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ABSTRACT
New Brunswick Scientific has introduced a new device that automatically counts yeast cells in about 30 seconds, making manual counting obsolete. Like the original NuceloCounter, an automated device for counting mammalian cells, the new NucleoCounter YC-100 for yeast combines a fluorescence microscope with CCD camera and integrated image analysis software to perform rapid and accurate cell counts.
Monitoring the cell count during a fermentation run allows the scientist to activate inducible systems, or harvest at optimal times for the best product yield. The NucleoCounter YC- 100 can provide a fast and accurate DNA-based volumetric cell count within seconds, independent of debris or substrate aggregates in the media.
This system uses a disposable sampling device, the NucleoCassette. The small, plastic cassette is internally pre-coated with a fluorescent dye, propidium iodide (PI), which stains the cells' nuclei. Because the NucleoCounter detects signals from the stained nuclei, and not the cell, the system operates without regard to cell size or morphology.
MATERIALS AND METHODS
Withdraw 100 μL of culture to ensure a representative sample, and place 50 μL into a 1.5 mL tube. Mix thoroughly by inverting and shaking to ensure homogeneity. Treat with 450 μL of Reagent YC-100, a lysis/disaggregation buffer, which permeates the yeast cell wall and membranes, allowing the cellular DNA to be stained. Reagent YC-100 has the ability to dissociate minor clusters of yeast cells, but not to dissolve or dissociate cells that are in the process of reproductive division. Vortex or pipette vigorously for a few seconds to mix. The total volume is now 500 μL, with a 10-fold dilution. If necessary, repeat the dilution process to yield a sample 1 x 105 cells/mL to 2 x 106 cells/mL. (In general, a 100 to 1,000-fold dilution is generally suitable.)
Load the sample into the NucleoCassette, using the built-in piston to aspirate a pre-defined volume of approximately 50 μL. Place the NucleoCassette into the NucleoCounter and press "Run". In about 30 seconds, the total cell count will be calculated and shown on a built-in display.
RESULTS AND DISCUSSION
Using optional NucleoView software connected to your PC, you may additionally store and export results, as well as view an image of the stained cells. Results may be plotted as Optical Density (OD600) against Cell concentration (cells/mL) or Dry Cellular Weight (g/L) against Cell concentration (cells/mL).
Figure 2: NucleoCounter YC-100 raw image of yeast cells in suspension ( Saccharomyces cerevisiae ) after Reagent YC-100 treatment using 100-fold dilution. Cell count is approx. 8 x 107/mL. The objects have similar fluorescence intensities and are uniform in size, indicating minimal cell disruption. The yeast cells are uniformly dispersed throughout the entire image. The yeast cells may be manually monitored in the NucleoCassette measurement chamber using fluorescence microscopy with appropriate filters for propidium iodide.
Clean up is simple. After use, the NucleoCassette can be disposed of as biological waste, with the PI dye safely enclosed. Unlike flow systems and hemacytometers, the system requires no cleaning, maintenance, or calibration. For in-lab demo, contact New Brunswick Scientific. For additional information contact Mike Sattan, Director of Marketing via email at sattan@nbsc.com
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