The Coulter Principle technology is employed in breweries for yeast cell enumeration and sizing. The Multisizer™ 3 and the Z™ Series instrument models are based on this technology. The article highlights the Z Series models, including the Z2 Analyzer, the Z1 Dual Threshold, and the Z1 Single Threshold.
Role of Z Series Models in Yeast Cell Enumeration and Sizing
The analysis of yeast cells is performed during fermentation, where the yeast strains convert the carbohydrates in the wort into alcohol and by-products such as carbon dioxide. The fermentation process involves several stages, during which the analysis of the yeast cells is carried out.
The pitching process is a key step for cell enumeration, starting the fermentation process by introducing the live yeast culture into the cooled wort. Pitching is a critical to ensure a successful fermentation by delivering the proper cell concentration.
The beer taste will also be influenced by the yeast concentration. The pitch concentration is typically 30.3 x 106 cells/mL. The initial yeast cell concentration can be determined with the help of either a Z1 single or dual threshold. A Z2 model is required to measure the size distribution.
The sampling is often performed during the fermentation process, when the yeast cells may be in the budding stage. A budding cell will be enumerated as a single unit by the Z Series instruments. The samples collected at the end of the fermentation process likely to show the presence of a proteinaceous material during the subsequent analysis. A Z2 analyzer is recommended for the analysis of these samples.
The size-distribution histogram may reveal a bi-modal size population, showing the yeast cell population and the flocculated protein (Figure 1). Some amount of 2M sodium hydroxide (NaOH) is added to the yeast sample for the removal of the protein material, and the resulting sample is analyzed on the Z2 model.
Figure 1. A yeast sample with a bi-modal peak. The first peak is yeast, while the second peak is that of flocculated protein. The secondary peak can be larger than the yeast cell peak. Image credit: Beckman Coulter
Instrumentation and Experimental Procedure
The choice of instruments available based on the requirement include Zl Single Threshold P/N 6605698, Zl Dual Threshold P/N 6605699, Z2 Analyzer P/N 6605700 or Accuvette™ Cups BCI P/N 8320592. The reagents for this experiment include 2M NaOH Sigma, Z pak, L3 Beads, L5 Beads, L10 Beads, P/N S8263, BCI P/N 8320312, BCI P/N 6602793, BCI P/N 6602794, and BCI P/N 6602796.
For Z1 Dual Threshold and Z2 Analyzer, around 10 µL of yeast sample is added to 20 mL Isoton II. The experimental setup involves a 100 µm aperture tube with a setting of 3 µm as lower threshold and 10µm as upper threshold. Count mode is set between TL and TU, with dilution factor as 2000.
For Z1 Single Threshold, the sample is prepared by adding 10 µL of yeast sample to 20 mL Isoton II. A 100µm aperture tube is used with a lower threshold value set to 3µm. Count mode is set above TL, with a dilution factor of 2000.
The next step is the calibration of the Z system, which involves addition of five drops of L10 Beads to 20 mL of Isoton II, followed by gentle stirring by inversion to avoid the introduction of air bubbles. Then, the ‘Cal’ button on the keypad is pressed to go into the C1 page. After setting units to microns, Number Mode is entered in the calibration size, and the ‘Start’ button is pressed to record the calibration factor. The same procedure is repeated for three times to get the average results, which are then entered in new calibration factor.
For running size standard, the ‘Set Up’ is selected to set the lower threshold as 3 µm and upper threshold as 10µm. The count mode is set between the TL and TU. Now, five drops of L5 Beads are added to Isoton II and gently mixed by inversion. The ‘Start’ button is then pressed to begin the run. The resulting data is then acquired by Accucomp software after the run (Figure 2). If sample controls are there, they need to be run at this time.
Figure 2. L3 and L5 beads used to set size parameters. Image credit: Beckman Coulter
The samples are run, the output is selected, and the dilution factor is set to 2000. The dilution factor value may differ with the sample density. The result type is set to concentration, and around 10 µL of sample is added to 20 mL Isoton II and gently mixed. Now, the ‘Start’ button is pressed to begin the run. The results after the run are acquired with Accucomp™ software (Figure 3).
Figure 3. Brewers Yeast, which was grown at 37ºC 2 hours in distilled water. Average yeast cell size is 3.5µm. Aggregates show as 7µm or higher. Image credit: Beckman Coulter
For samples having a bi-modal peak as shown in Figure 1, four drops of 2M NaOH is added to the sample and gently mixed. The next step is the incubation of the resulting solution for five minutes. Then, the ‘Start’ button is pressed to begin the run. The Accucomp™ software is used to acquire the data after the run.
The results clearly demonstrate the advantage of using Z Series models for yeast cell enumeration and sizing in the brewing industry.
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.