Title: Tomorrows Results Today
1D- CountApplicationsfor the Food and Cosmetic
IndustryElke Kohler, Chemunex SA, Paris
2D-Count Non-Filterable Product Testing
Real-time Microbiology Analysis in Food and
Cosmetics
3D-Count Technology
Viability substrate
Bacteria
Enzyme
Cell Labelling 12 minutes
Free fluorochrome
Flow Cell
Detector
Cell Counting 1 minutes
Laser
Detector
4FluorassureÒ Viability Markers
- Criteria of Viability
- Enzyme activity
- Membrane integrity
- Applicable for
- Total Viable Count
- Yeast Moulds
5Cell Labelling Technology
Fluorescent Signal
Fluorescence Based Cell Labels
515 nm
IncidentLight
488 nm
6D-Count Counting Principle
- Flow Cell
- Linear fluid stream
- Cells separated into
- single entities
- Sensitive detectors
7D-Count Optical Bench
Sample
Detectors
Flow cell
Objective Lens
Red emission
Dichroic filter
Beam splitter
Laser beam
8Data Processing
PMT Detector
Data Processing
Digital Signal Processor collects blocks of data
- Colour fingerprint
- Light intensity
- Signal shape
Rejected Background
Results
9D-Count Main Menu
10Sample identification
11Presentation of results
12Presentation of results
13Dedicated 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
14D-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
15D-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
16B a c t i F l o w
Compact Microbial Analyser
Real-time microbiology analysis in Food,
beverages cosmetics
17Based 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
18FluorassureÒ 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)
19Quantitative 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
20R 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.
21S 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
22E 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
23B 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
24D-COUNT and BACTIFLOW DISCRIMINANTS
25Fingerprint Colour
Fluorochrome Emission Spectrum
Green channel
Transmission
Red channel
Wavelength (nm)
26Ratio at Primary Peak
Intensity
H2
Green channel
Peak ratio
H1
H1
Intensity
Samples
Red channel
H2
Samples
27Signal
Fluorescence Intensity
Fluorescence Intensity
Baseline
Samples
(Signal) - (Baseline)
Samples
28Signalauswahl
Ausgewählte Signale
Schwellenwert
Hintergrund
29D-COUNT and BACTIFLOW APPLICATIONS
30Applications
- Beverages
- Fruit Juices
- Soft drinks concentrates
- Food
- Milk products
- Fruit products
- Frozen vegetables
- Salads
31Applicable to Range of Products
- Non sterile pharmaceuticals
- Syrups
- Ointments
- Tablets
- Lotions
- Cosmetic products
- Shampoos and hair conditioner
- Creams
- Make-up and Foundation
- Toothpaste
- Lotions
32Applications
- 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
33 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
34Food 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
35D-COUNT Sample Background Spiking Experiments
36D-Count Evaluation
- Pure culture correlation studies
- Non contaminated product analysis
- Inoculated product analysis
37Pure 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
38D-Count Evaluation
- Pure culture correlation studies
- Non contaminated product analysis
- Inoculated product analysis
39Pure Culture Correlation
Enterobacter cloacae Candida albicans ATCC
10231
40Pure Culture Correlation
Staph. aureus ATCC 6538P Bacillus circulans
41Pure Culture Correlation
E. coli ATCC 8739 Bacillus licheniformis
42Pure Culture Correlation
Aspergillus niger Penicillium decumbeus
43Pure Culture Correlation
Combined plot of all micro-organisms analysed
Staphylococcus aureus Escherichia coli Bacillus
circulans Bacillus licheniformis Enterobacter
cloacae Candida albicans
44Conclusions
- 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