The Rapid Microbiology Company D- Count ® Applications for the Food and Cosmetic Industry Elke Kohler, Chemunex SA, Paris

The Rapid Microbiology Company

D- D- CountCount®®

ApplicationsApplicationsfor the Food and Cosmetic for the Food and Cosmetic

IndustryIndustry

Elke Kohler, Chemunex SA, ParisElke Kohler, Chemunex SA, Paris


D-D-Count Count ®® : : Non-Filterable Product TestingNon-Filterable Product Testing

Real-time Microbiology Analysis in Food and Cosmetics


D-D-Count Count ®® Technology Technology

EnzymeViability substrate

Free fluorochrome

Bacteria

Detector

Detector

Laser

Flow Cell

Cell Labelling

12 minutes

Cell Counting

1 minutes


FluorassureFluorassure Viability Markers Viability Markers

Criteria of Viability :

Enzyme activity Membrane integrity

Enzyme

Viability substrate

Free fluorochrome

Bacteria

Applicable for :

Total Viable Count Yeast & Moulds


Cell Labelling TechnologyCell Labelling Technology

Fluorescent Signal

IncidentLight

515 nm

488 nm

Fluorescence Based Cell Labels


D-D-Count Count ® : Counting Principle : Counting Principle

Detector 1

Detector 2

Labelled Sample

Laser

Sample

Flow Cell :

Linear fluid stream Cells separated into single entities Sensitive detectors


D-D-Count Count ®® : Optical Bench: Optical Bench

Sample

Dichroic filter

Flow cell

Objective Lens

Beam splitter

Red emission

Detectors

Laser beam


Data ProcessingData Processing

Data Processing

Colour fingerprint

Light intensity

Signal shape

Results

RejectedBackground

PMT Detector

Digital Signal Processor collects blocks of data


D-D-Count Count ®® Main Menu Main Menu


Sample identificationSample identification


Presentation of resultsPresentation of results


Presentation of resultsPresentation of results


Dedicated Automation 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


D-D-Count 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


D-CountD-CountBenefits from speed and sensitivityBenefits 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


B a c t i B a c t i F l o wF l o w

Real-time microbiology analysis in Food, beverages & cosmetics

Compact Microbial AnalyserCompact Microbial Analyser


Based on Proven TechnologiesBased 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


FluorassureFluorassure Viability Markers Viability Markers

Criteria of Viability :

Enzyme activity Membrane integrity

Enzyme

Viability substrate

Free fluorochromeBacteria

Applicable for :

Total Viable CountTotal Count Yeast & Moulds Specifics (on going)


Quantitative Microbial CountingQuantitative Microbial Counting

Detector 1

Detector 2

Labelled Sample

Laser

Sample

Flow Cell :

Linear fluid stream

Cells Separated into single entities

Sensitive detectors

B a c t i B a c t i F l o wF l o w


R a p i dR 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

Labelling 10 min. Counting 1 min.

No cell multiplication required


S e n s i t i v eS 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


E a s y t o U s eE 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


B a c t i B a c t i F l o w F l o w A p p l i c a t i o n sA p p l i c a t i o n s

Fruit based Products Dairy Products Cosmetics

WaterPure cultures


D-D-COUNTCOUNT ®® and BACTI and BACTIFLOW FLOW DISCRIMINANTSDISCRIMINANTS


Fingerprint ColourFingerprint ColourT

rans

mis

sion

Wavelength (nm)

Fluorochrome Emission Spectrum

Green channel

Red channel


Ratio at Primary PeakRatio at Primary Peak

Green channel

Red channel

« Peak ratio »

Intensity

Intensity Samples

Samples

=

H1

H2

H2

H1


SignalSignal

(Signal) - (Baseline)

Fluorescence Intensity

Samples

Baseline

Samples

Fluorescence Intensity


SignalauswahlSignalauswahl

Hintergrund

Schwellenwert

Ausgewählte Signale


D-D-COUNT COUNT ® ® and BACTI and BACTIFLOWFLOW APPLICATIONSAPPLICATIONS


ApplicationsApplications

Beverages

Fruit Juices

Soft drinks concentrates

Food

Milk products

Fruit products

Frozen vegetables

Salads


Applicable to Range of ProductsApplicable to Range of Products

Non sterile pharmaceuticals

• Syrups

• Ointments• Tablets• Lotions

Cosmetic products• Shampoos and hair conditioner

• Creams• Make-up and Foundation• Toothpaste• Lotions


Applications Applications

D-Count

In Process

Raw Materials Bioburden Cleaning

validation Trending

Final product Microbial Limit Test Presence / Absence test

Environmental Surface Personnel

T V C

Yeast & Moulds

E. coli (on going)

Other specifics(on going)


ApplicationsApplications

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


Food & BeveragesFood & 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


D-COUNT D-COUNT SampleSample Background + Spiking ExperimentsBackground + Spiking Experiments


D-Count EvaluationD-Count Evaluation

Pure culture correlation studies

Non contaminated product analysis

Inoculated product analysis


Pure Cultures StudiesPure Cultures Studies

Range of micro-organisms tested

Staphylococcus aureus Escherichia coli Bacillus circulans Bacillus licheniformis

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

Enterobacter cloacae Candida albicans Aspergillus niger Penicillium decumbeus


D-D-Count Count ®® Evaluation Evaluation

Pure culture correlation studies

Non contaminated product analysis

Inoculated product analysis


Pure Culture CorrelationPure Culture Correlation

Enterobacter cloacae Candida albicans ATCC 10231

R2 = 0,9992

10

100

1000

10000

100000

1000000

10 100 1000 10000 100000 1000000

Plate Count

D-C

ount

( b

acte

ria /

100µ

l)

R2 = 0,9964

1

10

100

1000

10000

1 10 100 1000 10000Plate Count

D-C

ount

(yea

st /1

00µl

)



R2 = 0,996

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000

Plate Count

D-C

ou

nt

( b

ac

teri

a /

10

0µ

l) R2 = 0,9961

1

10

100

1000

1 10 100 1000

Plate count

D-C

ount

( ba

cter

ia /

100µ

l)

Staph. aureus ATCC 6538P Bacillus circulans



E. coli ATCC 8739 Bacillus licheniformis

R2 = 0,9961

1

10

100

1000

1 10 100 1000

Plate count

D-C

ou

nt

( b

acte

ria

/ 100

µl)

R2 = 0,9949

1

10

100

1000

10000

1 10 100 1000

Plate count

D-C

ou

nt

( b

acte

ria

/ 100

µl)



Aspergillus niger Penicillium decumbeus

R2 = 0,9968

1

10

100

1000

1 10 100 1000

Plate count

D-C

ount

(Mou

lds

/ 100

µl) R2 = 0,9571

1

10

100

1000

1 10 100 1000

Plate count D

-Cou

nt (M

ould

s / 1

00 µ

l)



R2 = 0,9919

1

10

100

1000

10000

100000

1000000

1 10 100 1000 10000 100000 1000000Plate Count

D-C

ou

nt

( M

icro

-org

anis

ms

/ 10

0µl)

Combined plot of all micro-organisms analysed

Staphylococcus aureusEscherichia coliBacillus circulansBacillus licheniformisEnterobacter cloacaeCandida albicans


ConclusionsConclusions

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

Documents

The Rapid Microbiology Company D- Count ® Applications for the Food and Cosmetic Industry Elke Kohler, Chemunex SA, Paris