Fast Cell Counting and Viability Measurement of Yeast Cells with the LUNA-FL™ Fluorescence Cell Counter
LUNA-FL™ DUAL FLUORESCENCE CELL COUNTERdownload pdf
The current method of counting yeast is the ASBC (American Society of Brewing Chemists) method. In this protocol, yeast cells are stained with Methylene blue which is a cell membrane-impermeable dye. Dead yeast is stained in blue due to the compromised membrane integrity while live yeast is not. Because of the small size, yeast cells need to be observed in a high magnification (eg., 40X or higher objective lens). The magnification is inversely proportional to FOV (Field Of View). Researchers have to count both live and dead yeasts in the small square of the hemocytometer, move the microscope stage to cover a neighboring square and count live and dead yeasts in the next square. Counting continues until total 0.1µl counting volume is reached. In case of the central big tile of the hemocytometer, it corresponds to 25 small squares. It is tedious and painful (It can take up to 30 min!). The statistical significance of the ASBC method is low (25% error is typical) due to human errors, human interpretation and low counting volume (0.1µl). Moreover, in case of messy cultures (beer and wine brewing and bioethanol production), the manual counting is even difficult. Researchers need to distinguish yeast cells and non-cellular debris (hop in the beer brewing, grape in the wine brewing and corn mash in the bioethanol production) one particle at a time.
In contrast, it takes about 30 sec for LUNA-FL™ fluorescence cell counter to count yeast cells in the counting volume of 0.5µl which is five-fold more than the ASBC method.
- Higher statistical significance can be obtained because no human error and no human interpretation are involved besides the larger counting volume. Users do not need to distinguish yeast cells from non-cellular debris (hops, grape and corn mash). In addition, cell viability can be measured by different criteria if two kind of dyes are used and their fluorescence excitation/emission wavelength are compatible with the optics of LUNA-FL™ fluorescence cell counter.
- Figure 1. The working principle of yeast counting using FDA.
Yeast viability kit (FDA and PI staining solution, Yeast dilution buffer, Fluorescence signal enhancer 1)
2. Dilute the confluent yeast at 1:100 and culture for additional 3h for mid-log phase cells (optional).
3. Dilute the yeast culture at 1:100 with the yeast dilution buffer (O/N culture) depending on the concentration of the yeast cells. Centrifuge, discard the supernatant and suspend the pellet with the yeast dilution buffer (the mid-log phase yeast). (optional) 4. Heat the yeast sample at 70℃ for 30 min to prepare 0% viability yeast cells. (optional)
5. Mix 1 µl of the fluorescence signal enhancer 1 and 17 µl of the yeast sample. (optional)
6. Incubate for 10 min at room temperature. (optional)
7. Add 1 µl of FDA and 1 µl of PI to the yeast sample.
8. Incubate for 10 min at room temperature.
9. Load 10 µl of the stained yeast sample on the counting slide.
10. Wait for about 1 min or until all yeast cells are immobile.
11. Turn on LUNA-FLTM and press the “Yeast Cell Counting (FDA/PI)”.
12. Perform the cell counting using the following protocol
- Unlike AO/PI staining, in which two nucleic acid binding dyes bind instantly to genomic DNA, FDA staining make use of the enzymatic reactions for generating fluorescence. So it takes some time for the fluorescence intensity to reach a detectable level. To measure the optimal time required for the fluorescein detection, FDA was added to 100% live yeast cells, and the average fluorescence intensity of objects (yeast cells) was measured at 0, 5, 10, 15, 20 and 30min after the addition of the dye. As shown in Fig 3A, without the incubation time, the FDA fluorescence is not distinguishable from background (around 40). Within the first five minutes, the yeast cells rapidly gained the fluorescence signal. The rate of the fluorescein accumulation dropped significantly during the next five minutes and reached a plateau at 10min after the reaction. We decided to incubate yeast cells and FDA for 10min for optimal yeast counting.
- Figure 2. Automated yeast cell counting with the LUNA-FL™
- Figure 3. The kinetics of yeast staining by FDA
- Figure 4. The YPD medium is auto-fluorescent and contains esterase activity.
- Figure 5. The fluorescence signal enhancer 1 dercreases the background fluorescene level and increases the fluorescence intensity of yeast cells.
- Using the LUNA Reusable Slide for accurate cell counting with automated cell counters
- Fast automated yeast cell counting algorithm using bright-field and fluorescence microscopic images
Quick Start Guide2018-12-05
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