knockout” when the yeast and wort are initially combined in the fermentation tank, and then every 24 hours after during fermentation. Tanks were then cooled to 0°C to precipitate yeast flocculation, then readied for processing and packaging at a time point called “crash”. Viability and vitality analyses were carried out daily until the tank was crash cooled. All samples were taken from the middle of the tank, where active yeast are present but does not include the yeast cells that had already flocculated and were therefore no longer in suspension.
The fermentation of ale yeast was maintained at a constant 18°C until the beer was roughly 70% attenuated, at which the temperature was raised to 22°C to permit diacetyl rest. This temperature alteration took place approximately 30 hours after the “last knockout” time point, in which the entire volume of wort was mixed with the yeast in the fermentation tank. After cooling, yeast cell counts fell dramatically as yeast began to flocculate. The fermentation of lager yeast was kept at 13°C until 70% attenuation, at which the temperature was raised to 18°C to permit diacetyl rest. For the lager yeast, this temperature fluctuation occurred much later during fermentation, at approximately 115 hours after the last knock out.
Image Cytometric Analysis
The appropriate FOMs were used to detect the emission wavelengths for each stain. The nucleic acid viability stains used: VB-450-302 (EX: 375 nm, EM: 450 nm), VB-595-502 (EX: 525 nm, EM: 595 nm)/ VB-660-503 (EX: 540nm, EM: 660 nm) were used to detect DAPI, PI/EB, and 7-AAD, respectively. For membrane potential, intracellular, and enzymatic viability stains: VB-535-402 (EX: 470 nm, EM: 535 nm) was used to detect CFDA-AM and oxonol. VB-535-302 (EX: 375 nm, EM: 535 nm) was used to detect MgANS. For dual staining methods, VB-535-402 and 660-503 were used for CFDA-AM/AO and PI, respectively. Three equations were used by the Nexcelom software for viability calculations (see below). Nucleic acid membrane integrity, membrane potential, and intracellular staining methods utilized equation 1. Enzymatic staining viability utilized equation 2. In equations 1 and 2, FL represents nonviable and viable cells, respectively. Dual staining methods utilized equation 3, where FL1 and FL2 represent the total number of viable and nonviable cells, respectively.
Equation 1: Viability = (BR-FL)/FL x 100%
Equation 2: Viability = FL/BR x 100%
Equation 3: Viability = FL1/(FL1+FL2) x 100%