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 Y100, a lysis/disaggregation buffer, which permeates the yeast cell wall and membranes, allowing the cellular DNA to be stained. Reagent Y100 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.

For a viability count, you load the original cell culture into a cassette and this way you can calculate a viability.

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 Y100 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 or calibration.