1. D- Count® Applications for the Food and Cosmetic Industry Elke Kohler, Chemunex SA, Paris

2. D-Count ® : Non-Filterable Product Testing
Real-time Microbiology Analysis
in Food and Cosmetics

3. D-Count ® Technology Cell Labelling Cell Counting 12 minutes 1 minutes
Bacteria
Viability substrate
Enzyme
Cell Labelling
12 minutes
Free fluorochrome
Flow Cell
Detector
Cell Counting
1 minutes
Laser
Detector

4. FluorassureÒ Viability Markers
Enzyme
Viability substrate
Free fluorochrome
Bacteria
Criteria of Viability :
Enzyme activity
Membrane integrity
Applicable for :
Total Viable Count
Yeast & Moulds

5. Cell Labelling Technology
Fluorescent Signal
Fluorescence Based Cell Labels
515 nm
Incident Light
488 nm

6. D-Count ® : Counting Principle
Flow Cell :
Linear fluid stream
Cells separated into
single entities
Sensitive detectors
Detector 1
Detector 2
Labelled Sample
Laser
Sample

7. D-Count ® : Optical Bench
Sample
Blue light
Green emission
Detectors
Flow cell
Objective
Lens
Red emission
Beam splitter
Dichroic filter
Laser beam

8. Data Processing Data Processing Results PMT Detector
Digital Signal Processor collects blocks of data
Colour fingerprint
Light intensity
Signal shape
Rejected
Background
Results

9. D-Count ® Main Menu

10. Sample identification

11. Presentation of results

12. Presentation of results

13. Dedicated Automation Capacity High Throughput Automate
50 samples / batch
High Throughput Automate
up to 60 samples per hour
Simple sample pre-treatment
Product sample directly loaded onto automate
samples containing big particles are centrifiltrated before loading

14. D-Count ® Combining Speed with Sensitivity
Designed for non filterable products
Direct Counting in less than 30 minutes
Automated system
Automate capacity samples
High throughput
Sensitivity down to 5x102/ml sample
Linearity between 5x102 to 5x105/ml

15. 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

16. Real-time microbiology analysis in Food, beverages & cosmetics
B a c t i F l o w
Compact Microbial Analyser
Real-time microbiology analysis
in Food, beverages & cosmetics

17. Based on Proven Technologies
Fluorescent cell labelling (Fluorassure®)
Same reagent technologies as used on D-Count® & ChemScan® RDI
Validated on more than 500 strains
Bench Top Microbial Analyser
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

18. FluorassureÒ Viability Markers
Viability substrate
Criteria of Viability :
Enzyme activity
Membrane integrity
Enzyme
Bacteria
Free fluorochrome
Applicable for :
Total Viable Count
Total Count
Yeast & Moulds
Specifics (on going)

19. Quantitative Microbial Counting
Flow Cell :
Linear fluid stream
Cells Separated into
single entities
Sensitive detectors
Labelled Sample
Detector 1
Laser
Detector 2
B a c t i F l o w
Sample

20. No cell multiplication
R a p i d
Time to result:
10 minutes labelling prior to analysis
1 minute analysis per sample
Simple sample preparation adapted to product matrices
No cell multiplication
required
Labelling 10 min.
Counting 1 min.

21. S e n s i t i v e 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

22. E a s y t o U s e 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

23. B a c t i F l o w A p p l i c a t i o n s
Dairy Products
Fruit based Products
Cosmetics
Water
Pure cultures

24. D-COUNT ® and BACTIFLOW DISCRIMINANTS

25. Fingerprint Colour Transmission Wavelength (nm)
Fluorochrome Emission Spectrum
Green channel
Transmission
Red channel
Wavelength (nm)

26. Ratio at Primary Peak H2 Green channel « Peak ratio » = H1 H1 Red
Intensity H2
Green
channel
« Peak ratio » = H1 H1
Intensity
 Samples
Red
channel H2
Samples

27. Signal (Signal) - (Baseline) Fluorescence Intensity
Samples
(Signal) - (Baseline)
 Samples

28. Signalauswahl
Ausgewählte Signale
Schwellenwert
Hintergrund

29. D-COUNT ® and BACTIFLOW APPLICATIONS

30. Applications Beverages Food Fruit Juices Soft drinks concentrates
Milk products
Fruit products
Frozen vegetables
Salads

31. 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

32. Applications Final product In Process D-Count Environmental T V C
Microbial Limit Test
Presence / Absence test
In Process
Raw Materials
Bioburden
Cleaning
validation
Trending
T V C
Yeast & Moulds
E. coli (on going)
Other specifics
(on going)
D-Count
Environmental
Surface
Personnel

33. Applications Presence / Absence Test TVC - Direct Counting
24 hours
TVC - Direct Counting
90 minutes
Yeast / Moulds - Direct Counting
E. coli and coliform tests (availabilty sept. - oct. 01)
20 hours

34. 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

35. D-COUNT Sample Background + Spiking Experiments

36. D-Count Evaluation Pure culture correlation studies
Non contaminated product analysis
Inoculated product analysis

37. Pure Cultures Studies Range of micro-organisms tested Methods
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

38. D-Count ® Evaluation Pure culture correlation studies
Non contaminated product analysis
Inoculated product analysis

39. Pure Culture Correlation
Enterobacter cloacae Candida albicans ATCC 10231

40. Pure Culture Correlation
Staph. aureus ATCC 6538P Bacillus circulans

41. Pure Culture Correlation
E. coli ATCC Bacillus licheniformis

42. Pure Culture Correlation
Aspergillus niger Penicillium decumbeus

43. Pure Culture Correlation
Combined plot of all micro-organisms analysed
Staphylococcus aureus
Escherichia coli
Bacillus circulans
Bacillus licheniformis
Enterobacter cloacae
Candida albicans

44. 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