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Counting of Somatic Cells in Raw Milk
NucleoCounter®SCC-100TM
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NucleoCounter SCC-100
Stand-alone fluorescence microscope with built in computer for image analysis
Disposable sampling and measuring device, preloaded with nuclei staining dye
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Easy operation
1. Lyse 2. Load 3. Count
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Fast Result
+ = 30 seconds
= 30 seconds
------------------
max 1 minute
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Base technology of ChemoMetec
Fluorescence Microscopy
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Integrated Fluorescence Microscope
Lenses: magnification <1X.
Excitation filter: sorts out green light.
Emission filter: sorts out long wave length including red light.
LensesCCD
Emission Filter
LED’s
Excitation Filter
fixture
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NucleoCassette
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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
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1 dye molecule per 4-5 bp
Genome between 2.5 x 109 – 4.0 x 109 bp. Propidium Iodide
DNA base pair (bp):
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
C27H34N42+.2I- Mr 668.4
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Reagent C-100• Disrupts the plasma membranes of the mammalian cells, rendering the nuclei
susceptible to staining with PI
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Total cell count
Viable Non-viable
Free nucleus
PI
PI-stained nucleus
Lysis buffer
(Reagents C)
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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
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Calibrated SCC-cassettes
W
T (
co
de
)
Dots for thickness code (T)
TAF0.01TAreaTWLVolume
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Highly reliable LEDs
Thermal drift in optical intensity is approximately 0.2 %/°C
Optical intesity versus number of Number of analysis for the NucleoCounter SCC-100
0.90
0.95
1.00
1.05
1.10
0 250000 500000 750000 1000000
Number of analysis
Optica
l inte
nsi
ty (
a.u
.) v
Test conditions:t = 60°C, If(LED) = 100mASCC-100 Operating conditions max:t = 40°C, If(LED) = 30mA
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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
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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.
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Repeatability•Repeatability error dominated by count statistics (Poisson distribution) •CV = 1/ x 100%
CV% - NucleoCounter SCC-100
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
14.0%
16.0%
18.0%
0 200 400 600 800 1000 1200
SCC
Nucle
oC
ounte
r SC
C-1
00 C
V%
NucleoCounter SCC-100 Poisson
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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
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Reproducibility•Comparison of two instruments from different production series - Factory Settings!
NucleoCounter SCC-100 B vs A
y = 1.0047x - 0.0285
0
200
400
600
800
1000
1200
0 200 400 600 800 1000 1200
NucleoCounter SCC-100 Inst. A
Nucle
oC
ounte
r SC
C-1
00 I
nst.
B
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Reproducibility
•Only significant method contribution is repeatability–In test more than 95% of the intra-laboratory reproducibility explained by repeatability
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Routine Milk Samples
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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
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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
• (≈5% @ 500,000/ml)– Sample throughput – About 100 samples/hour– SCC - 10,000/ml to 1,000,000/ml