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D- Count® Applications for the Food and Cosmetic Industry Elke Kohler, Chemunex SA, Paris 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 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 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 Ò Viability substrate Criteria of Viability : Enzyme è Enzyme activity Fluorassure Viability Markers Ò Viability substrate Criteria of Viability : Enzyme è Enzyme activity è Membrane integrity Bacteria Free fluorochrome Applicable for : è Total Viable Count è Yeast & Moulds The Rapid Microbiology Company

Cell Labelling Technology Fluorescence Based Cell Labels Fluorescent Signal 515 nm Incident Light 488 Cell Labelling Technology Fluorescence Based Cell Labels Fluorescent Signal 515 nm Incident Light 488 nm The Rapid Microbiology Company

D-Count ® : Counting Principle Flow Cell : Labelled Sample è Linear fluid stream D-Count ® : Counting Principle Flow Cell : Labelled Sample è Linear fluid stream è Cells separated into single entities è Sensitive detectors Detector 1 Laser Detector 2 Sample The Rapid Microbiology Company

D-Count ® : Optical Bench Sample Detectors Flow cell Objective Lens Red emission Beam D-Count ® : Optical Bench Sample Detectors Flow cell Objective Lens Red emission Beam splitter Dichroic filter Laser beam The Rapid Microbiology Company

Data Processing PMT Detector Data Processing Digital Signal Processor collects blocks of data è Data Processing PMT Detector Data Processing Digital Signal Processor collects blocks of data è Colour fingerprint è Light intensity è Signal shape Rejected Background Results The Rapid Microbiology Company

D-Count ® Main Menu The Rapid Microbiology Company D-Count ® Main Menu The Rapid Microbiology Company

Sample identification The Rapid Microbiology Company Sample identification The Rapid Microbiology Company

Presentation of results The Rapid Microbiology Company Presentation of results The Rapid Microbiology Company

Presentation of results The Rapid Microbiology Company Presentation of results The Rapid Microbiology Company

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

D-Count ® Combining Speed with Sensitivity l Designed for non filterable products l Direct D-Count ® Combining Speed with Sensitivity l Designed for non filterable products l Direct Counting in less than 30 minutes l Automated system l Automate capacity 50 -64 samples l High throughput l Sensitivity down to 5 x 102/ml sample l Linearity between 5 x 102 to 5 x 105/ml The Rapid Microbiology Company

D-Count Benefits from speed and sensitivity l Final products testing in hours not days D-Count Benefits from speed and sensitivity l 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 ä ä Decrease inventory holding and warehouse costs Decrease product losses Minimise production disruption Increase production flexibility l l The Rapid Microbiology Company

B a c t i F l o w Compact Microbial Analyser Real-time microbiology B a c t i F l o w Compact Microbial Analyser Real-time microbiology analysis in Food, beverages & cosmetics The Rapid Microbiology Company

Based on Proven Technologies l Fluorescent cell labelling (Fluorassure®) è è l Bench Top Based on Proven Technologies l Fluorescent cell labelling (Fluorassure®) è è l Bench Top Microbial Analyser è è è l Same reagent technologies as used on D-Count® & Chem. Scan® 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 è è Automatic data interpretation Quantitative results in minutes The Rapid Microbiology Company

Fluorassure Viability Markers Ò Viability substrate Criteria of Viability : Enzyme è Enzyme activity Fluorassure Viability Markers Ò Viability substrate Criteria of Viability : Enzyme è Enzyme activity è Membrane integrity Bacteria Free fluorochrome Applicable for : è Total Viable Count è Total Count è Yeast & Moulds è Specifics (on going) The Rapid Microbiology Company

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

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

S e n s i t i v e Combining Speed with Sensitivity l S e n s i t i v e Combining Speed with Sensitivity l l Quantitative counting cell by cell Sensitivity of detection less than 100 cells/ml labelled solution l Linear response up to 100, 000 micro-organisms l Direct Counting in less than 15 minutes l Protocol available for Presence / Absence within 24 hours The Rapid Microbiology Company

E a s y t o U s e l Ready to use Reagents E a s y t o U s e l Ready to use Reagents l Simple preparation for sample products l Validated protocols for a full range of applications l Fully automated detection and counting l No data interpretation required l Full data traceability l PC with Windows XP interface The Rapid Microbiology Company

B a c t i F l o w Fruit based Products Pure cultures B a c t i F l o w Fruit based Products Pure cultures A p p l i c a t i o n s Dairy Products Cosmetics Water The Rapid Microbiology Company

D-COUNT ® and BACTIFLOW DISCRIMINANTS The Rapid Microbiology Company D-COUNT ® and BACTIFLOW DISCRIMINANTS The Rapid Microbiology Company

Fingerprint Colour Fluorochrome Emission Spectrum Transmission Green channel Red channel Wavelength (nm) The Rapid Fingerprint Colour Fluorochrome Emission Spectrum Transmission Green channel Red channel Wavelength (nm) The Rapid Microbiology Company

Ratio at Primary Peak Intensity Green channel « Peak ratio » = H 1 Ratio at Primary Peak Intensity Green channel « Peak ratio » = H 1 Intensity Red channel H 2 H 1 Samples H 2 Samples The Rapid Microbiology Company

Signal Fluorescence Intensity Baseline Samples (Signal) - (Baseline) The Rapid Microbiology Company Signal Fluorescence Intensity Baseline Samples (Signal) - (Baseline) The Rapid Microbiology Company

Signalauswahl Ausgewählte Signale Schwellenwert Hintergrund The Rapid Microbiology Company Signalauswahl Ausgewählte Signale Schwellenwert Hintergrund The Rapid Microbiology Company

D-COUNT ® and BACTIFLOW APPLICATIONS The Rapid Microbiology Company D-COUNT ® and BACTIFLOW APPLICATIONS The Rapid Microbiology Company

Applications Beverages l Fruit Juices l Soft drinks concentrates Food l Milk products l Applications Beverages l Fruit Juices l Soft drinks concentrates Food l Milk products l Fruit products l Frozen vegetables l Salads The Rapid Microbiology Company

Applicable to Range of Products Non sterile pharmaceuticals • Syrups • Ointments • Tablets 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 The Rapid Microbiology Company

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

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

Food & Beverages Applications l Yeast detection in Fermented milk products l TVC or Food & Beverages Applications l Yeast detection in Fermented milk products l TVC or Yeast counts in Juice concentrates l “Sterility test” in Desserts l l TVC or Yeast & mould detection in Fruit juices & beverages TVC in Process Water The Rapid Microbiology Company

D-COUNT Sample Background + Spiking Experiments The Rapid Microbiology Company D-COUNT Sample Background + Spiking Experiments The Rapid Microbiology Company

D-Count Evaluation l Pure culture correlation studies l Non contaminated product analysis l Inoculated D-Count Evaluation l Pure culture correlation studies l Non contaminated product analysis l Inoculated product analysis The Rapid Microbiology Company

Pure Cultures Studies l Range of micro-organisms tested è è Staphylococcus aureus Escherichia coli Pure Cultures Studies l Range of micro-organisms tested è è Staphylococcus aureus Escherichia coli Bacillus circulans Bacillus licheniformis è è Enterobacter cloacae Candida albicans Aspergillus niger Penicillium decumbeus l Methods è Micro-organisms isolated from plates è Selected colony incubated for 24 hours at 30°C in TSB è Serial dilution in physiological water è Analysis by D-Count & plate The Rapid Microbiology Company

D-Count ® Evaluation l Pure culture correlation studies l Non contaminated product analysis l D-Count ® Evaluation l Pure culture correlation studies l Non contaminated product analysis l Inoculated product analysis The Rapid Microbiology Company

Pure Culture Correlation Enterobacter cloacae Candida albicans ATCC 10231 The Rapid Microbiology Company Pure Culture Correlation Enterobacter cloacae Candida albicans ATCC 10231 The Rapid Microbiology Company

Pure Culture Correlation Staph. aureus ATCC 6538 P Bacillus circulans The Rapid Microbiology Company Pure Culture Correlation Staph. aureus ATCC 6538 P Bacillus circulans The Rapid Microbiology Company

Pure Culture Correlation E. coli ATCC 8739 Bacillus licheniformis The Rapid Microbiology Company Pure Culture Correlation E. coli ATCC 8739 Bacillus licheniformis The Rapid Microbiology Company

Pure Culture Correlation Aspergillus niger Penicillium decumbeus The Rapid Microbiology Company Pure Culture Correlation Aspergillus niger Penicillium decumbeus The Rapid Microbiology Company

Pure Culture Correlation Combined plot of all micro-organisms analysed Staphylococcus aureus Escherichia coli Bacillus Pure Culture Correlation Combined plot of all micro-organisms analysed Staphylococcus aureus Escherichia coli Bacillus circulans Bacillus licheniformis Enterobacter cloacae Candida albicans The Rapid Microbiology Company

Conclusions l Good correlation with all micro-organisms tested l Sensitivity of detection less than Conclusions l Good correlation with all micro-organisms tested l Sensitivity of detection less than 100 cells per ml l Linear response up to 5 x 10 e 5 micro-organisms per ml The Rapid Microbiology Company