Fast and Precise Analysis of Yeast with the NucleoCounter YC-100 for Beer !
Yeast Cell (Saccharomyces cerevisiae)
The NucleoCounter YC-100 offers simple and
effective direct estimate of cell count on routine bases.
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
any other method for the estimation of cell concentration
and the method can be performed by operators
with only limited training in laboratory work.
It is therefore the obvious choice of methods
for any laboratory or production facility
where the speed and reliability is of importance.
When working with cell cultures, determination of the number of cells per volume using microscopic techniques is a part of the daily routine. The manual microscopic method commonly used today is relatively time consuming and generally provides results,
which depend on the subjective judgement of the operator.
system is a fast alternative to these manual counting methods.
-based image analysis.
Prior to analysis the sample is mixed with a reagent, which effectively disrupts the cell membrane and dissolves any cell aggregates.
A suitable volume of the sample is mixed with a volume of a lysis buffer, which effectively dissolves cell aggregates and mixed for
a few seconds. Due to the lysing of the cell
membrane the cell nuclei is susceptible to staining with nuclei staining
The NucleoCassette is a highly specialised device,
which holds the key to several of the unique features of
NucleoCounter. The cassette is pre-loaded with a dye
and its construction is opotimised for the mixing of sample
and dye. It contains a measurement chamber where
the fluorescent image is recorded.
Approximately 50 ul of the sample mixture is loaded into
the NucleoCassette. Within the NucleoCassette a nuclei staining dye has been immobilised (Propidium iodide). The sample dissolves the dye and after mixing the mixture
reaches the transparent measurement chamber where
the fluorescent image is recorded.
The NucleoCassette is analysed
in the NucleoCounter Instrument.
The estimate of the number of cells per ml
is presented after about 30 seconds.
The excitation source
are green Light Emitting Diodes (LED's).
focused onto the detector
One of the key features of the NucleoCounter is the low
magnification of the microscope. The effect of the low
magnification is the large area of the sample which is
represented in each image.
The section which is blown-up in the figure illustrates approximately the field of view in traditional microscopy methods. The effect of low magnification is clearly visible in the limited spatial representation of each cell,
which only expands few pixels.
The result of a comparison test between
a manual total cell count and the NucleoCounter
using NSO mouse myeloma cell samples
from a 10-Liter bioreactor.
The observed correlation coefficient was r = 0.96 indicating a good correlation between the two methods.
Repeatability test based on triplicate measurements
of samples of a CHO HIR cell line.
The figure illustrates that the NucleoCounter shows less repeatability error than the manual microscopy method.
The observed CV of the NucleoCounter method is less than 5%.
The NucleoCounter Instrument offers several unique benefits when compared to other manual
and automated methods for cell analysis.
For the analysis a small volume of the sample mixture is loaded into the NucleoCassette.
This volume is only about 50 ul.
Therefore it is possible to perform an accurate an a precise estimate of the cell concentration
with only a very small sample consumption.
The measurement volume of the NucleoCounter is as large as 2 ul.
This volume is at least 10 times as large as the volume normally used for the manual cell counting methods.
Therefore the precision of the NucleoCounter method is generally better than 5 % expressed
as Coefficient of Variance (CV).
The measurement range of the NucleoCounter Instrument is between 5x10E3 (5,000) and 2x10E6 (2,000,000)
cells/ml. The optimal range in the sample mixture is between 1x10E5 (100,000) and 2x10E6 (2,000,000) cells/ml.
Assuming normal sample preparation the optimal measurement range is equivalent to between3.0x10E5 (300,000)
and 6x10E6 (6,000,000) cells/ml in the original cell suspension.
The effective view area (the surface area of the sample recorded in each image) of each NucleoCounter
Instrument is determined by the optical system of the fluorescence microscope and it will be unchanged
throughout the lifetime of the instrument.
during production. This information is written on each NucleoCassette and read by the NucleoCounter Instrument
each NucleoCassette renders a precise assessment of the
volume of sample or sample mixture being analysed.
volume. Finally, it takes less than 60 seconds to perform the entire analysis, including the time needed for sample
The NucleoCounter is a versatile instrument offering a simple and a reliable solution to many of those tasks carried out
on a daily bases in a typical laboratory working with cells.
The inherent property of the NucleoCounter is to measure cells, which have a nuclei which is permeable with respect
to the dye being used. Since non-viable cells are permeable to the dye this can be used to estimate the viability of
a cell sample.
for the counting of various cell species without any adjustment of calibration.
One application where the NucleoCounter offers substantial advantage compared to other methods is when cells are
immobilised, either in cell aggregates or on the surface of Cytodex microcarriers.
The intended use of the NucleoCounter system is for research use only - not for diagnostic use.
Since the detection principle is based on staining nuclei with a dye
it becomes evident that in order to stain the nuclei it has to be permeable to the dye. The dye can not penetrate a viable cell and
thus it is necessary to lyse the cell membrane prior to staining. Non-viable cells, on the other hand, are permeable and can therefore
be stained with the nuclei dye. Using this property of non-viable cells
and combining results obtained from samples which have not be
lysed (non-viable cell count) and the result obtain by lysing the cells
(a total cell count) it becomes possible to use the NucleoCounter to estimate the viability of a cell sample.
The cell samples were analysed on the NucleoCounter before and after treatment with a lysis buffer, giving an estimate of non-viable and total cells, respectively. The manual counting method was based on the Trypan blue exclusion procedure.
Mammalian cells can differ with respect to many physical and/or morphological properties.
Many methods for the counting of mammalian cells are therefore dependent on a calibration or adjustment
in order to count reliable
the number of cells present in a sample.
Since the principle used in NucleoCounter for the counting of mammalian cells is based on staining the DNA
within the nuclei it becomes evident that the method is reliable in the measurement of various cell species
adjustment or calibration.
CHO cells and
Many mammalian cells have affinity to grow on a surface or to form aggregates or clusters. This can make the sample
treatment of such sample very complicated when it is necessary to retain the physical and/or morphological property
of the cells as is the case with most commonly used
methods for cell counting.
to preserve the cells. In fact, the sample preparation used in the NucleoCounter method largely dissolves the cell
membrane releasing the nuclei to the exterior of the cell. This is done quickly and effectively and to such an extend
aggregates are dissolved in a matter of few seconds.
aggregates or immobilised on a surface.
- Click on the photos for large view -
The NucleoCounter is an instrument suited for the counting of cells and estimate of viability. Optionally it can be connected to a PC for documentation of results and images.
The technique that the NucleoCounter is based on offers great advantages
compared to existing methods. High precision, fast analysis, small sample
consumption, simple operation, reliable operation.
One of the inventive aspects of the NucleoCounter is the NucleoCassette.
It is a disposable sampling and measurement unit, which comes pre-loaded
with nuclei staining dye, Propidium-iodide.
After analysis the NucleoCassette containing the sample, can be safely
disposed of in the same manner as biological laboratory waste.
Heterogeneous cell lines such as adipocytes, CHO cells and
hybridomas have successfully been measured without any adjustment
of the instrument.
The NucleoCounter is also well suited for the measurement of cells,
which are grown on the surface of Cytodex Micro carriers.
of the Cytodex microcarires are easily analysed.
The sample pre-treatment involves the lysing of the cell plasma membrane
thus dissolving any cell aggregates.
This releases the cell nuclei to the solution and it becomes available for staining.
The sample consumption of the NucleoCounter is very low.
Volumes as small as 30 to 50 ul can be used for total cell count and volumes
between 75 and 100 ul are needed for the non-viable cell count.
These are the smallest usable volumes and due to dosage error it is generally recommended that volumes of between 200 and 400 ul are used.
When estimating total count the sample pre-treatment involves the addition of
two lysing buffers. Normally the buffers are added to the sample in volume
equal to the sample volume.
After the addition of the first buffer the sample is mixed for few seconds.
The effect of the first buffer (Reagent A100) is the lysing of the cell plasma membrane.
This lysing is completed virtually instantaneously.
As soon as Reagent A100 has been mixed with the sample the second buffer
(Reagent B) is added and the sample mixture thoroughly mixed again.
The effect of Reagent B is partly to stabilise the nuclei but also to provide optimal
conditions for the nuclei staining dye. T
he sample mixture is stable for considerable time after the addition of the reagents,
it only needs to be mixed prior to analysis.
To analyse the sample it is loaded into the NucleoCassette where it dissolves
the immobilised Propidium iodide nuclei staining dye.
About 50 ul are loaded in the cassette by pressing the piston.
loaded with the sample without any sample pre-treatment.
For the estimate of total count it is the sample/reagent mixture which is loaded I
nto the cassette.
When the sample or sample mixture has been loaded into the NucleoCassette
it is ready to be analysed without further delay.
The cassette is inserted into the NucleoCounter instrument where the final mixing
of the sample or sample mixture and the nuclei staining dye takes place.
The instrument needs no warming-up or preparation.
It is ready to measure few seconds after it has been turned on.
When the NucleoCassette has been inserted into the NucleoCounter the operator
simply has to press the "Run" button to initiate the analysis.
the resulting cell count is presented in the built-in display of the instrument.
For visual inspection of the collected image and for documentation purposes
The NucleoView is a software which allows the user to generate and print tables
with the results as well as doing viability calculations.