Counting of Somatic Cells in Raw Milk NucleoCounter ® SCC-100 TM
NucleoCounter SCC-100 Stand-alone fluorescence microscope with built in computer for image analysis Disposable sampling and measuring device, preloaded with nuclei staining dye
Easy operation 1. Lyse2. Load3. Count
Fast Result + = 30 seconds max 1 minute
Base technology of ChemoMetec Fluorescence Microscopy
Integrated Fluorescence Microscope Lenses: magnification <1X. Excitation filter: sorts out green light. Emission filter: sorts out long wave length including red light. fixture
NucleoCassette
Propidium iodide (PI) Immobilized in the interior of the NucleoCassette, 2.8 ug in each A fluorescence dye, which intercalates with DNA of cell nuclei Cell membrane permeable
1 dye molecule per 4-5 bp Genome between 2.5 x 10 9 – 4.0 x 10 9 bp. Propidium Iodide DNA base pair (bp): A: Adenine T: Thymine G: Guanine C: Cytosine C 27 H 34 N I - Mr 668.4
Reagent C-100 Disrupts the plasma membranes of the mammalian cells, rendering the nuclei susceptible to staining with PI
Total cell count Viable Non-viable Free nucleus PI PI-stained nucleus Lysis buffer (Reagents C)
Sensitive parts, moving parts and “wear and tear” parts have been taken out of the instrument and integrated in the disposable NucleoCassette - Pump - Tubing’s - Mixer - Precision optics - Chemicals - Calibration - Waste container The NucleoCassette
Calibrated SCC-cassettes
Highly reliable LEDs Thermal drift in optical intensity is approximately 0.2 %/°C
Key Advantages Simple Operation –Ready in 5 seconds –5 minutes operator training –No cleaning Calibration Free –LED's, CCD and fixed optics assure stable factory calibration –Individually calibrated cassettes assure long term stability Maintenance Free –Robust and reliable parts used in design –2 years product guarantee Operator Independent –Objective cell counting Safe sample handling –PI immobilized inside NucleoCassette
Good reproducibility Results from different NucleoCounters can be directly compared: –All NucleoCounters are calibrated to respond as the same Master NucleoCounter at ChemoMetec. –The initial calibration will maintain throughout the lifetime of a NucleoCounter.
Repeatability Repeatability error dominated by count statistics (Poisson distribution) CV = 1/ x 100%
Repeatability Repeatability*: - CV < 8% at 150,000 cells/ml - CV < 6% at 300,000 cells/ml - CV < 4% at 500,000 cells/ml - CV < 3% at 1,000,000 cells/ml CV = Repeatability error Coefficient of Variance
Reproducibility Comparison of two instruments from different production series - Factory Settings!
Reproducibility Only significant method contribution is repeatability –In test more than 95% of the intra-laboratory reproducibility explained by repeatability
Routine Milk Samples
Milk Application SCC Stability –The inherent stability of the NucleoCounter SCC-100 offers tools to monitor and assure stability of SCC results obtained by routine instruments Pilot Samples –The NucleoCounter SCC-100 allows fast and reliable determination of SCC pilot samples
Somatic Cell Counting NucleoCounter SCC-100 –Stand-alone fluorescence microscope –Maintenance free –Calibration free NucleoCassette –Pre-loaded with nuclei staining dye –Analysed volume 1µl (working factor 1,000) –Calibrated measurement chamber (Dot code) Cell Counting –Operator independent –Reproducibility - Appr. Equal to Repeatability! –Repeatability Error - Appr. Poisson statistics 500,000/ml) –Sample throughput – About 100 samples/hour –SCC - 10,000/ml to 1,000,000/ml