09/07/2005 • Focus on Products at Analytica • Laboratory appliances

Yeast Cells Counted in 30 Seconds

The NucleoCounter YC-100 offers simple and effective direct estimate of cell concentration on routine basis. Compared to many methods used for the estimate of cell concentration the value of direct cell counting improves greatly the value of the results, since no consideration has to be given to the strain of cells, nor the sample condition. Further, the NucleoCounter YC-100 is truly calibration free instrument!

The NucleoCounter YC-100 is as simple as any other method for the estimation
of cell concentration and can be used by operators without substantial laboratory training. It is therefore the obvious choice of methods for any laboratory or production facility where speed and reliability is of importance

The NucleoCounter method provides a fast alternative to the manual methods, yielding objective and reliable results. Additionally the NucleoCounter technology provides superior safety for the user compared to other cell counting methods.

The NucleoCounter system comprises NucleoCassette for sample handling,
the NucleoCounter instrument for particle analysis and lysis buffer for
cell lysis. Basics of the system are explained below.

NucleoCounter YC-100 is based on a fluorescent microscopy technique for counting yeast cells. This instrument is optimized to count Saccharomyces cerevisiae cells in research and production applications including beer production. The integrated fluorescence microscope of the NucleoCounter is designed to detect signals from the fluorescent dye, Propidium Iodide (PI), which intercalates to DNA in the cell. Excitation of PI occurs at ~540 nm (green light) and strong emission (fluorescence) occurs at ~600 nm (red light). The signal intensity is enhanced by a factor of 20–30 when PI is bound to DNA which improves the signal to noise ratio.

The NucleoCounter detects "PI-stained" DNA rather than individual cells. Since the amount of stainable DNA is relatively constant regardless of strains, no calibration for varying cell size or morphology is required. The instrument can be set up to measure either haploid or diploid cells.

Combined with a charged coupled device (CCD) camera and integrated image analysis, the compact microscope is designed to permit small and large cell culture facilities to perform fast, efficient, and reproducible cell counts.

Determination of Total Cell Count

The determination of the total cell count involves sample preparation and sample analysis. During sample preparation, a representative cell sample from the cell suspension (e.g. 50 µl) is mixed with a volume of lysis buffer Reagent Y100 (e.g. 450 µl or more depending on cell concentration) and vortexed for few seconds. This subsequent cell lysate is loaded into a NucleoCassette, where the DNA is "stained" with PI. By immersing the tip of the cassette into the sample mixture and depressing the loading piston; this loads a predefined volume (~50 ml) of the sample mixture into the cassette.

The NucleoCassette is then placed into the NucleoCounter for analysis. The lysate dissolves the Propidium Iodide, which is immobilized in the cassette, and a mechanical drive in the instrument presses the piston further, thereby transferring the sample into the measuring area of the cassette. During analysis the fluorescent signal is registered and correlated to a total cell count. The total cell concentration in the NucleoCassette is presented in the NucleoCounter display as cells per ml. Optionally, the data can be presented on a PC using the NucleoView software or printed on an optional external printer.

Determination of Viability

As indicated above, the total cell determination is achieved by deliberately disrupting the plasma membranes of all cells in a sample by pre-treatment with Reagent Y100. All nuclei are therefore accessible to PI staining, independent of whether cells initially were viable or non-viable prior to cell lysis. However, by loading a cell sample, without pre-treatment other than dilution by PBS depending on cell concentration, directly into the NucleoCassette only cells with pre-impaired plasma membranes are "PI-stained" Analysis in the NucleoCounter then gives an estimate of the concentration of non-viable cells in the original cell suspension. Based on these two measurements the viability can be calculated and displayed via the optional NucleoView software.

Discussion and Conclusion

One of the major disadvantages of standard manual cell counting (haemocytometer) is the subjective nature of the method. It has been demonstrated that the manual method is often severely affected by operator effect. Such a variation from specialist-to-specialist, can unfortunately lead to complications in downstream applications, especially when accuracy is critical for the validity of the analysis.

Other automated methods of yeast cell counting currently in use have not entirely solved the critical points of reproducibility and speed to a satisfactory level. To date, manual and automated viability determinations have been based on exclusion methods (e.g. Methylene Violet) or on differentiation between cells sizes (e.g. by using impedance). Many of these systems require frequent calibration for cell size or morphology prior to use and often these requirements compromise the accuracy of the final result. As indicated above, the NucleoCounter YC-100 offer high precision, low maintenance and an instrument that does not require calibration for individual cell types or morphology.
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