Tomorrows Results Today

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D- CountApplicationsfor the Food and Cosmetic
IndustryElke Kohler, Chemunex SA, Paris
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D-Count Non-Filterable Product Testing
Real-time Microbiology Analysis in Food and
Cosmetics
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D-Count Technology
Viability substrate
Bacteria
Enzyme
Cell Labelling 12 minutes
Free fluorochrome
Flow Cell
Detector
Cell Counting 1 minutes
Laser
Detector
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FluorassureÒ Viability Markers

• Criteria of Viability
• Enzyme activity
• Membrane integrity

• Applicable for
• Total Viable Count
• Yeast Moulds

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Cell Labelling Technology
Fluorescent Signal
Fluorescence Based Cell Labels
515 nm
IncidentLight
488 nm
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D-Count Counting Principle

• Flow Cell
• Linear fluid stream
• Cells separated into
• single entities
• Sensitive detectors

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D-Count Optical Bench
Sample

• Green emission

• Blue light

Detectors
Flow cell
Objective Lens
Red emission
Dichroic filter
Beam splitter
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
• 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

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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

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D-CountBenefits 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

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

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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)

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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
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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.
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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

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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

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B a c t i F l o w A p p l i c a t i o n s
Dairy Products
Cosmetics
Fruit based Products
Water
Pure cultures
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D-COUNT and BACTIFLOW DISCRIMINANTS
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Fingerprint Colour
Fluorochrome Emission Spectrum
Green channel
Transmission
Red channel
Wavelength (nm)
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Ratio at Primary Peak
Intensity
H2
Green channel

 Peak ratio 
H1
H1
Intensity
 Samples
Red channel
H2
Samples
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Signal
Fluorescence Intensity
Fluorescence Intensity
Baseline
Samples
(Signal) - (Baseline)
 Samples
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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
• Raw Materials
• Bioburden
• Cleaning
• validation
• Trending

T V C Yeast Moulds E. coli (on going) Other
specifics (on going)
D-Count

• Environmental
• Surface
• Personnel

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Applications

• Presence / Absence Test
• 24 hours
• TVC - Direct Counting
• 90 minutes
• Yeast / Moulds - Direct Counting
• 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 30C
  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
Enterobacter cloacae Candida albicans ATCC
10231
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Pure Culture Correlation
Staph. aureus ATCC 6538P Bacillus circulans
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Pure Culture Correlation
E. coli ATCC 8739 Bacillus licheniformis
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Pure Culture Correlation
Aspergillus niger Penicillium decumbeus
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Pure Culture Correlation
Combined plot of all micro-organisms analysed
Staphylococcus aureus Escherichia coli Bacillus
circulans Bacillus licheniformis Enterobacter
cloacae Candida albicans
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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