D- Count®
Applications
for the Food and Cosmetic
Industry
Elke Kohler, Chemunex SA, Paris
The Rapid Microbiology Company
D-Count ® : Non-Filterable Product Testing
Real-time Microbiology Analysis
in Food and Cosmetics
The Rapid Microbiology Company
D-Count ® Technology
Bacteria
Enzyme
Viability substrate
Cell Labelling
12 minutes
Free fluorochrome
Flow Cell
Detector
Cell Counting
Laser
1 minutes
Detector
The Rapid Microbiology Company

Fluorassure Viability Markers
Viability substrate
Criteria of Viability :
Enzyme
Enzyme activity
 Membrane integrity

Bacteria
Free fluorochrome
Applicable for :
Total Viable Count
 Yeast & Moulds

The Rapid Microbiology Company
Cell Labelling Technology
Fluorescence Based Cell Labels
Fluorescent
Signal
515 nm
Incident
Light
488 nm
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D-Count ® : Counting Principle
Flow Cell :
Labelled Sample
Linear fluid stream
 Cells separated into
single entities
 Sensitive detectors

Detector 1
Laser
Detector 2
Sample
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D-Count ® : Optical Bench
Sample
Detectors
Flow cell
Objective
Lens
Red emission
Beam splitter
Dichroic filter
Laser beam
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Data Processing
PMT Detector
Data Processing
Digital Signal Processor
collects blocks of data

Colour fingerprint

Light intensity

Signal shape
Rejected
Background
Results
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D-Count ® Main Menu
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Sample identification
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Presentation of results
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Presentation of results
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Dedicated Automation

Capacity


High Throughput Automate


50 samples / batch
up to 60 samples per hour
Simple sample pre-treatment

Product sample directly loaded onto automate

samples containing big particles are centrifiltrated before
loading
The Rapid Microbiology Company
D-Count ®
Combining Speed with Sensitivity

Designed for non filterable products

Direct Counting in less than 30 minutes

Automated system

Automate capacity 50 -64 samples

High throughput

Sensitivity down to 5x102/ml sample

Linearity between 5x102 to 5x105/ml
The Rapid Microbiology Company
D-Count
Benefits from speed and sensitivity

Final products testing in hours not days
Fast detection of problems (Raw materials  Final product)
Immediate response to contamination incidents
Rapid confirmation of corrective action effectiveness
Real time trending of the production process




Decrease inventory holding and warehouse costs
Decrease product losses
Minimise production disruption
Increase production flexibility




The Rapid Microbiology Company
BactiFlow
Compact Microbial Analyser
Real-time microbiology analysis
in Food, beverages & cosmetics
The Rapid Microbiology Company
Based on Proven Technologies

Fluorescent cell labelling (Fluorassure®)



Bench Top Microbial Analyser




Same reagent technologies as used on D-Count® & ChemScan® RDI
Validated on more than 500 strains
Compact Flow cytometer specifically designed for microbial detection
Laser based detection
Simple to operate
Data acquisition, analysis and real time result display


Automatic data interpretation
Quantitative results in minutes
The Rapid Microbiology Company

Fluorassure Viability Markers
Viability substrate
Criteria of Viability :
Enzyme
Enzyme activity
 Membrane integrity

Bacteria
Free fluorochrome
Applicable for :
Total Viable Count
Total Count
 Yeast & Moulds
 Specifics (on going)

The Rapid Microbiology Company
Quantitative Microbial Counting
Flow Cell :

Linear fluid stream

Cells Separated into
single entities
Labelled Sample
Detector 1
Laser

Sensitive detectors
Detector 2
BactiFlow
Sample
The Rapid Microbiology Company
Rapid

Time to result:
 10
1
minutes labelling prior to analysis
minute analysis per sample
 Simple
sample preparation adapted to product
matrices
No cell multiplication
required
Labelling 10 min.
Counting 1 min.
The Rapid Microbiology Company
Sensitive
Combining Speed with Sensitivity

Quantitative counting cell by cell

Sensitivity of detection less than 100 cells/ml labelled
solution

Linear response up to 100,000 micro-organisms

Direct Counting in less than 15 minutes

Protocol available for Presence / Absence within 24 hours
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Easy to Use

Ready to use Reagents

Simple preparation for sample products

Validated protocols for a full range of applications

Fully automated detection and counting

No data interpretation required

Full data traceability

PC with Windows XP interface
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BactiFlow
Fruit based Products
Pure cultures
Applications
Dairy Products
Cosmetics
Water
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D-COUNT ® and BACTIFLOW
DISCRIMINANTS
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Fingerprint Colour
Fluorochrome Emission Spectrum
Transmission
Green channel
Red channel
Wavelength (nm)
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Ratio at Primary Peak
Intensity
H2
Green
channel
« Peak ratio » =
H1
H1
Intensity
Red
channel
Samples
H2
Samples
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Signal
Fluorescence Intensity
Fluorescence Intensity
Baseline
Samples
Samples
(Signal) - (Baseline)
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Signalauswahl
Ausgewählte Signale
Schwellenwert
Hintergrund
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D-COUNT ® and BACTIFLOW
APPLICATIONS
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Applications
Beverages
 Fruit Juices
 Soft drinks concentrates
Food
 Milk products
 Fruit products
 Frozen vegetables
 Salads
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Applicable to Range of Products
Non sterile pharmaceuticals
• Syrups
• Ointments
• Tablets
• Lotions
Cosmetic products
• Shampoos and hair conditioner
• Creams
• Make-up and Foundation
• Toothpaste
• Lotions
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Applications
Final product

Microbial Limit Test
 Presence / Absence test
In Process
TVC

Raw Materials
 Bioburden
 Cleaning
validation
 Trending
Yeast & Moulds
D-Count
E. coli (on going)
Other specifics
(on going)
Environmental


Surface
Personnel
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Applications

Presence / Absence Test


TVC - Direct Counting


90 minutes
Yeast / Moulds - Direct Counting


24 hours
90 minutes
E. coli and coliform tests

(availabilty sept. - oct. 01)
20 hours
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Food & Beverages
Applications

Yeast detection in Fermented milk products

TVC or Yeast counts in Juice concentrates

“Sterility test” in Desserts

TVC or Yeast & mould detection in Fruit juices &
beverages

TVC in Process Water
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D-COUNT
Sample Background + Spiking Experiments
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D-Count Evaluation

Pure culture correlation studies

Non contaminated product analysis

Inoculated product analysis
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Pure Cultures Studies

Range of micro-organisms tested





Staphylococcus aureus
Escherichia coli
Bacillus circulans
Bacillus licheniformis




Enterobacter cloacae
Candida albicans
Aspergillus niger
Penicillium decumbeus
Methods
Micro-organisms isolated from plates
 Selected colony incubated for 24 hours at 30°C in TSB
 Serial dilution in physiological water
 Analysis by D-Count & plate

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D-Count ® Evaluation

Pure culture correlation studies

Non contaminated product analysis

Inoculated product analysis
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Pure Culture Correlation
Candida albicans ATCC 10231
1000000
10000
D-Count (yeast /100µl)
D-Count ( bacteria / 100µl)
Enterobacter cloacae
2
R = 0,9992
100000
10000
1000
100
10
10
100
1000
10000
Plate Count
100000 1000000
R2 = 0,9964
1000
100
10
1
1
10
100
Plate Count
1000
10000
The Rapid Microbiology Company
Pure Culture Correlation
Staph. aureus ATCC 6538P
Bacillus circulans
D-Count ( bacteria / 100µl)
100000
D-Count ( bacteria / 100µl)
R2 = 0,996
10000
1000
100
10
1
1
10
100
1000
Plate Count
10000
100000
1000
R2 = 0,9961
100
10
1
1
10
100
Plate count
The Rapid Microbiology Company
1000
Pure Culture Correlation
Bacillus licheniformis
1000
D-Count ( bacteria / 100µl)
D-Count ( bacteria / 100µl)
E. coli ATCC 8739
R2 = 0,9961
100
10
1
1
10
100
Plate count
1000
10000
R2 = 0,9949
1000
100
10
1
1
10
100
1000
Plate count
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Pure Culture Correlation
Penicillium decumbeus
1000
D-Count (Moulds / 100 µl)
D-Count (Moulds / 100 µl)
Aspergillus niger
R2 = 0,9968
100
10
1
1
10
100
Plate count
1000
1000
R2 = 0,9571
100
10
1
1
10
100
Plate count
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1000
Pure Culture Correlation
Combined plot of all micro-organisms analysed
Staphylococcus aureus
Escherichia coli
Bacillus circulans
Bacillus licheniformis
Enterobacter cloacae
Candida albicans
D-Count ( Micro-organisms /
100µl)
1000000
R2 = 0,9919
100000
10000
1000
100
10
1
1
10
100
1000
10000
Plate Count
100000 1000000
The Rapid Microbiology Company
Conclusions

Good correlation with all micro-organisms tested

Sensitivity of detection less than 100 cells per ml

Linear response up to 5 x 10e5 micro-organisms per
ml
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